WO2019049663A1

WO2019049663A1 - Method for assessing suitability of artificial protein including amino acid residue derived from non-naturally-occurring amino acid, for synthesis by ribosomes

Info

Publication number: WO2019049663A1
Application number: PCT/JP2018/030934
Authority: WO
Inventors: 賢三小俣
Original assignee: 賢三小俣
Priority date: 2017-09-08
Filing date: 2018-08-22
Publication date: 2019-03-14

Abstract

The present invention addresses the problem of providing a method for assessing whether or not an artificial protein having a specific introduced non-naturally-occurring amino acid can be synthesized by the synthesis system provided by ribosomes. This method comprises steps a and b. (Step a): A step for performing transition state structure optimization calculation (TS optimization calculation) by a molecular orbital method or performing approximation calculation which can reproduce the transition state optimization structure, using, as an initial structure, a reaction transition state (general formula (3-1), (3-2), (3-3), (3-4), or (3-5)) in a peptidyl group transfer reaction between aminoacyl-tRNA of the non-naturally-occurring amino acid represented by general formula (1), and peptidyl-tRNA having any amino acid sequence represented by general formula (2-1) or (2-2). (Step b) A step for determining that the non-naturally-occurring amino acid has high enough suitability to be introduced into a protein in the protein synthesis system provided by ribosomes if the structure obtained as a result of step a can be assumed to be a transition state structure.

Description

Method for estimating suitability for synthesis by ribosomes of artificial proteins containing amino acid residues derived from unnatural amino acids

The present invention relates to a method of estimating the suitability of artificial proteins for synthesis by ribosomes by molecular orbital method.

Proteins are widely used in medical and various industrial fields. The biological synthesis method by E. coli etc. which is the main manufacturing method has greatly developed in recent years as protein engineering. In protein engineering, it is also possible to arbitrarily design the sequence order of natural 20 kinds of amino acids and to produce non-naturally occurring proteins accordingly. Such techniques for producing non-naturally occurring proteins can significantly improve the functions and physical properties of proteins.

In conventional protein engineering methods, only 20 natural amino acids can be used as materials. However, in recent years, techniques for synthesizing artificial proteins in which non-natural amino acids are incorporated into proteins have been developed. According to the technology of synthesizing an artificial protein incorporating a non-natural amino acid, it is possible to dramatically increase the variety of physical properties and functions that can be imparted to the artificial protein.
As a technique for incorporating a non-naturally occurring amino acid into an artificial protein, there is a method of making a non-naturally occurring amino acid correspond to an amber codon which is one of the stop codons (Patent Document 1) or a method using four base codons (eg Patent Document 2) ), A reprogramming method of the genetic code (non-patent documents 1 to 3) of assigning a non-natural amino acid newly by emptying a specific codon (non-patent documents 1 to 3), a method of creating artificial codons using non-natural bases (non-patent document) 4) is known.

JP, 2006-180701, A Japanese Patent Application Publication No. 2006-280250

As described above, various techniques for introducing unnatural amino acids into proteins have been developed.
Here, unnatural amino acids are naturally different in structure from natural amino acids. Therefore, it is easy to imagine that unnatural amino acids far from natural amino acid structures, such as those having a large structure in the side chain, can not be synthesized by ribosomal protein synthesis systems.

Thus, when a non-natural amino acid is introduced into a protein by a ribosomal protein synthesis system, the structure and properties of the non-natural amino acid have certain limitations. However, it was impossible to predict what kind of structure of unnatural amino acids could be synthesized by a ribosomal protein synthesis system.

In view of these problems, the problem to be solved by the present invention is to provide a method for estimating whether an artificial protein into which a specific non-natural amino acid has been introduced can be synthesized by a ribosomal synthesis system. .

As described above, it was impossible to predict what kind of structure of unnatural amino acid could be synthesized by a ribosomal protein synthesis system. The biggest reason for this unpredictability is that the details of the amidation reaction on the ribosome are unknown.

The present inventor has clarified the details of the amidification reaction mechanism on the ribosome by the three-dimensional structure information by X-ray analysis of the ribosome and the knowledge of the amidization transition state structure by molecular orbital calculation. From this, the relationship between the amidation reaction on the ribosome and the amino acid which is the raw material is elucidated, and the condition of the non-natural amino acid suitable for the ribosomal protein synthesis system is found, and the present invention has been made.

The present invention which solves the above-mentioned subject is as follows.

<1> A method for estimating the adaptability of an unnatural amino acid to a protein in a ribosomal protein synthesis system, comprising the following steps a and b:
(Step a)
An aminoacyl-tRNA of the unnatural amino acid represented by the general formula (1);
A peptidyl tRNA having any amino acid sequence represented by the general formula (2-1) or (2-2),
Reaction transition state (general formula (3-1), (3-2), (3-3), (3-4) or (3-5)) or its structure reaction in the peptidyl group transfer reaction of A step of performing transition state structure optimization calculation (TS optimization calculation) by the molecular orbital method with a structure omitted as appropriate in order to reduce calculation load from a part far from the center as an initial structure.
(Step b)
A step of judging that the adaptability of the unnatural amino acid to the protein in the protein synthesis system by ribosome is high when it can be confirmed that the structure obtained as a result of the step a is a transition state structure.

(A ^a1 represents a structural moiety other than an amino group and a carboxyl group involved in the peptide bond in the non-natural amino acid.
B ^a1 represents an arbitrary group or a group which forms a cyclic structure of B ^a1 -A ^a1 -N ^t1 together with the above-mentioned A ^a1 .
The tRNA ^a1 represents a tRNA covalently linked to O ^{a12 at} the 3 'end. )

(TRNA ^P1 represents a tRNA covalently bound to ^{O P1} in the 3 'end.
P ^P1 represents the peptidyl group. )

(Structural portion of the ^tRNA ^P12 ^-O P12 ^-H P12 has the general formula and tRNA ^P1 in (2-1) represents the same structure. ^Namely, the oxygen ^{O P12} is bound to 2 'carbon of the 3'-terminal sugar of tRNA ^P1 The atom, ^HP12 is a hydrogen atom bonded to ^OP12 , and tRNA ^P12 is a portion of tRNA ^P1 from which ^{OP 12} and ^{HP 12} have been removed.
The meaning of the symbols in the general formula (2-2) is the same as in the general formula (2-1). )

(Structure N ^t1 (H ^t1 ) -C ^t1 (O ^t1 ) moiety is a reaction center in the reaction transition state structure of the first step of stepwise amidification reaction,
The distance between N ^t1 and C ^t1 is 1.53 Å to 1.77 Å,
The distance between N ^t1 and H ^t1 is 1.10 Å to 1.35 Å.
The distance between H ^t1 and O ^t1 is 1.35 Å to 1.45 Å.
The meaning of the symbols in the general formula (3-1) is the same as in the general formulas (1) and (2-1). )

(H ^t12 is a hydrogen atom which protonated O ^t1 , which was carbonyl oxygen in the first step of the stepwise amidation reaction.
The moiety C ^t1 -O ^p1 -H ^t12 -O ^t1 is the reaction center in the reaction transition state structure of the second step of the stepwise amidification reaction,
The distance between C ^t1 and O ^p1 is 1.45 Å to 1.70 Å.
Distance of O ^p1 and ^{H t12} is 1.05Å ~ 1.30Å,
The distance between H ^t12 and O ^t1 is 1.10 Å to 1.40 Å,
The distance between O ^t1 and C ^t1 is 1.22 Å to 1.42 Å.
The meaning of the symbols in the general formula (3-2) is the same as in the general formulas (1) and (2-1). )

(Structure N ^t1 (H ^t1 ) -C ^t1 (O ^p1 ) moiety is a reaction center in the transition state structure of a concerted amidation reaction having a four-membered ring structure,
The distance between N ^t1 and C ^t1 is 1.40 Å to 1.65 Å,
The distance between N ^t1 and H ^t1 is 1.00 Å to 1.24 Å,
The distance between C ^t1 and O ^p1 is 1.35 Å to 2.40 Å.
Distance O ^p1 and ^{H t1} is 1.00Å ~ 1.84Å.
The meaning of the symbols in the general formula (3-3) is the same as in the general formulas (1) and (2-1). )

(Structure N ^t1 -C ^t1 -O ^p1 -H ^p12 -O ^p12 -H ^t1 moiety is the reaction center in the transition state structure of the concerted amidation reaction having a six-membered ring structure,
The distance between N ^t1 and C ^t1 is 1.40 Å to 2.10 Å.
The distance between N ^t1 and H ^t1 is 1.00 Å to 1.20 Å,
The distance between C ^t1 and O ^p1 is 1.35 Å to 2.30 Å.
Distance of O ^p1 and ^{H p12} is 1.00Å ~ 1.56Å,
The distance between H ^p12 and O ^p12 is 1.00 Å to 1.67 Å,
The distance between Op ¹² and H ^t1 is 1.20 Å to 1.96 Å.
The meaning of the symbols in the general formula (3-4) is the same as in the general formulas (1) and (2-2). )

(H ^s12 -O ^s1 -H ^s11 is one of the solvent water molecules, and the structure N ^t1 -C ^t1 -O ^p1 -H ^s11 -O ^s1 -H ^p12 -O ^p12 -H ^t1 moiety has an eight-membered ring structure A reaction center in the transition state structure of the concerted amidification reaction,
The distance between N ^t1 and C ^t1 is 1.40 Å to 1.79 Å.
The distance between N ^t1 and H ^t1 is 1.00 Å to 1.26 Å,
The distance between C ^t1 and O ^p1 is 1.40 Å to 2.21 Å,
Distance of O ^p1 and ^{H s12} is 1.13Å ~ 1.43Å,
The distance between H ^s12 and O ^s1 is 1.00 Å to 1.37 Å,
The distance between O ^s1 and H ^p12 is 1.30 Å to 1.69 Å,
The distance between H ^p12 and O ^p12 is 0.98 Å to 1.24 Å,
The distance between Op ¹² and H ^t1 is 1.17 Å to 1.76 Å.
The meaning of the symbols in the general formula (3-5) is the same as in the general formulas (1) and (2-2). )

<2> The method according to Item 1, wherein the general formula (3-1) is the following general formula (3-1-1).

(The meaning of the symbol in the general formula (3-1-1) is the same as in the general formula (3-1).)

The structure represented by <3> said General formula (1) is a structure represented by following General formula (4),
The structures represented by the general formulas (3-1), (3-2), (3-3), (3-4), and (3-5) have the following general formulas (5-1) and (5-3), respectively. 5. The method according to item 1, wherein the structure is represented by 5-2), (5-3), (5-4), (5-5).

(C ^α1 is an α carbon of the non-natural amino acid, and R ^a1 and R ^a2 each independently represent a side chain structure of the non-natural amino acid (however, R ^a2 may be a hydrogen atom).
Other symbols are the same as in the general formula (1). )

(The distances between N ^t1 and C ^t1 , N ^t1 and H ^t1 , and H ^t1 and O ^t1 are the same as in the general formula (3-1).
The meaning of the symbols in the general formula (5-1) is the same as in the general formulas (1), (2-1) and (4). )

(The meanings of the symbols R ^a1 , C ^α1 and R ^a2 are the same as in the general formula (4). The meanings of the symbols in the general formula (5-2) and the interatomic distance are the same as in the general formula (3-2). )

(The meanings of the symbols R ^a1 , C ^α1 and R ^a2 are the same as in the general formula (4). The meaning of the symbols in the general formula (5-3) and the interatomic distance are the same as in the general formula (3-3). )

(The meanings of the symbols R ^a1 , C ^α1 and R ^a2 are the same as in the general formula (4). The meanings of the symbols in the general formula (5-4) and the interatomic distance are the same as in the general formula (3-4). )

(The meanings of the symbols R ^a1 , C ^α1 and R ^a2 are the same as in the general formula (4). The meaning of the symbols in the general formula (5-5) and the interatomic distance are the same as in the general formula (3-5). )

<4> A method for estimating the adaptability of an unnatural amino acid to a protein in a protein synthesis system by ribosome, comprising the following steps of c to e.
(Step c)
Prepare three-dimensional structural data obtained by X-ray structural analysis of ribosome in which tRNA or its derivative is bound to A and P sites,
In the three-dimensional structure data, according to the following conditions:
An aminoacyl-tRNA of the unnatural amino acid represented by the general formula (1);
A peptidyl tRNA having any amino acid sequence represented by the general formula (2-1) or (2-2),
A step of incorporating the assumed reaction transition state (general formula (3-1), (3-2), (3-3), (3-4) or (3-5)) in the peptidyl group transfer reaction of
(Condition 1) The general formula (3) is such that the atomic coordinates of the 3 'terminal carbon of tRNA ^a1 are located at least near the 3' terminal carbon of the tRNA at the position of the tRNA on the A site in the three-dimensional structure data. 1) Incorporate the assumed structure represented by (3-2), (3-3), (3-4) or (3-5).
(Condition 2) The atomic formula of the 3 'terminal carbon of tRNA ^P1 is located at least near the 3' terminal carbon of the tRNA at the position of the tRNA on the P site in the three-dimensional structure data, 1) Incorporate the assumed structure represented by (3-2), (3-3), (3-4) or (3-5).
(Step d)
Transition state structure optimization calculation (TS optimization calculation) by molecular orbital method with a structure obtained by appropriately removing the structure far from the reaction center of the structure obtained by the step c or the structure, in order to reduce the calculation load as an initial structure Process of
(Step e)
A step of judging that the adaptability of the unnatural amino acid to the protein in the protein synthesis system by ribosome is high when the structure obtained as a result of the step d is confirmed to be a transition state structure.

(Structural portion of the ^tRNA P12 ^-O P12 ^{-HP 12} has the general formula and tRNA ^P1 in (2-1) represents the same structure. ^Namely, the oxygen ^{O P12} is bound to 2 'carbon of the 3'-terminal sugar of tRNA ^P1 The atom, ^HP12 is a hydrogen atom bonded to ^OP12 , and tRNA ^P12 is a portion of tRNA ^P1 from which ^{OP 12} and ^{HP 12} have been removed.
The meaning of the symbols in the general formula (2-2) is the same as in the general formula (2-1). )

(H ^t12 is a hydrogen atom that protonated O ^t1 , which was carbonyl oxygen in the second step of the stepwise amidation reaction.
The moiety C ^t1 -O ^p1 -H ^t12 -O ^t1 is the reaction center in the reaction transition state structure of the second step of the stepwise amidification reaction,
The distance between C ^t1 and O ^p1 is 1.45 Å to 1.70 Å.
Distance of O ^p1 and ^{H t12} is 1.05Å ~ 1.30Å,
The distance between H ^t12 and O ^t1 is 1.10 Å to 1.40 Å,
The distance between O ^t1 and C ^t1 is 1.22 Å to 1.42 Å.
The meaning of the symbols in the general formula (3-2) is the same as in the general formulas (1) and (2-1). )

<5> The method according to Item 4, comprising the following e ′ step after the e step.
(E 'process)
The structure confirmed to be a transition state structure in the step e) is
The cyclic structure containing the reaction center (in a form incorporating the structure represented by the general formula (3-4) or the general formula (3-5), the cyclic structure containing tRNA ^P12 of the general formula is included ) By adjusting the twist angle of the bond axis of the other part within the range that steric hindrance does not occur, the three-dimensional carbon corresponding atom of tRNA ^{a1 and} the three-terminal carbon corresponding atom of tRNA ^P1 are respectively three-dimensional In the case where the displacement between the terminal 3 'carbon of tRNA on the A site and the terminal 3' carbon of tRNA on the P site is within 0.3 Å in structural data, and fitting to the three-dimensional structural data is possible:
It is judged that the adaptability of the unnatural amino acid to the protein is higher.

When incorporating the assumed structure represented by General Formula (3-1), (3-2), (3-3), (3-4), or (3-5) in the <6> step c above,
A part of the structure of a similar structural compound from which a transition state optimization structure is obtained, or a method according to item 1 or 2, when it is judged that the aptitude for introducing the unnatural amino acid into a protein is high The method according to Item 4 or 5, wherein a part of the structure obtained as a result of the a step is incorporated, which is confirmed to be a transition state structure in the b step.

<7> The method according to any one of Items 4 to 6, further comprising the following f step:
(Step f)
It is confirmed that the structure obtained as a result of the step d is a transition state structure, and
When the three-dimensional structural data is an X-ray analysis structure of a Thermus thermophilus ribosome, the side chain of the unnatural amino acid is formed by 2451A, 2452C, 2506U and 2585U in the ribosome When it fits in space without steric hindrance,
Alternatively, when the three-dimensional structural data is an X-ray analysis structure of a ribosome other than Thermus thermophilus, the side chain of the unnatural amino acid is in the ribosome of Thermus thermophilus. In the space formed by the bases corresponding to 2451A, 2452C, 2506U and 2585U of
And a step of judging that the adaptability of the unnatural amino acid to the protein in the protein synthesis system by ribosome is high.

<8> A method for estimating the adaptability of an unnatural amino acid to a protein in a ribosomal protein synthesis system, comprising the following g and h steps:
(Step g)
An aminoacyl-tRNA of any amino acid represented by the general formula (6):
A peptidyl tRNA having as a constituent an amino acid residue of the non-natural amino acid represented by the general formula (7-1) or (7-2);
Reaction transition state (general formula (8-1), (8-2), (8-3), (8-4) or (8-5)) or reaction of its structure in the peptidyl group transfer reaction of A step of performing transition state structure optimization calculation (TS optimization calculation) by the molecular orbital method with a structure omitted as appropriate in order to reduce calculation load from a part far from the center as an initial structure.
(Step h)
A step of judging that the adaptability of the unnatural amino acid to the protein in the protein synthesis system by ribosome is high when it can be confirmed that the structure obtained as a result of the step g is a transition state structure.

(A ^a2 represents a structural moiety other than an amino group and a carboxyl group involved in peptide bond in any of the amino acids.
B ^a2 represents an arbitrary group or a group which forms a cyclic structure of B ^a2 -A ^a2 -N ^t2 together with the above-mentioned A ^a2 .
tRNA ^a2 represents a tRNA covalently linked to O ^{a22 at} the 3 'end. )

(A ^P represents a structural moiety other than an amino group and a carboxyl group involved in a peptide bond in the non-natural amino acid.
B ^P represents any group, or the ^{A P} and integral with it ^B P ^-A P -N group to form a cyclic structure of ^P.
tRNA ^P2 represents a tRNA covalently bound to ^{O P2} in the 3 'end.
P ^P2 represents the peptidyl group. )

(The structural portion of tRNA ^P22 -OP ²² -HP ²² has the same structure as tRNA ^P2 in the general formula (7-1). That is, ^OP22 is an oxygen bonded to the 2 'carbon of the 3' terminal sugar of tRNA ^P2 The atom, ^HP22 is a hydrogen atom bonded to ^OP22 , and tRNA ^P22 is a portion of tRNA ^P2 excluding ^OP ²² and ^{HP 22} .
The meaning of the symbols in the other general formula (7-2) is the same as in the general formula (7-1). )

(Structure N ^t2 (H ^t2 ) -C ^t2 (O ^t2 ) moiety is a reaction center in the reaction transition state structure of the first step of the stepwise amidation reaction,
The distance between N ^t2 and C ^t2 is 1.53 Å to 1.77 Å,
The distance between N ^t2 and H ^t2 is 1.10 Å to 1.35 Å.
The distance between H ^t2 and O ^t2 is 1.35 Å to 1.45 Å.
The meaning of the symbols in the general formula (8-1) is the same as in the general formulas (6) and (7-1). )

(H ^t22 is a hydrogen atom which protonated O ^t2 , which was carbonyl oxygen in the second step of the stepwise amidation reaction.
The moiety C ^t2 -O ^p2 -H ^t22 -O ^t2 moiety is the reaction center in the reaction transition state structure of the second stage of the stepwise amidation reaction,
The distance between C ^t2 and O ^p2 is 1.45 Å to 1.70 Å.
Distance of O ^p2 and ^{H t22} is 1.05Å ~ 1.30Å,
The distance between H ^t22 and O ^t2 is 1.10 Å to 1.40 Å,
The distance between O ^t2 and C ^t2 is 1.22 Å to 1.42 Å.
The meaning of the symbols in the general formula (8-2) is the same as in the general formulas (6) and (7-1). )

(Structure N ^t2 (H ^t2 ) -C ^t2 (O ^p2 ) moiety is a reaction center in the transition state structure of a concerted amidation reaction having a four-membered ring structure,
The distance between N ^t2 and C ^t2 is 1.40 Å to 1.65 Å,
The distance between N ^t2 and H ^t2 is 1.00 Å to 1.24 Å,
The distance between C ^t2 and O ^p2 is 1.35 Å to 2.40 Å,
Distance O ^p2 and ^{H t2} is 1.00Å ~ 1.84Å.
The meaning of the symbols in the general formula (8-3) is the same as in the general formulas (6) and (7-1). )

(Structure N ^t2 -C ^t2 -O ^p2 -H ^p22 -O ^p22 -H ^t2 moiety is the reaction center in the transition state structure of the concerted amidation reaction having a six-membered ring structure,
The distance between N ^t2 and C ^t2 is 1.40 Å to 2.10 Å,
The distance between N ^t2 and H ^t2 is 1.00 Å to 1.20 Å,
The distance between C ^t2 and O ^p2 is 1.35 Å to 2.30 Å.
Distance of O ^p2 and ^{H p22} is 1.00Å ~ 1.56Å,
The distance between H ^p22 and O ^p22 is 1.00 Å to 1.67 Å,
The distance between Op ²² and H ^t2 is 1.20 Å to 1.96 Å.
The meaning of the symbol in the general formula (8-4) is the same as in the general formulas (6) and (7-2). )

(H ^s21 -O ^s2 -H ^s22 is one of the solvent water molecules, and the structure N ^t2 -C ^t2 -O ^p2 -H ^s21 -O ^s2 -H ^p22 -O ^p22 -H ^t2 moiety has an eight-membered ring structure A reaction center in the transition state structure of the concerted amidification reaction,
The distance between N ^t2 and C ^t2 is 1.40 Å to 1.79 Å.
The distance between N ^t2 and H ^t2 is 1.00 Å to 1.26 Å,
The distance between C ^t2 and O ^p2 is 1.40 Å to 2.21 Å,
Distance of O ^p2 and ^{H s21} is 1.13Å ~ 1.43Å,
The distance between H ^s21 and O ^s2 is 1.00 Å to 1.37 Å,
The distance between O ^s2 and H ^p22 is 1.30 Å to 1.69 Å,
The distance between H ^p22 and O ^p22 is 0.98 Å to 1.24 Å,
The distance between Op ²² and H ^t2 is 1.17 Å to 1.76 Å.
The meaning of the symbol in the general formula (8-5) is the same as in the general formulas (6) and (7-2). )

9. The method according to item 8, wherein the general formula (8-1) is the following general formula (8-1-1).

(The meaning of the symbol in the general formula (8-1-1) is the same as in the general formula (8-1).)

<10> The structure represented by the general formula (7-1) or (7-2) is a structure represented by the following general formula (9-1) or (9-2),
The structures represented by the general formulas (8-1), (8-2), (8-3), (8-4), and (8-5) have the following general formulas (10-1) and (10), respectively. 9. A method according to item 8, which is characterized in that it has a structure represented by -2), (10-3), (10-4) or (10-5).

(C ^α2 is the α carbon of the non-natural amino acid, and R ^P1 and R ^P2 each independently represent the side chain structure of the non-natural amino acid (however, R ^P2 may be a hydrogen atom).
The meaning of the other symbols is the same as in the general formula (7-1). )

(TRNA ^p22 , ^OP ²² , and ^{HP 22} have the same meaning as in general formula (7-2). The meanings of other symbols are the same as in general formula (9-1).)

((Structure N ^t2 (H ^t2 ) -C ^t2 (O ^t2 ) part, the distances between N ^t2 and C ^t2 , N ^t2 and H ^t2 , and H ^t2 and O ^t2 are the same as in the general formula (8-1).
The meaning of the symbols in the general formula (10-1) is the same as in the general formulas (6) and (7-1). )

(The meanings of the symbols R ^P1 , C ^α2 and R ^P2 are the same as in the general formula (9-1). The meaning of the symbols in the general formula (10-2) and the interatomic distance are the same as in the general formula (8-2) the same.)

(The meanings of the symbols R ^P1 , C ^α2 and R ^P2 are the same as in the general formula (9-1). The meaning of the symbols in the general formula (10-3) and the interatomic distance are the same as in the general formula (8-3) the same.)

(The meanings of the symbols R ^P1 , C ^α2 and R ^P2 are the same as in the general formula (9-2). The meaning of the symbols in the general formula (10-4) and the interatomic distance are the same as in the general formula (8-4) the same.)

(The meanings of the symbols R ^P1 , C ^α2 and R ^P2 are the same as in the general formula (9-2). The meanings of the symbols in the general formula (10-5) and the interatomic distance are the same as in the general formula (8-5) the same.)

<11> A method for estimating the adaptability of an unnatural amino acid to a protein in a protein synthesis system by ribosome, comprising the following ik steps:
(Step i)
Prepare three-dimensional structural data obtained by X-ray structural analysis of ribosome in which tRNA or its derivative is bound to A and P sites,
In the three-dimensional structure data, according to the following conditions:
An aminoacyl-tRNA of any amino acid represented by the general formula (6):
A peptidyl tRNA having as a constituent an amino acid residue of the non-natural amino acid represented by the general formula (7-1) or (7-2);
A step of incorporating an assumed reaction transition state (general formula (8-1), (8-2), (8-3), (8-4) or (8-5)) in the peptidyl group transfer reaction of
(Condition 3) The general formula (8) is such that the atomic coordinates of the 3 'terminal carbon of tRNA ^a2 are located at least near the 3' terminal carbon of the tRNA at the position of the tRNA on the A site in the three-dimensional structure data. Incorporate the assumed structure represented by -1), (8-2), (8-3), (8-4) or (8-5)).
(Condition 4) The general formula (8) is such that atomic coordinates of the 3 'terminal carbon of tRNA ^P2 are located at least near the 3' terminal carbon of the tRNA at the position of the tRNA on the P site in the three-dimensional structure data. Incorporate the assumed structure represented by -1), (8-2), (8-3), (8-4) or (8-5)).
(Step j)
Transition state structure optimization calculation (TS optimization calculation) by molecular orbital method with the structure obtained by appropriately removing the structure far from the reaction center of the structure obtained by the step i or the structure omitted to reduce the calculation load as an initial structure Process of
(Step k)
A step of judging that the adaptability of the unnatural amino acid to the protein in the protein synthesis system by ribosome is high when the structure obtained as a result of the step j is confirmed to be a transition state structure.

<12> The method according to item 11, comprising the following k ′ step after the k step.
(K 'process)
The structure confirmed to be a transition state structure in the k step is
The cyclic structure including the reaction center (in a form incorporating the structure represented by the general formula (8-4) or the general formula (8-5), includes a cyclic structure containing the tRNA ^P22 of the general formula ) By adjusting the twist angle of the bond axis of the other part within the range where steric hindrance does not occur, the three-dimensional carbon corresponding atom of tRNA ^{a2 and} the three-terminal carbon corresponding atom of tRNA ^P2 are respectively three-dimensional In the case where the displacement between the terminal 3 'carbon of tRNA on the A site and the terminal 3' carbon of tRNA on the P site is within 0.3 Å in structural data, and fitting to the three-dimensional structural data is possible:
It is judged that the adaptability of the unnatural amino acid to the protein is higher.

When incorporating the assumed structure represented by the general formula (8-1), (8-2), (8-3), (8-4), or (8-5) in the <13> step i,
The case where it is judged that the aptitude for introducing the unnatural amino acid into a protein is high by a part of the structure of the analogous structural compound from which the transition state optimization structure is obtained, or the method according to item 8 or 9. The method according to Item 11 or 12, wherein a part of the structure obtained as a result of the g step is incorporated, which has been confirmed to be a transition state structure in the h step.

<14> The method according to any one of Items 11 to 13, further comprising the following 1 step:
(L process)
It is assumed that the structure obtained as a result of the j step is a transition state structure, and
When the three-dimensional structural data is an X-ray analysis structure of a Thermus thermophilus ribosome, the side chain of the unnatural amino acid is formed by 2451A, 2452C, 2506U and 2585U in the ribosome When it fits in space without steric hindrance,
Alternatively, when the three-dimensional structural data is an X-ray analysis structure of a ribosome other than Thermus thermophilus, the side chain of the unnatural amino acid is in the ribosome of Thermus thermophilus. In the space formed by the bases corresponding to 2451A, 2452C, 2506U and 2585U of
And a step of judging that the adaptability of the unnatural amino acid to the protein in the protein synthesis system by ribosome is high.

<15>
The structure represented by the general formula (7-1) is a structure represented by the following general formula (9-1),
The structure represented by the general formula (8-1) is a structure represented by the following general formula (10-1),
The method according to any one of Items 11 to 14, characterized by comprising the following m steps:
(M process)
In the i step, the structure represented by the general formula (10-1) is incorporated, and in the structure obtained as a result of the j step, the twist angle ∠N ^t2 -C ^t2 -C ^α2 -R ^P1 is 0 ° to 108 ° Determining that the adaptability of the unnatural amino acid to the protein in the protein synthesis system by ribosome is high.

(C ^α2 is the α carbon of the non-natural amino acid, and R ^P1 and R ^P2 each independently represent the side chain structure of the non-natural amino acid (however, R ^P2 may be a hydrogen atom)).

(Structure N ^t2 (H ^t2 ) -C ^t2 (O ^t2 ) portion, the distances of N ^t2 and C ^t2 , N ^t2 and H ^t2 , and H ^t2 and O ^t2 are the same as in the general formula (8-1).
The meaning of the symbols in the general formula (10-1) is the same as in the general formulas (6) and (7-1). )

<16> the general formula (7-1) or when the hydrophilic atoms are present in the ^{A P} in (7-2), characterized in that it comprises the following n steps, any one of claim 11 to 13 Method described in Section.
(N steps)
In the structure obtained as a result of the j step, the hydrophilic atom is a group represented by the general formula (8-1), (8-2), (8-3), (8-4) or (8-5). A step of judging that the adaptability of the unnatural amino acid to a protein is high in a ribosomal protein synthesis system, when it is presumed that a hydrogen bond can be formed with ^Ht2 and / or ^Ot2 .

The method according to any one of items 11 to 16, wherein the following o to q steps are provided.
(Step o)
In the structure obtained by the i step,
Furthermore, the reaction of the peptidyl group transfer reaction in the ribosome is a substance which is present in the reaction system of the biological synthesis method by the ribosome and which is confirmed or predicted to promote or not inhibit the peptidyl group transfer reaction by the ribosome A step of setting as an initial structure a structure which is disposed at the center or its periphery and a structure obtained by adding this or a portion far from the reaction center of the structure is omitted to reduce the calculation load.
(Step p)
A step of performing TS optimization calculation by a molecular orbital method on the initial structure obtained by the step o.
(Step q)
It is confirmed that the structure obtained as a result of the p step is a transition state structure, and ribosomal transfer reaction which proceeds by taking the transition state structure is not inhibited by the steric hindrance by the substance, the ribosome by the ribosome A step of judging that the adaptability of the unnatural amino acid to the protein in the protein synthesis system is high.

<18> The method according to item 15, further comprising the following r step:
(R process)
It is confirmed that the structure obtained as a result of the p step is a transition state structure, and the general formula (8-1), (8-2), (8-3), (8-4), or 8-4), when it is possible to form a hydrogen bond between the molecule of the transition state optimization structure and the substance, the ribosome synthesis system of ribosomes has the adaptability to introduce the unnatural amino acid into a protein A step of determining high.

<19> The three-dimensional structure data is atomic coordinates registered as Accession number 4V5C or 4V5D in Protein Data Bank (PDB), any one of items 4 to 6 and 10 to 16 Method described in Section.

<20> The method according to any one of items 1 to 17, wherein the molecular orbital method is a semi-empirical molecular orbital method.

<21> An estimation step of estimating adaptability of a non-natural amino acid to a protein in a ribosomal protein synthesis system by the method according to any one of items 1 to 20;
A process for producing an artificial protein, comprising the step of producing a protein by a ribosomal protein synthesis system using as a raw material a non-natural amino acid determined to have high introduction suitability in the estimation step.

<22> A method of improving the performance of a protein or peptide, comprising
21. An estimation step of estimating adaptability of an unnatural amino acid to a protein in a ribosomal protein synthesis system by the method according to any one of Items 1 to 20;
A production process for producing a protein or a peptide by a protein synthesis system by ribosome using as a raw material a non-natural amino acid determined to have high introduction suitability in the estimation step;
Assessing the performance of the protein or peptide produced in the production step.

<23> A method for improving the productivity or production efficiency of a protein or peptide by a protein synthesis system by ribosome,
21. An estimation step of estimating adaptability of an unnatural amino acid to a protein in a ribosomal protein synthesis system by the method according to any one of Items 1 to 20;
A production process for producing a protein or a peptide by a protein synthesis system by ribosome using as a raw material a non-natural amino acid determined to have high introduction suitability in the estimation step;
And e) evaluating the productivity or production efficiency of the protein or peptide in the production step.

<24> A drug design method for a drug comprising an artificial protein or artificial peptide into which a non-natural amino acid has been introduced,
Selecting a non-naturally occurring amino acid that improves efficacy or reduces side effects by introducing it into a lead compound protein or peptide according to Ligand Based Drug Design (LBDD) and / or Structure-Base Drug Design (SBDD);
Item 19. An inferring step of inferring the adaptability of the unnatural amino acid to the protein or peptide in a ribosomal protein synthesis system by the method according to any one of items 1 to 18;
A drug design method characterized in that

According to the present invention, it is possible to infer the aptitude for introducing a non-natural amino acid into a protein using a ribosomal protein synthesis system.

Although a brief description of each drawing will be added below, what has the notation "S" at the upper left of the drawing is a three-dimensional figure (stereogram). Stereoscopically view the left image with the right eye and the right image with the left eye.

The X-ray structural-analysis photograph (PDB 4V5C) of 70s ribosome as described in the nonpatent literatures 6 and 7. FIG. The space filling model represents the tRNA at the A site on the center left, the tRNA at the P site on the center right, and the mRNA at the center bottom. Represent ribosomes in a wire model. It is an image showing the periphery of the reaction site of a ribosome. (Non-patent Documents 6 and 7; PDB 4V5C) The tRNA at the A site in the lower left and the tRNA at the P site in the lower right are represented by a stick model. 50S RNA is represented by a wire model. X-ray analysis structure of amidation reaction (Non-patent documents 6 and 7; PDB 4V5C) X-ray analysis structure of amidation reaction (Non-patent documents 6 and 7; PDB 4V5D) The state in which the T1'-2 structure is connected to the X-ray analysis structure is shown. The X-ray analysis structure is represented by a rod model, and the T1'-2 structure is represented by a ball and rod model. The state in which the T3 structure is connected to the X-ray analysis structure is shown. The X-ray analysis structure is represented by a rod model, and the T3 structure is represented by a ball and rod model. 2 represents the initial structure of the As1 Ps1 type. As1Ps1 type structure optimized by TS optimization calculation. As1Ps2 type structure optimized by TS optimization calculation. As1Ps3 type structure optimized by TS optimization calculation. As2Ps1 type structure optimized by TS optimization calculation. As2Ps2 type structure optimized by TS optimization calculation. As2Ps3 type structure optimized by TS optimization calculation. It represents an optimized cross-linked structure. FIG. 6 is a diagram showing a process of generating an initial structure for transition state structure optimization calculation in ribosomes. The initial structure of the amidation reaction on the ribosome for the structure of As2Ps1 type is shown. It shows the structure of As1 Ps1 type. It shows a structure of As1 Ps2 type. It shows the structure of As1Ps3 type. It shows the structure of As2Ps1 type. It shows the structure of As2Ps2. It shows the structure of As2Ps3 type. It shows the structure of As1 Ps1 type. It shows a structure of As1 Ps2 type. It shows the structure of As1Ps3 type. It shows the structure of As2Ps1 type. It shows the structure of As2Ps2. It shows the structure of As2Ps3 type. The reaction center of the structure of As2Ps1 type | mold and the initial structure which arrange | positions six water molecules around it are represented. The structure obtained as a result of carrying out structure optimization calculation is shown with the reaction center of the structure of As 2 Ps type 1 and a structure in which six water molecules are arranged around it as an initial structure. It shows a state in which the structure of FIG. 30 obtained as a result of the structure optimization calculation is superimposed on the X-ray analysis structure.

The present invention is a method for estimating the adaptability of unnatural amino acids to proteins using a ribosomal protein synthesis system.
In the present invention, the “protein synthesis system by ribosome” includes all synthesis systems utilizing peptidyl group transfer reaction by the catalytic activity of ribosome, and includes both in vivo synthesis system and in vivo synthesis system.

The present invention comprises steps a and b as essential steps. Each will be described in detail below.

(Step a)
In step a, transition state structure optimization calculation (TS optimization calculation) in a peptidyl group transfer reaction between an aminoacyl tRNA of an unnatural amino acid whose ability to be introduced into a protein is to be estimated and a peptidyl tRNA having an arbitrary amino acid sequence is performed.

The aminoacyl tRNA of the unnatural amino acid is a structure represented by the general formula (1).

(In the general formula (1), A ^a1 represents a structural moiety other than an amino group and a carboxyl group involved in a peptide bond in the non-natural amino acid.
B ^a1 represents an arbitrary group or a group which forms a cyclic structure of B ^a1 -A ^a1 -N ^t1 together with the above-mentioned A ^a1 .
The tRNA ^a1 represents a tRNA covalently linked to O ^{a12 at} the 3 'end. )

As shown in the general formula (1), non-naturally occurring amino acids whose introduction suitability can be estimated by the method of the present invention are not limited to α-amino acids. Naturally, it is also possible to infer the aptitude for introducing an α-amino acid having a structure as shown in the following general formula (4).

In the general formula (4), C ^α1 is the α carbon of the unnatural amino acid, and R ^a1 and R ^a2 each independently represent the side chain structure of the unnatural amino acid (however, R ^a2 may be a hydrogen atom) .
The meaning of the other symbols is the same as in the general formula (1).

In the present specification, C, N, O and H to which superscripts are attached respectively represent a carbon atom, a nitrogen atom, an oxygen atom and a hydrogen atom.

On the other hand, peptidyl-tRNA having any amino acid sequence is represented by the general formula (2-1) or (2-2).

(In the general formula (2-1), tRNA ^P1 is, ^{.P P1} representing a tRNA covalently bound to ^{O P1} in the 3 'end represents the peptidyl group.)

In the general formula (2-2), the structural part of tRNA ^P12 -O ^P12 -H ^P12 has the same structure as tRNA ^P1 in the general formula (2-1). That is, ^OP12 represents an oxygen atom bonded to the 2 'carbon of 3' terminal sugar of tRNA ^P1 , ^HP12 represents a hydrogen atom bonded to ^OP12 , and tRNA ^P12 represents a portion of tRNA ^P1 excluding ^OP12 and ^HP12. .
The meaning of the symbols in the general formula (2-2) is the same as in the general formula (2-1).

In the present specification, “peptidyl group” refers to a group formed by polymerizing a plurality of amino acids by peptide bond. As the bonding mode of the moiety in which the peptidyl group bonds to another structure (X in the formula below), six types shown in formulas (i) to (vi) below can be assumed. In this case, a portion surrounded by a dashed dotted line in the following formula corresponds to a peptidyl group.
However, P ^P1 in the general formula (2-1) or (2-2), the general formulas (3-1) to (3-5) and the general formulas (5-1) to (5-5) is (v) In the general formula (7-1) or (7-2), the general formulas (8-1) to (8-5), the general formula (9) and the general formulas (10-1) to (10-5) ^P2 is a peptidyl group attached in the manner of (iv).

A in (i) to (vi) represents a structural moiety other than an amino group and a carboxyl group involved in a peptide bond in an amino acid residue constituting a peptidyl group. X represents another structure to which a peptidyl group is attached.

In the peptidyl transfer reaction at the ribosome, transfer of the peptidyl group from the peptidyl tRNA located at the P site of the ribosome to the aminoacyl tRNA located at the A site occurs.
In step a, a transition state by a molecular orbital method in a peptidyl group transfer reaction from a peptidyl tRNA represented by General Formula (2-1) or (2-2) to an aminoacyl tRNA represented by General Formula (1) Perform structure optimization calculation (TS optimization calculation).

In performing TS optimization calculation, the structure represented by the general formula (3-1), (3-2), (3-3), (3-4) or (3-5) is used as an initial structure.

In the general formula (3-1), the structure N ^t1 (H ^t1 ) -C ^t1 (O ^t1 ) moiety is a reaction center in the reaction transition state structure of the first step of the stepwise amidation reaction,
The distance between N ^t1 and C ^t1 is 1.53 Å to 1.77 Å,
The distance between N ^t1 and H ^t1 is 1.10 Å to 1.35 Å.
The distance between H ^t1 and O ^t1 is 1.35 Å to 1.45 Å.
The meaning of the symbols in the general formula (3-1) is the same as in the general formulas (1) and (2-1).

In addition, as a structure represented by General Formula (3-1), a stereoisomer structure represented by the following General Formula (3-1-1) can be used. The meaning of the symbols in the general formula (3-1-1) is the same as in the general formula (3-1).

In the general formula (3-2), H ^t12 is a hydrogen atom obtained by protonating O ^t1 , which was carbonyl oxygen in the second step of the stepwise amidation reaction.
The moiety C ^t1 -O ^p1 -H ^t12 -O ^t1 is the reaction center in the reaction transition state structure of the second step of the stepwise amidification reaction,
The distance between C ^t1 and O ^p1 is 1.45 Å to 1.70 Å.
Distance of O ^p1 and ^{H t12} is 1.05Å ~ 1.30Å,
The distance between H ^t12 and O ^t1 is 1.10 Å to 1.40 Å,
The distance between O ^t1 and C ^t1 is 1.22 Å to 1.42 Å.
The meaning of the symbols in the general formula (3-2) is the same as in the general formulas (1) and (2-1).

In the general formula (3-3), the structure N ^t1 (H ^t1 ) -C ^t1 (O ^p1 ) is a reaction center in the transition state structure of a concerted amidation reaction having a four-membered ring structure,
The distance between N ^t1 and C ^t1 is 1.40 Å to 1.65 Å,
The distance between N ^t1 and H ^t1 is 1.00 Å to 1.24 Å,
The distance between C ^t1 and O ^p1 is 1.35 Å to 2.40 Å.
Distance O ^p1 and ^{H t1} is 1.00Å ~ 1.84Å.
The meaning of the symbols in the general formula (3-3) is the same as in the general formulas (1) and (2-1).

In the general formula (3-4), the structure N ^t1 -C ^t1 -O ^p1 -H ^p12 -O ^p12 -H ^t1 moiety is a reaction center in the transition state structure of a concerted amidation reaction having a six-membered ring structure ,
The distance between N ^t1 and C ^t1 is 1.40 Å to 2.10 Å.
The distance between N ^t1 and H ^t1 is 1.00 Å to 1.20 Å,
The distance between C ^t1 and O ^p1 is 1.35 Å to 2.30 Å.
Distance of O ^p1 and ^{H p12} is 1.00Å ~ 1.56Å,
The distance between H ^p12 and O ^p12 is 1.00 Å to 1.67 Å,
The distance between Op ¹² and H ^t1 is 1.20 Å to 1.96 Å.
The meaning of the symbols in the general formula (3-4) is the same as in the general formulas (1) and (2-2).

In the general formula (3-5), H ^s12 -O ^s1 -H ^s11 is one of solvent water molecules, and the structure N ^t1 -C ^t1 -O ^p1 -H ^s11 -O ^s1 -H ^p12 -O ^p12 -H ^t1 part is the reaction center in the transition state structure of the concerted amidation reaction having an 8-membered ring structure,
The distance between N ^t1 and C ^t1 is 1.40 Å to 1.79 Å.
The distance between N ^t1 and H ^t1 is 1.00 Å to 1.26 Å,
The distance between C ^t1 and O ^p1 is 1.40 Å to 2.21 Å,
Distance of O ^p1 and ^{H s12} is 1.13Å ~ 1.43Å,
The distance between H ^s12 and O ^s1 is 1.00 Å to 1.37 Å,
The distance between O ^s1 and H ^p12 is 1.30 Å to 1.69 Å,
The distance between H ^p12 and O ^p12 is 0.98 Å to 1.24 Å,
The distance between Op ¹² and H ^t1 is 1.17 Å to 1.76 Å.
The meaning of the symbols in the general formula (3-5) is the same as in the general formulas (1) and (2-2).

When an α-amino acid is used as the non-natural amino acid, it is particularly possible to use the following general formulas (5-1), (5-2), (5-3), (5-4), (5-5) The structure represented is the initial structure.

In the general formula (5-1), the distances between N ^t1 and C ^t1 , N ^t1 and H ^t1 , and H ^t1 and O ^t1 are the same as in the general formula (3-1).
The meaning of the symbols in the general formula (5-1) is the same as in the general formulas (1), (2-1) and (4).

In addition, as a structure represented by General Formula (5-1), a stereoisomer structure represented by the following General Formula (5-1-1) can be used. The meaning of the symbols in the general formula (5-1-1) is the same as in the general formula (5-1).

In the general formula (5-2), the meanings of the symbols R ^a1 , C ^α1 and R ^a2 are the same as in the general formula (4). The meaning of the symbols in the general formula (5-2) and the interatomic distance are the same as in the general formula (3-2).

In the general formula (5-3), the meanings of the symbols R ^a1 , C ^α1 and R ^a2 are the same as in the general formula (4). The meaning of the symbols in the general formula (5-3) and the interatomic distance are the same as in the general formula (3-3).

In the general formula (5-4), the meanings of the symbols R ^a1 , C ^α1 and R ^a2 are the same as in the general formula (4). The meaning of the symbols in the general formula (5-4) and the interatomic distance are the same as in the general formula (3-4).

In the general formula (5-5), the meanings of the symbols R ^a1 , C ^α1 and R ^a2 are the same as in the general formula (4). The meaning of the symbols in the general formula (5-5) and the interatomic distance are the same as in the general formula (3-5).

General formulas (3-1), (3-2), (3-3), (3-4), (3-5), (5-1), (5-2), (5-3) Since the structure represented by (5-4) or (5-5) includes a tRNA structure, it is expected that computational load will be high when performing TS optimization calculation. Therefore, in the present invention, a structure in which a portion far from the reaction center of the structure represented by the general formula may be omitted as appropriate to reduce the calculation load may be used as the initial structure. The omission can be done by the usual method.

The general structures (3-1), (3-2), (3-3), (3-4), (3-5), (5-1), (5-2), (5), which are initial structures. The setting of more detailed parameters in the structure represented by -3), (5-4) or (5-5) can be performed by a conventional method. For example, the maximum point of the energy profile obtained by the minimum energy path calculation can be assumed to be a structure near the transition state, and can be used as an initial structure of the transition state.
Further, details of the initial structure may be set with reference to a known amidated transition state structure (for example, a report of Oie et al. (Non-patent Document 8)).

The method of TS optimization calculation by molecular orbital method is not particularly limited, and either semiempirical molecular orbital method or non-empirical molecular orbital method may be used. From the balance of simplicity (calculation time) and accuracy, it is preferable to use a semiempirical molecular orbital method.

As molecular orbital calculation software, known ones can be used without limitation. For example, Gaussian, HONDO, etc. by non-empirical molecular orbital method, MOPAC by semi-empirical molecular orbital method, etc. can be mentioned.

(Step b)
In step b, it is confirmed whether the structure obtained as a result of TS optimization calculation in step a is a transition state structure of peptidyl transfer reaction (amidation reaction). When it is possible to confirm that the structure obtained as a result of TS optimization calculation in step a is a transition state structure, it is judged that the aptitude for introducing the unnatural amino acid into a protein in a protein synthesis system by ribosome is high.

Here, as a method of confirming whether or not it is a transition state structure, vibration calculation, IRC calculation, or a combination of these may be mentioned.

In a preferred embodiment of the present invention, the following c to e steps are further provided.

The present invention also relates to a method for estimating the adaptability of an unnatural amino acid to a protein in a ribosomal protein synthesis system, and relates to a method comprising the following steps of ce.

(Step c)
In step c, first, three-dimensional structure data (hereinafter, also simply referred to as three-dimensional structure data) obtained by X-ray structural analysis of a ribosome in which tRNA or its derivative is bound to the A and P sites is prepared.

Here, "derivatives" of tRNA on the A and P sites include aminoacyl-tRNA, peptidyl aminoacyl-tRNA, and derivatives of these.

As three-dimensional structure data, data registered in Protein Data Bank (PDB) can be used. Specifically, data registered as Accession number 4V5C or 4V5D can be used.
In addition, although the said data are the analysis result of the ribosome of the thermophile (Thermus thermophilus (Thermus thermophilus (Thermus thermophilus)), the structure of the reaction center of a ribosome has few differences between species. Therefore, in the present invention, even such thermophilic bacteria data can be used for examination in ribosomal synthesis systems such as E. coli used for protein engineering.

In step c, the structure represented by the general formula (3-1), (3-2), (3-3), (3-4) or (3-5) is incorporated under the following conditions.

(Condition 1) The atomic formula of the 3 'terminal carbon atom of tRNA ^a1 is located at least near the 3' terminal carbon of the tRNA at the position of the tRNA on the A site in the three-dimensional structure data. 1) Incorporate the assumed structure represented by (3-2), (3-3), (3-4) or (3-5).

(Condition 2) The atomic formula of the 3 'terminal carbon of tRNA ^P1 is located at least near the 3' terminal carbon of the tRNA at the position of the tRNA on the P site in the three-dimensional structure data, 1) Incorporate the assumed structure represented by (3-2), (3-3), (3-4) or (3-5).

Here, the term "nearby" means a distance of 1 Å or less.

The incorporation of atoms into three-dimensional structure data can be performed according to a conventional method using the function of molecular orbital calculation software.
At the time of integration, the bond between the 3 'carbon and o ^a12 of the 3' end of tRNA ^a1, also, the bond between the 3 'carbon and o ^P1 of the 3' end of tRNA ^P1, the normal coupling conditions An operation may be added to converge. This operation can be carried out by performing structure optimization calculation as appropriate according to a conventional method.

In addition, at the time of incorporation, part of the structure of a similar structural compound from which a transition state optimization structure is obtained may be incorporated. As such a structure, a known amidated transition state structure according to the report of Oie et al. (Non-Patent Document 8) can be mentioned.

In addition, when incorporated, it is determined that the aptitude for introducing the unnatural amino acid into a protein is high according to the method of the present invention comprising steps a and b described above, and the transition state in step b A part of the structure obtained as a result of the step a) which has been confirmed to be a structure may be incorporated.

In addition, when taking in these structures, the electronic data of TS optimization structure may be input as it is, and you may adjust by manual input.

(Step d)
Step d is a step of performing TS optimization calculation with the structure obtained in step c as an initial structure.
The molecular orbital calculation in step d includes not only aminoacyl-tRNA and peptidyl-tRNA but also the structure of a large ribosome. The ribosomal molecule is huge, and it is difficult to obtain the transition state structure in the peptidyl transfer reaction on the ribosome as it is, including the entire ribosomal molecule, by molecular orbital calculation as it is. Therefore, in order to reproduce the true aspect of the peptidyl group transfer reaction as much as possible without losing as much as possible, the widest possible calculation area around the transfer reaction center of the ribosome is taken out, and the optimization calculation is carried out. It is preferable to devise appropriate restraints or the like so that the peripheral structure does not diverge.
That is, a structure in which a portion far from the reaction center of the structure obtained in step c may be omitted as appropriate to reduce the calculation load may be used as the initial structure. The omission can be performed by the usual method as described above.

As for the details of the molecular orbital method, the matters described in the description of step a above can be applied.

(Step e)
The step e is a step of evaluating the structure obtained as a result of the TS optimization calculation in the step d. That is, when it is confirmed that the obtained structure is a transition state structure, it is judged that the suitability for introduction of the non-natural amino acid into a protein in a protein synthesis system by ribosome is high.
The confirmation of whether or not it is a transition state structure can apply the matter described in the explanation of step b.

Moreover, it is particularly preferable to include the following e ′ step after the e step.
(E 'process)
The structure confirmed to be a transition state structure in the step e) is
The cyclic structure containing the reaction center (in a form incorporating the structure represented by the general formula (3-4) or the general formula (3-5), the cyclic structure containing tRNA ^P12 of the general formula is included ) By adjusting the twist angle of the bond axis of the other part within the range that steric hindrance does not occur, the three-dimensional carbon corresponding atom of tRNA ^{a1 and} the three-terminal carbon corresponding atom of tRNA ^P1 are respectively three-dimensional In the case where the displacement between the terminal 3 'carbon of tRNA on the A site and the terminal 3' carbon of tRNA on the P site is within 0.3 Å in structural data, and fitting to the three-dimensional structural data is possible:
It is judged that the adaptability of the unnatural amino acid to the protein is higher.

The e ′ step is a step of confirming that the structure that has been confirmed to be a transition state structure is a structure that can be taken inside the ribosome. According to the e ′ step, it is possible to more accurately estimate the adaptability of the unnatural amino acid to the protein.

(Step f)
In Thermus thermophilus ribosomes, there is a space formed by 2451A, 2452C, 2506U and 2585U. As described above, since the structure of the reaction center of the ribosome is small among species, the probability that the space corresponding to the space is present is also high in the ribosomes of species other than Thermus thermophilus.
In the present invention, in addition to the steps c to e, it is preferable to further include an step f for evaluating the space in the ribosome.

In step f, the structure obtained as a result of step d is confirmed to be a transition state structure, and the space formed by 2451A, 2452C, 2506U and 2585U in the ribosome of Thermus thermophilus (or this When the side chain of the non-natural amino acid fits into the space corresponding to (1) without steric hindrance, it is judged that the protein synthesis system by ribosome has high suitability for introducing the non-natural amino acid into protein.

The presence or absence of steric hindrance is evaluated on the basis of the interatomic distance between the side chain of the amino acid residue of the unnatural amino acid and the base forming the space.

The f step is preferably carried out in combination with the e 'step which also performs an evaluation on the space in the ribosome. This makes it possible to evaluate the adaptability of the unnatural amino acid to the protein with higher accuracy.

By the way, in the protein synthesis reaction by ribosome, translocation occurs after peptidyl transfer reaction. That is, when the aminoacyl-tRNA located at the A site prior to the reaction undergoes peptidyl transfer, the mRNA moves to the P site.

The method of the present invention comprising the steps a and b and the steps c to f relates to the evaluation of the adaptability of the unnatural amino acid to a protein in the state where the aminoacyl tRNA of the unnatural amino acid is located at the A site. there were.
In the following, a form comprising g and h steps for evaluating the ability to introduce an unnatural amino acid into a protein in a state where the aminoacyl tRNA of the unnatural amino acid located at the A site undergoes peptidyl transfer and translocates to the P site The description of the invention of

In addition, evaluation of the state where the aminoacyl tRNA of the unnatural amino acid is located at the A site (steps a and b) and evaluation of the state where it is located at the P site (steps g and h) Thus, it is possible to more accurately estimate the aptitude for introducing a non-natural amino acid into a protein.

(Step g)
The g step is a step of performing TS optimization calculation in a peptidyl group transfer reaction between an aminoacyl tRNA of any amino acid and a peptidyl tRNA having a residue of an unnatural amino acid.

The aminoacyl-tRNA of any of the above amino acids is represented by the general formula (6).

In the general formula (6), A ^a2 represents a structural moiety other than an amino group and a carboxyl group involved in peptide bond in any natural amino acid.
B ^a2 represents an arbitrary group or a group which forms a cyclic structure of B ^a2 -A ^a2 -N ^t2 together with the above-mentioned A ^a2 .
tRNA ^a2 represents a tRNA covalently linked to O ^{a22 at} the 3 'end.

The general formula (6) is not limited to the aminoacyl-tRNA of the α-amino acid, but of course it may be an aminoacyl-tRNA of the α-amino acid.

On the other hand, the above peptidyl-tRNA is represented by the general formula (7-1) or (7-2).

In the general formula (7-1), A ^P is in the unnatural amino acid, represents a structural part other than the amino group and a carboxyl group participating in peptide bonds.
B ^P represents any group, or the ^{A P} and integral with it ^B P ^-A P -N group to form a cyclic structure of ^P. tRNA ^P2 represents a tRNA covalently bound to ^{O P2} in the 3 'end. P ^P2 represents the peptidyl group.

In general formula (7-2), the structural part of tRNA ^P22 -OP ²² -HP ²² has the same structure as tRNA ^P2 in general formula (7-1). That is, ^OP22 is an oxygen atom bonded to the 2 'carbon of the 3' terminal sugar of tRNA ^P2 , ^HP22 is a hydrogen atom bonded to ^OP22 , and tRNA ^P22 is a portion of tRNA ^P2 excluding ^OP ²² and ^{HP 22} .
The meaning of the symbols in the other general formula (7-2) is the same as in the general formula (7-1).

Further, in the general formula (7-1) or (7-2), it is not limited that the non-natural amino acid is an α-amino acid, but of course, the non-natural amino acid may be an α-amino acid. In that case, the structure represented by the general formula (7-1) or (7-2) is represented by the general formula (9-1) or (9-2).

In the general formula (9-1), C ^α2 is the α-carbon of the non-natural amino acid, and R ^P1 and R ^P2 each independently represent the side chain structure of the non-natural amino acid (however, R ^P2 is also a hydrogen atom) Good).
The meaning of the other symbols is the same as in the general formula (7-1).

In the general formula (9-2), tRNA ^p22 , ^OP ²² and ^{HP 22} have the same meaning as in the general formula (7-2). The meaning of the other symbols is the same as in the general formula (9-1).

In step g, TS optimization calculation in the peptidyl group transfer reaction between the aminoacyl tRNA of general formula (6) and the peptidyl tRNA of general formula (7-1) or (7-2) is performed.
The items described in the step a can be applied to the embodiment of the TS optimization calculation.

(Step h)
In step h, it is confirmed whether or not the structure obtained as a result of TS optimization calculation in step g is a transition state structure of the amidation reaction. When the structure obtained as a result of TS optimization calculation in step a is confirmed to be a transition state structure, it is determined that the ribosomal protein synthesis system has high adaptability for introducing the unnatural amino acid into a protein.
The description in the step b can be applied as it is to a method of confirming whether or not it is a transition state structure.

The present invention also relates to a method for estimating the adaptability of a non-natural amino acid to a protein in a ribosomal protein synthesis system, and also relates to a method comprising the following ik steps:

(Step i)
In step i, three-dimensional structure data is prepared as in step c. For details of the three-dimensional structure data, the contents described in step c can be applied as they are.

In the step i, the structure represented by the general formula (8-1), (8-2), (8-3), (8-4) or (8-5) is incorporated under the following conditions.

In the general formula (8-1), the structure N ^t2 (H ^t2 ) -C ^t2 (O ^t2 ) moiety is a reaction center in the reaction transition state structure of the first step of the stepwise amidation reaction,
The distance between N ^t2 and C ^t2 is 1.53 Å to 1.77 Å,
The distance between N ^t2 and H ^t2 is 1.10 Å to 1.35 Å.
The distance between H ^t2 and O ^t2 is 1.35 Å to 1.45 Å.
The meaning of the symbols in the general formula (8-1) is the same as in the general formulas (6) and (7-1).

As the structure represented by the general formula (8-1), one having an optical isomer structure represented by the following general formula (8-1-1) can also be used. The meaning of the symbol of the general formula (8-1-1) is the same as that of the general formula (8-1).

In the general formula (8-2), H ^t22 is a hydrogen atom obtained by protonating O ^t2 , which was carbonyl oxygen in the second step of the stepwise amidation reaction.
The moiety C ^t2 -O ^p2 -H ^t22 -O ^t2 moiety is the reaction center in the reaction transition state structure of the second stage of the stepwise amidation reaction,
The distance between C ^t2 and O ^p2 is 1.45 Å to 1.70 Å.
Distance of O ^p2 and ^{H t22} is 1.05Å ~ 1.30Å,
The distance between H ^t22 and O ^t2 is 1.10 Å to 1.40 Å,
The distance between O ^t2 and C ^t2 is 1.22 Å to 1.42 Å.
The meaning of the symbols in the general formula (8-2) is the same as in the general formulas (6) and (7-1).

In the general formula (8-3), the structure N ^t2 (H ^t2 ) -C ^t2 (O ^p2 ) moiety is a reaction center in the transition state structure of a concerted amidation reaction having a four-membered ring structure,
The distance between N ^t2 and C ^t2 is 1.40 Å to 1.65 Å,
The distance between N ^t2 and H ^t2 is 1.00 Å to 1.24 Å,
The distance between C ^t2 and O ^p2 is 1.35 Å to 2.40 Å,
Distance O ^p2 and ^{H t2} is 1.00Å ~ 1.84Å.
The meaning of the symbols in the general formula (8-3) is the same as in the general formulas (6) and (7-1).

In the general formula (8-4), the structure N ^t2 -C ^t2 -O ^p2 -H ^p22 -O ^p22 -H ^t2 moiety is the reaction center in the transition state structure of the concerted amidation reaction having a six-membered ring structure ,
The distance between N ^t2 and C ^t2 is 1.40 Å to 2.10 Å,
The distance between N ^t2 and H ^t2 is 1.00 Å to 1.20 Å,
The distance between C ^t2 and O ^p2 is 1.35 Å to 2.30 Å.
Distance of O ^p2 and ^{H p22} is 1.00Å ~ 1.56Å,
The distance between H ^p22 and O ^p22 is 1.00 Å to 1.67 Å,
The distance between Op ²² and H ^t2 is 1.20 Å to 1.96 Å.
The meaning of the symbols in the general formula (8-4) is the same as in the general formulas (6) and (7-2).

In the general formula (8-5), H ^s21 -O ^s2 -H ^s22 is one of solvent water molecules, and the structure N ^t2 -C ^t2 -O ^p2 -H ^s21 -O ^s2 -H ^p22 -O ^p22 -H ^t2 part is the reaction center in the transition state structure of the concerted amidation reaction having an 8-membered ring structure,
The distance between N ^t2 and C ^t2 is 1.40 Å to 1.79 Å.
The distance between N ^t2 and H ^t2 is 1.00 Å to 1.26 Å,
The distance between C ^t2 and O ^p2 is 1.40 Å to 2.21 Å,
Distance of O ^p2 and ^{H s21} is 1.13Å ~ 1.43Å,
The distance between H ^s21 and O ^s2 is 1.00 Å to 1.37 Å,
The distance between O ^s2 and H ^p22 is 1.30 Å to 1.69 Å,
The distance between H ^p22 and O ^p22 is 0.98 Å to 1.24 Å,
The distance between Op ²² and H ^t2 is 1.17 Å to 1.76 Å.
The meaning of the symbols in the general formula (8-5) is the same as in the general formulas (6) and (7-2).

Also, in the general formulas (8-1), (8-2), (8-3), (8-4) and (8-5), it is not limited that the non-natural amino acid is an α-amino acid, Naturally, the non-naturally occurring amino acid may be an alpha amino acid. In that case, the structures represented by general formulas (8-1), (8-2), (8-3), (8-4), and (8-5) have the general formula (10-1), respectively. And (10-2), (10-3), (10-4) and (10-5).

In the general formula (10-1), the distance between the structure N ^t2 (H ^t2 ) -C ^t2 (O ^t2 ) part, N ^t2 and C ^t2 , N ^t2 and H ^t2 , and H ^t2 and O ^t2 is a general formula (8- Same as 1).
The meaning of the symbols in the general formula (10-1) is the same as in the general formulas (6) and (7-1).

As the structure represented by the general formula (10-1), a stereoisomer structure represented by the following general formula (10-1-1) can be used. The meaning of the symbols in the general formula (10-1-1) is the same as in the general formula (10-1).

In the general formula (10-2), the meanings of the symbols R ^P1 , C ^α2 and R ^P2 are the same as in the general formula (9-1). The meaning of the symbols in the general formula (10-2) and the interatomic distance are the same as in the general formula (8-2).

In the general formula (10-3), the meanings of the symbols R ^P1 , C ^α2 and R ^P2 are the same as in the general formula (9-1). The meaning of the symbols in the general formula (10-3) and the interatomic distance are the same as in the general formula (8-3).

In the general formula (10-4), the meanings of the symbols R ^P1 , C ^α2 and R ^P2 are the same as in the general formula (9-2). The meaning of the symbols in General Formula (10-4) and the interatomic distance are the same as in General Formula (8-4).

In the general formula (10-5), the meanings of the symbols R ^P1 , C ^α2 and R ^P2 are the same as in the general formula (9-2). The meaning of the symbols in the general formula (10-5) and the interatomic distance are the same as in the general formula (8-5).

(Condition 3) The general formula (8) is such that the atomic coordinates of the 3 'terminal carbon of tRNA ^a2 are located at least near the 3' terminal carbon of the tRNA at the position of the tRNA on the A site in the three-dimensional structure data. Incorporate the assumed structure represented by -1), (8-2), (8-3), (8-4) or (8-5)).

(Condition 4) The general formula (8) is such that atomic coordinates of the 3 'terminal carbon of tRNA ^P2 are located at least near the 3' terminal carbon of the tRNA at the position of the tRNA on the P site in the three-dimensional structure data. Incorporate the assumed structure represented by -1), (8-2), (8-3), (8-4) or (8-5)).

For incorporation of the structure represented by general formula (8-1), (8-2), (8-3), (8-4) or (8-5) into three-dimensional structure data, in step c The contents described can be applied as they are.

(Step j)
Step j is a step of performing TS optimization calculation with the structure obtained in step i or a portion far from the reaction center of the structure omitted as appropriate for reducing the calculation load in order to reduce the calculation load.
The matters described in the description of the steps a and d can be applied to the structure optimization calculation by the molecular orbital method in the step j.

(Step k)
The k step is a step of evaluating the structure obtained as a result of the TS optimization calculation in the j step. That is, when it is confirmed that the obtained structure is a transition state structure, it is judged that the suitability for introduction of the non-natural amino acid into a protein in a protein synthesis system by ribosome is high.
The confirmation of whether or not it is a transition state structure can apply the matter described in the explanation of step b.

In addition, it is preferable that the following k 'process is included after k process.
(K 'process)
The structure confirmed to be a transition state structure in the k step is
The cyclic structure including the reaction center (in a form incorporating the structure represented by the general formula (8-4) or the general formula (8-5), includes a cyclic structure containing the tRNA ^P22 of the general formula ) By adjusting the twist angle of the bond axis of the other part within the range where steric hindrance does not occur, the three-dimensional carbon corresponding atom of tRNA ^{a2 and} the three-terminal carbon corresponding atom of tRNA ^P2 are respectively three-dimensional In the case where the displacement between the terminal 3 'carbon of tRNA on the A site and the terminal 3' carbon of tRNA on the P site is within 0.3 Å in structural data, and fitting to the three-dimensional structural data is possible:
It is judged that the adaptability of the unnatural amino acid to the protein is higher.

The k 'step is a step of confirming that the structure confirmed to be a transition state structure in the k step is a structure that can be taken within the ribosome. Therefore, by performing the k ′ step, it is possible to more accurately estimate the aptitude for introducing a non-natural amino acid into a protein.

(L process)
In Thermus thermophilus ribosomes, there is a space formed by 2451A, 2452C, 2506U and 2585U. As described above, since the structure of the reaction center of the ribosome is small among species, the probability that the space corresponding to the space is present is also high in the ribosomes of species other than Thermus thermophilus.
In the present invention, in addition to the h to k steps, it is preferable to further include an l step for evaluating the space in the ribosome.

In step 1, the structure obtained as a result of step j is confirmed to be a transition state structure, and the space formed by 2451A, 2452C, 2506U and 2585U in the ribosome of Thermus thermophilus (or When the side chain of the non-natural amino acid fits into the space corresponding to (1) without steric hindrance, it is judged that the protein synthesis system by ribosome has high suitability for introducing the non-natural amino acid into protein.

The l step is preferably carried out in combination with the k 'step which also carries out an evaluation on the space in the ribosome. This makes it possible to evaluate the adaptability of the unnatural amino acid to the protein with higher accuracy.

The preferred embodiment of the present invention further comprises the following m steps.

(M process)
In the m step, the structure represented by the general formula (10-1) is incorporated in the i step, and the twist angle ∠N ^t2 -C ^t2 -C ^α2 -R ^P1 in the structure obtained as a result of the j step is 0 ° When it is ̃108 °, it is judged that the adaptability of the unnatural amino acid to the protein in the protein synthesis system by ribosome is high.

Also, A ^P in the general formula (7-1) or ^(7-2), when the hydrophilic atoms are present in the side chains of unnatural amino acids, in particular contained in A ^P, comprises the following n steps Is preferred.

(N steps)
In the n step, in the structure obtained as a result of the j step, the hydrophilic atom is a group represented by the general formula (8-1), (8-2), (8-3), (8-4), or (8-5) The ability to introduce the unnatural amino acid into a protein in a protein synthesis system by ribosomes is considered to be high when it is presumed that a hydrogen bond can be formed with ^Ht2 and / or ^{Ot2 in}
Whether or not a hydrogen bond can be formed can be estimated based on the distance between atoms and the like. For example, when the distance between the two atoms mentioned above is 1.65 to 1.85 Å, it can be estimated that a hydrogen bond can be formed between the two atoms.

Further, in the present invention, if there is a substance which is present in a protein synthesis reaction system by ribosome and which is confirmed or predicted to promote or do not inhibit the peptidyl transfer reaction by ribosome, the substance is It can be added to the target of orbit calculation.
That is, the present invention may comprise the following steps o to q.

(Step o)
In the step o, as in the step i, three-dimensional structure data is prepared, and the data represented by the general formula (8-1), (8-2), (8-3), (8-4), or (8) is prepared. Incorporate the structure represented by -5). Then, the above-mentioned substance is placed at or around the reaction center of the peptidyl transfer reaction in the ribosome.

As the substance to be disposed in the step o, water molecules as a solvent, various ions present in the reaction system and the like can be mentioned.
In the case of arranging water molecules, 2 to 8 molecules, more preferably 4 to 7 molecules, still more preferably 5 to 7 molecules, still more preferably 6 molecules of water are formed around the reaction center of the peptidyl group transfer reaction and its periphery. Deploy. In this case, it is preferable to arrange so that a network of hydrogen bonds by water molecules is formed at or around the reaction center.

Then, a structure in which the above-mentioned substance is disposed at or around the reaction center of the peptidyl group transfer reaction in the ribosome, or a structure far from the reaction center of the structure is set as an initial structure .

(Step p)
In the p process, structure optimization calculation by the molecular orbital method is performed on the initial structure set in the o process.

For the details of the molecular orbital method, the matters described in the description of the steps a, d and j can be applied.

(Step q)
The q step is a step of evaluating the structure obtained as a result of the TS optimization calculation in the p step. That is, it is confirmed that the obtained structure is a transition state structure, and the ribosomal protein synthesis system is used when the peptidyl group transfer reaction which proceeds by taking the transition state structure is not inhibited by the steric hindrance caused by the substance. It is determined that the aptitude for introducing the unnatural amino acid into a protein is high.
The confirmation of whether or not it is a transition state structure can apply the matter described in the explanation of step b.

The judgment as to whether the peptidyl transfer reaction is inhibited by the substance due to the steric hindrance by the substance or not can be made by the general formulas (8-1), (8-2), (8-3), (8-4), Alternatively, the distance between the molecule represented by (8-5) and the substance can be used as an index.

In step q, it was evaluated whether steric hindrance caused by the substance would inhibit the peptidyl group transfer reaction, but it is equipped with an r step to determine whether the substance can promote the peptidyl group transfer reaction. It is also good. The r process is as described below.

(R process)
The r step is a step of evaluating the structure obtained as a result of the TS optimization calculation in the p step.
That is, it is confirmed that the structure obtained as a result of the p step is a transition state structure, and the general formulas (8-1), (8-2), (8-3), (8-4), Or introduction of the unnatural amino acid into a protein in a protein synthesis system by ribosomes, when hydrogen bonding can be formed between the substance and the molecule of the transition state optimization structure represented by (8-5) or I judge that the aptitude is high.
The confirmation of whether or not it is a transition state structure can apply the matter described in the explanation of step b.

Moreover, the contents described in the description of the n step can be applied to the determination of whether or not hydrogen bonding is possible.

The invention also relates to a method of producing an artificial protein. The production method of the present invention comprises an estimation step and a step of producing a protein.

The inference step is a step of evaluating the introduction suitability of a non-natural amino acid which is being studied for introduction into an artificial protein by a ribosomal protein synthesis system. The estimation of the suitability for introducing unnatural amino acids into proteins can be performed by the method of the present invention described above.

Next, in the step of producing a protein, a protein is produced by a protein synthesis system using ribosomes, using as a raw material a non-natural amino acid which is judged to be high in the introduction suitability in the estimation step.

The present invention also relates to a method of improving the performance of a protein or peptide, and a method of improving productivity or production efficiency.
These methods comprise the steps of guessing and protein or peptide production of the embodiments described above.
And it has the evaluation process of evaluating the performance of the protein or peptide manufactured at the manufacturing process, or the productivity or manufacturing efficiency of the protein or peptide at the manufacturing process.

In addition, the present invention can be applied to a drug design method of an artificial protein preparation into which non-natural amino acids are introduced.

Most pharmaceuticals currently in use are developed by optimizing lead compounds. Lead compounds also include natural proteins and peptides from plants, microorganisms and higher organisms, including hormones and transmitters.

In recent years, in the molecular design of these lead compound drug discovery, computer-predicted virtual screening technology has become an important item in a series of drug discovery screening research.
In the background, in addition to the drastic improvement of computer performance, the amount of information on structure-activity relationship accumulated by high-throughput screening and combinatorial synthesis technology has dramatically increased, and is targeted by the progress of genome research The structural information of proteins has been dramatically increased.

Broadly speaking, molecular design techniques that realize virtual screening can be divided into two: Ligand Based Drug Design (LBDD) based on known structure-activity relationship information, and Structure-Base Drug Design (SBDD) using conformational information of target proteins. It can be mentioned.

In particular, when multiple drugs are bound in the vicinity of the active site of a target protein, the SBDD technology has a complementary relationship with each drug, and the amount of free energy change in each binding process represents the strength of pharmacological activity. It is based on the knowledge that This is a computer to estimate the binding state of the target protein and ligand and its pharmacological activity value, and expect highly accurate activity value prediction even though it does not require foresight information of structure activity relationship It has the advantage of being able to

On the other hand, the drug discovery technology LBDD (superposition analysis, quantitative structure activity relationship (QSAR), etc.) is a drug discovery technology focusing on the fact that homology is seen in its physicochemical parameters among drugs bound to a common site. In, although it is an approximate solution, it has a high rate of correct answers to various target systems, and has an advantage that it does not require a receptor structure (binding model) unlike SBDD.

The drug design method of the present invention is characterized by combining it with a drug efficacy or side effect estimation technique of such a drug.
In the first step, by introducing LBPD and / or SBDD into a protein or peptide which is a lead compound, non-naturally occurring amino acids are selected which improve drug efficacy or reduce side effects.

Specifically, the efficacy or side effect may be estimated when an unnatural amino acid is introduced into the pharmacophore portion of a protein or peptide to be a lead compound.

In the second step, estimation is performed by the estimation method of the present invention described above. That is, according to the above-described estimation method of the present invention, an estimation step of estimating the adaptability of the unnatural amino acid to the protein or peptide in a ribosomal protein synthesis system is performed.

Thus, by combining the molecular design technology of lead compound drug discovery and the estimation method of the present invention, it is possible to comprehensively evaluate and screen from the viewpoint of drug efficacy or side effects and the possibility of synthesis by ribosome synthesis system. It is possible to provide a throughput drug design method.

Hereafter, the process which came to find the natural law which this invention utilizes is explained in full detail.

(1) Purpose of the test In the literature (non-patent documents 6, 7) (PDB 4V5C), RNA corresponding to aminoacyl-tRNA in ribosome and RNA corresponding to peptidyl-tRNA bind to A site and P site respectively, The structure of X-ray analysis with bound mRNA is described (FIG. 1, FIG. 2).

However, from the structure described in the document, detailed information on the reaction center is not obtained, which enables specific discussion on the reaction mechanism of the peptidyl group transfer reaction.
Two types of X-ray analysis structures shown in FIG. 3 and FIG. 4 are described in the documents (Non-Patent Documents 6 and 7).
The structure shown in FIG. 3 lacks the terminal peptidyl group on the P site side. The structure shown in FIG. 4 has a peptidyl terminal similar structure to a phenylalanine similar structure at the terminal end on the P site side, but as shown in the drawing, it binds to the amino group at the A terminal tRNA end by an amidation reaction The carbon atom of the carbonyl group to be removed is separated by 3.32 Å and is not at a position where a reactive transition state can be formed. Therefore, no information is available to know the actual aspect of the amidation reaction on the ribosome.

We complemented this missing structure and resolved the contradiction of the distance between reaction substrates rationally, and examined the amidation reaction mechanism consistent with the X-ray analysis structure.

(2) Problems in the examination of the peptidyl transfer reaction mechanism There are several problems that need to be solved in order to study the amidification reaction mechanism not inconsistent with the X-ray analysis structure.

(2-a) Confirmation of Reaction Mechanism Underlying Amidation Reaction Peptidyl transfer reaction is, as an organic chemical reaction, an amidation reaction with an amino group and an ester group. When a peptidyl transfer reaction proceeds on a ribosome, the reaction proceeds in the same manner as in the basic amidation reaction, and the ribosome has a structure that promotes the formation of a transition state structure at this time, and this structure is The structure must be consistent with the X-ray analysis structure.

Findings of the basic amidation reaction mechanism include molecular orbital calculation results of stepwise and concerted amidation reactions of ammonia and formic acid reported by Oie et al. (See Non-Patent Document 8, Formulas 1 and 2).
However, these results are obtained by the MNDO method, STO-3G method, and 3-21G method, and it is impossible to study the amidation reaction mechanism on a large ribosome by these calculation methods.
Following this result, another means is needed to study the amidation reaction mechanism on a large ribosome.

[Equation 1]
Amidation reaction mechanism of formic acid and ammonia (Non-patent document 8 (Oie et al.))

[Formula 2]
Transition State of Amidation Reaction (Non-patent Document 8 (Oie et al.))

(2-b) Matching of Amidation Reaction Transition State Structure of Ammonia-Formic Acid with Peptidyl Transfer Reaction The amidization transition state structure reported by Oie et al. Has three kinds of T1, T2 and T3, and further Each of them also has isomers such as optical isomer. It is necessary to study the amidation reaction mechanism on the ribosome excluding the contradiction with the actually proceeding peptidyl transfer reaction.

(2-c) Extension to peptidyl transfer reaction The amidation reaction in (2-a) and (2-b) is an amidation reaction of ammonia-formic acid which is a simple molecule. It should be examined whether there is any contradiction between this and the reaction between amino acids and also the actual transfer of peptidyl group on the ribosome. For this purpose, the amidation reaction should be extended to a peptidyl transfer reaction such as alanylalanine, and the amidification reaction mechanism should be examined as close as possible to the actual peptidyl transfer reaction on the ribosome.

(3) Investigation of amidification reaction mechanism and examination results (3-a) Selection of calculation method In this study, MOAC-PM3 semi-empirical MO method: (hereinafter abbreviated as MOAC-PM3) was used for molecular orbital calculation. MOAC-PM3 has the disadvantage of being inferior in reproducibility of molecules containing atoms other than carbon, but has the following features.
・ Quickness that can calculate the number of atoms.
・ Quick and flexible response to expansion of calculation target structure.
• Computational power that can handle a wide range of structures away from the reaction center.

In this study, while taking advantage of this feature, the above-mentioned drawbacks were overcome by selecting conditions consistent with the structure obtained by X-ray analysis. As a result, the following amidification reaction mechanism of 3-b) to 3-f) became possible was studied (calculation methods a, c, d, e, f).

(3-b) Reproduction of transition state structure by MOPAC-PM3 described in the literature Oie et al. Analyze the reaction (formula 1) in which formamide is formed from ammonia and formic acid using MNDO, STO-3G, 3-21G, and the transition state The structure is obtained (see Non-Patent Document 8, Formula 1 and Formula 2). It was verified whether this transition state structure can be reproduced by MOAC-PM3.

First, with the result of MNie of Oie et al. As an initial structure, computational package of Winmostar (GE) V4.024; TS optimization calculation using X-Ability, transition state structure; T1, T2, T3 Although there is a difference between the two, it was basically confirmed that they can be reproduced (Method a, c, d, e, f).

(3-c) Verification of the structure described in the literature (3-c-1) Verification of the X-ray analysis structure as an amidification reaction site In particular, the amidation reaction of the X-ray analysis structure shown in Ramakrishnan et al. It was verified that there is no contradiction between the 3 'terminal structure of the site tRNA (FIG. 3, FIG. 4) and the reaction transition state structure obtained in the above (3-b). Those with contradictions were excluded, and only those without contradictions were used in the analysis described later.

In the case of FIG. 3, a phenylalanine analog structure is amide-bonded to the 3 'end of tRNA bound to the A site instead of an ester bond. Such a phenylalanine analog structure is not bound to the 3 'end of the tRNA bound to the P site.

On the other hand, in the case of FIG. 4, a phenylalanine analog structure is bound to both the A and P sites. Since a phenylalanine analog structure is bound to both sites, the structure here seems to be closer to the actual amidation transition state structure. However, as shown in FIG. 4, C = O, which is to form an amide bond, is separated from N by a distance of 3.32 Å and does not take a direction to easily form a reactive transition state. In order to form a reaction transition state, the conformation of the phenylalanine analog structure on the P site side must be largely changed, and it is considered that the X-ray analysis structure and the conformation of the reaction transition state are greatly different.

On the other hand, the benzene ring of the phenylalanine analog structure at the A site is contained in the pocket formed by 2451 Adenine-2452 Cytidine of 50s RNA in both FIG. 3 and FIG. 4 (FIG. 2). In addition, since N forming an amide bond is directed to the P site side, it is considered that there is no significant change with this conformation even in the reaction transition state.

Based on the above results, when conducting a study to connect the reaction transition state structure of alanylalanine to the X-ray analysis structure described later, the transition state to 3 'of the 3' terminal 3 'of tRNA at the P site side of FIG. The ester group of the structure is connected with respect to its twist angle without relying on the X-ray analysis structure, and the connection with the A site is verified.

(3-c-2) Verification of reaction transition state structure described in Article.
The validity of the reaction transition state structure corresponding to each of the plurality of possible reaction mechanisms obtained in (3-b) above was verified. When there was a contradiction with the X-ray analysis structure, its transition state structure and its reaction mechanism were excluded from consideration.

The amidation reaction in the peptidyl transfer reaction on the ribosome is considered to be common to the basic part of the reaction mechanism described in (2-a) above. If the actual reaction mechanism of the amidation reaction is Stepwise reaction mechanism, the X-ray analysis structure of ribosome is closely related to the structure of T1 (Formula 2) which is the first reaction step. It is thought that The concerted reaction mechanism is considered to be more closely related to the structure of T3 (Formula 2).

If the structures of T1, T2 and T3 shown in the formula 2 correspond to the actual peptidyl group transfer reaction, either one of the two H atoms other than H bonded to the N atom in the transition state structure is A It should correspond to the Cα atom (A ^{* of} Formula 3) of the terminal amino acid of the aminoacyl tRNA of the site (Formula 2, Formula 3).

[Equation 3] Other candidates for T1 structure

A ^* ; Aminoacyl-tRNA part
P ^* ; Peptidyl part
R ^* ; tRNA part of peptidyl-tRNA

Also, the H atom bonded to the C atom in the transition state structure (formula 3) corresponds to the Cα atom (P ^{* in} formula 3) of the amino acid bonded to the tRNA at the peptide end of the peptidyl tRNA of the P site; The H atom attached to the atom should correspond to the 3 'carbon atom (R ^{* in} Formula 3) of the terminal sugar of the tRNA. An isomer in which the H atom and the O2 atom bonded to the C atom in the formula 3 are inverted; T1 '(formula 3) is also present as a possibility of the transition state structure.

In the formulas 1 and 2, the H atom corresponding to the 3 'carbon atom of the terminal sugar of the tRNA bound to the O2 atom is inverted in direction by T1 and T2 in the structure by Oie et al. (Non-patent document 8) .
If this structure corresponds to the actual peptidyl transfer reaction, the relative orientations of the two tRNAs at the A and P sites will largely change between T1 and T2. Therefore, it can not be made to correspond to the actual peptidyl group transfer reaction in which the portion ahead of O2 is eliminated after transitioning continuously as T1 → tetrahedral structure; THI → T2. If the structure of T1 takes the structure of T1-2 or T1'-2 of Formula 3 instead, this problem is solved.

Transition state structure described in Oie et al. (Non-patent document 8); TS structure is confirmed by MOAC-PM3 using X-Ability with T1 and T3 as initial structures, and then the results in this T1 And T1 ', which is an optical isomer of T1 at C as initial structure, and further twist angles of T1 and T1'; N-C-O2-H inverted by 180 ° as initial structure and TS optimization as well I did the calculation.

As a result, although T1 'was confirmed, T1-2 and T1'-2 return to the structures of T1 and T1' respectively as a result of structural optimization, and T1-2 and T1'-2 are I could not confirm. However, when T1 is the initial structure and the amidation reaction of ammonia and formic acid is extended to the amidation reaction of ammonia and formate methyl ester, the transition state structures of T1-2 and T1'-2 are obtained, and the peptidyl group transfer reaction It was possible to confirm the transition state in the near state (Equation 4).

[Formula 4] T1 optimized structure confirmed by MOPAC-PM3

The structure of the confirmed transition state T3 (formula 2), and further, the structure of T1-2 and T1'-2 created from the structure of T1 at the P site of the structure of FIG. 3 of the X-ray analysis structure It was connected to the 3 'C atom of the terminal sugar of tRNA. The corresponding carbon atoms of T1-2 and T1'-2 methyl and O2 atoms are superposed on each of the 3 'C atom and the 3' O atom, and the twist angle is N-C-O2-3 'C, and The C-O2-3'C-2'C was examined by rotating it at various angles. As a result, the distance between the N atom of the T1 structure and the carbon atom corresponding to the Cα of the phenylalanine-like structure bonded to the terminal of the tRNA at the A site, which is the atom to be bonded thereto, is T1′-2. It was found that the structure could be as close as 1.73 Å (Figure 5).

With this structure, it was shown that fine adjustment of the structure of the end of the tRNA may exist a structure consistent with the X-ray analysis structure.
In a similar study for T1-2, steric hindrance between the H atom bonded to the C atom in the T1-2 structure and the 2'O atom of the terminal sugar of the P site tRNA leads to an inconsistency with the X-ray analysis structure It turned out that T1 transition state structure with aminoacyl-tRNA of not A site can not be taken.

Similarly, for T3 and its optical isomer, the O 1 atom is connected to the 3 'C atom of the terminal sugar of P sato tRNA in the same manner as in T 1-2 and T 1' -2 above, and the twist angle N-C-O1- The 3'C and C-O1-3'C-2'C were rotated at various angles for verification. As a result, it was found that the distance between the N atom of the T3 structure and the carbon atom corresponding to Cα, which is the atom to be bonded to this, can not be a distance closer than 2.17 Å (FIG. 6). At this time, the distance between the H1 atom and the 2′O atom of the terminal sugar of the A-site tRNA is only 1.54 Å, and if this steric hindrance is avoided, the above interatomic distance further becomes larger than 2.17 Å. It turned out that the T3 structure consistent with the line analysis structure can not be taken.

The same controversial conclusion is obtained for optical isomers in which the configuration of the C atom of T3 is reversed.
From this, the concerted reaction mechanism in which T3 is a transition state structure is excluded from the verification of the amidation reaction on the ribosome.
From the above, the investigation of the peptidyl group transfer reaction mechanism on the ribosome by MOAC-PM3 was focused on the transition state structure T1'-2 in the stepwise reaction mechanism (equation 5).

[Equation 5]

(3-d) Extension of the transition state T1'-2 to the formation reaction of AlanylAlanine by the examination of (3-c-2) The T1'-2 structure (Fig. 3-2) is obtained as the narrowed transition state structure. The Based on this structure, we examined whether there would be any contradiction when extending the amidation reaction from ammonia-formic acid to the reaction between amino acids by calculation with MOAC-PM3. Structures and conformations in which contradiction such as steric hindrance occurred were excluded from the examination.

The formation of Alanyl-alanine by amidification reaction in which ammonia in the transition state T1'-2 structure is an amino group of alanine methyl ester and formate methyl ester is acetylalanine methyl ester was examined. In the examination, the initial structure of TS optimization calculation was constructed with the following assumptions (3-d-1) to (3-d-3) taking account of the actual reaction field on the ribosome as much as possible. (See Equation 6, Equation 7, Calculation Methods a and c).

[Formula 6] Extension of T1'-2 structure to alanylalanine (See Formula 7 for T1'-2 structure)

The substitution positions of the (3-d-1) amine are as shown in Formula 7 in two ways; the possibilities of As1 and As2 are considered.

[Formula 7] Possible substitution position of A (aminoacyl portion of tRNA)

Since the alanine methyl ester corresponding to the peptidyl group at the (3-d-2) P site has no data of X-ray analysis structure, T1 'of the formula 8 is used to examine the conformation of Cα as comprehensively as possible. Twist angle in the structure of -2; NC * CαCβ is divided into categories of 0 ° -108 °, 108 ° -235 °, 235 ° -360 °, and 54 °; Ps1, 171 °; Ps2 as a representative of each category 297 °; Ps3 was the initial structure of the conformation of Cα (Equation 8).
(When twist angle NC ^* -Cα-Cβ = 108 ° Twist angle O1-C ^* -Cα-Cβ = 0 °, twist angle NC ^* -Cα-C-β = 235 ° Twist angle O2 Each corresponds to -C ^* -Cα-Cβ = 0 °.)

[Equation 8] Conformation of possible P (peptidyl moiety) at Cα

The conformation of Cα of alanine methyl ester corresponding to an aminoacyl-tRNA amino acid at (3-d-3) A site is the X-ray analysis structure of phenylalanine-like structure bound to the end of tRNA. Is referred to (equation 9).

[Expression 9] C α conformation of A

The initial structures of As1Ps1, As1Ps2, As1Ps3, As2Ps1, As2Ps2 and As2Ps3 were obtained as the six cases obtained on the premise of (3-d-1) to (3-d-3) above. Among them, As1Ps1 is shown in FIG.
Based on the six initial structures, optimization calculation of the TS structure was performed to obtain the corresponding transition state structures; FIGS. 8 to 13 were obtained (see calculation methods a, c, e, f).

(4) Examination of Transition State T1 on Ribosome (4-a) The examination of (3-d) gave the transition state structure of the alanylalanine formation reaction by the amidation reaction. Using this result, we examined the amidation reaction mechanism in the actual peptidyl transfer reaction on the ribosome.

The reactive transition state structure of alanylalanine was incorporated at the 3 'end of both the A and P sites of the X-ray analysis structure. The structural optimization calculation was performed on the part excluding the reaction center of this structure, and distortion of the structure around the reaction center was removed. The transition state structure was determined with the obtained optimized structure as an initial structure (calculation methods a, b, c, d, e, f).

(4-a-1) The ribosomal molecule is huge, and it is impossible to calculate the transition housing structure of the amidation reaction on this ribosome as it is. In order to reproduce the true aspect of the reaction as much as possible without losing as much as possible, the as wide and computable range as possible around the reaction transition state structure is taken out, and the surrounding structure does not diverge during optimization calculation. It is necessary to devise appropriate restraints.

In this study, the following structures were created as a basis for incorporating into the ribosome the transition state structure of the alanylalanine-producing reaction obtained by the examination of (3-d) above.
In the X-ray analysis structure, the phosphate ester closest to the amidization reaction center of tRNA bound to both A and P sites is deleted from the structural part on the opposite side of the reaction center from the remaining two phosphate ester moieties. 10 methylene groups are inserted and crosslinked (calculation method c).

Furthermore, structural optimization calculation (calculation method d) is performed on this crosslinked structure in a state in which portions other than the crosslinked structure portion are fixed. The result after optimization calculation is shown in FIG.
If the structure optimization calculation of (4-a-3) is performed without inserting the above-mentioned methylene group, the structure diverges and an optimized structure can not be obtained.

(4-a-2) Six TS-optimized structures of the amidation reaction obtained in the above (3-d) examination; each structural part of FIGS. 8 to 13; -O ^* -C (= O) C (CH ₃ ) H-NH--C (= O 1) O 2− is incorporated into the corresponding part of the structure of (4-a-1) above (FIG. 14) and the phenylalanine-like structural part is deleted (FIG. 4-) 2- (a).

The incorporation connects the O2 atom and the P site side C3 'atom, adjusts the twist angle of the C3'-O2 and O2-C bond, and the distance between the O ^* atom and the A site side C3' atom becomes shortest. In this structure, a bond is formed between O ^* -C3 '(the portion shown by the yellow broken line in FIG. 15 (b)). This structure is used as an initial structure for optimizing other than the TS part in consideration of the computer load (FIG. 15 (b)).

(4-a-3) Fix the structures of the amidation reaction center, the phosphate ester part, and the cross-linking part (the part shown in pink in Fig. 15 (b)), and make the other parts free to optimize the structure Calculation (calculation method d, FIG. 15 (b) → (c)).

(4-a-4) The base portion of tRNA to remove the methylene bridge portion from the structure obtained in the above (4-a-3) and reduce the load of the computer in the later TS optimization calculation. , And the phosphate ester portion at the A site are deleted, and this structure is taken as the initial structure of TS optimization calculation (FIG. 15 (c)).

By the above operation, six transition state structures obtained by the above operation (3-d); initial structures used for TS optimization calculation in ribosomes corresponding to each of FIGS. 8 to 13 were obtained. The initial structure obtained in the structure As2Ps1 is shown in FIG.

(4-b) Verification and consideration of TS optimization calculation results on ribosome TS optimization calculation is performed using the initial structure obtained in the above (4-a) (refer to calculation methods a, c, e, f) , An amidation reaction on the ribosome substantially consistent with the X-ray analysis structure; a transition state structure of a formation reaction of alanylalanine; FIGS. 17 to 22 were obtained.

When each of these transition state structures is superimposed on the X-ray analysis structure in the vicinity of the reaction site (FIGS. 23 to 28), As1 of two types of Cα-N bonds (As1, As2) at the A site In the structures (As1Ps1, As1Ps2, As1Ps3), the configuration of the aminoacyl terminus on the A site side is largely different from the Cα, Cβ corresponding position of the phenylalanine analog structure of the aminoacyl terminus of the X-ray analysis structure (FIG. 23). On the other hand, in the As2 structure (As2Ps1, As2Ps2, As2Ps3), they agree well (FIG. 26).

In Ps2 among Ps1, Ps2 and Ps3, the extension direction of the peptide chain in the peptidyl group transfer reaction is in the opposite direction, and it is inserted between the tRNA bound to both A and P sites. Therefore, the possibility of adopting the Ps2 structure is considered to be low (FIGS. 24 and 27).

These results suggest that the actual transition state of the peptidyl transfer reaction is likely to be the structure of As2Ps1 or As2Ps3 (FIGS. 26, 28).

Furthermore, it turned out that the amidation reaction site of the transition state structure can not have a hydrogen bondable distance with 2451 Adenine (the adenine of 50S ribosomal A chain 2451) located at the closest distance among ribosomal molecules in the X-ray analysis structure. (FIGS. 23-28), there appears to be no direct involvement (hydrogen bonding to reactive site atoms) of the 2451 Adenine amidation reaction.

Instead, considering the structure around the benzene ring of the side chain in the phenylalanine-like structure of the aminoacyl terminus of the X-ray analysis structure, 2451 Adenine, together with 2452 Cytidine (cytidine of 50S ribosomal A chain 2452), is an aminoacyl linked to the A site The side chain of the terminal amino acid of tRNA constitutes a pocket that fits loosely rather than strictly like "key and keyhole".

On the other hand, a structure corresponding to a pocket in which the side chain of the terminal amino acid of aminoacyl-tRNA bound to the P site is not found in the X-ray analysis structure. It is considered that this may be related to a mechanism having a unique selectivity that one enzyme, ribosome, can respond to the reaction of 20 amino acids (in the case of natural amino acids).

(4-c) Verification of influence of solvent water molecule Although a large space exists around the amidification reaction center, water of the solvent may be deeply involved in the reaction in this space.
In order to confirm this, the initial structure of the conformation As2Ps1 suggested to be highly likely; referring to FIG. 20, referring to Non-Patent Document 9, the water around the reaction center and the hydrophilic atom in the periphery thereof Were arranged in six molecules (FIG. 29). Using this as an initial structure, TS optimization calculation was performed to obtain a TS structure in the presence of water molecules (FIG. 30).

When the structure of FIG. 30 is superimposed on the X-ray analysis structure (FIG. 31), a good agreement is obtained such that the difference with the X-ray analysis structure is almost slight compared to FIG. .

Furthermore, as shown in FIG. 31, a water molecule hydrogen-bonded to a hydrogen atom bonded to a nitrogen atom at the reaction site is present at a position capable of hydrogen bonding with the N3 atom of the 2451 Adenine. It has been shown that 2451 Adenine may be involved in the amidation reaction via a hydrogen bond with a water molecule.

The hydrogen bond network arrangement of water molecules in the initial structure of the TS optimization calculation of FIG. 29 has many other possibilities, but can be obtained from the restriction of hydrogen bond to the reaction center and its surrounding hydrophilic atoms There is no significant difference between the calculated TS structures. From this, it is considered that the true aspect of the transition state of the amidation reaction in the presence of the solvent water molecule is not significantly different from FIG.

(5) Summary The present study revealed the transition state structure of aminoacyl-tRNA and peptidyl-tRNA in the peptidyl transfer reaction (amidation reaction) on the ribosome.
That is, in the transition state, the aminoacyl-tRNA and the peptidyl-tRNA are very likely to have the As2Ps1 structure (FIG. 26, FIG. 30 in the presence of a water molecule).

That is, it can be said that a protein composed of natural amino acids is synthesized by peptidyl group transfer reaction in which a transition state of As2Ps1 structure is taken on a ribosome. In other words, if the non-natural amino acids aminoacyl tRNA and peptidyl tRNA can adopt the transition state of the As2Ps1 structure on the ribosome, it is also possible to introduce the non-natural amino acid into the protein by the ribosome synthesis system. it can.

Therefore, perform TS optimization calculations for the peptidyl transfer reaction between the aminoacyl tRNA of the unnatural amino acid that is being studied for introduction into proteins and the peptidyl tRNA, and determine whether this takes the transition state of the As2Ps1 structure. The ability to introduce the non-natural amino acid into protein by the ribosomal synthesis system can be estimated by

(6) Calculation method (a) All calculations are done in TOSHIBA dynabook R731 / 37C.
I went on the computer.

(B) All coordinate data required for calculation by MOAC-PM3 were obtained from PDB. A graphic interface, RASMOL, was used to visualize the calculation results.

(C) MOAPC-PM3 A semi-empirical MO method was performed with the Winmostar (GE) V4.024 (X-Ability) calculation package. The initial structure required for calculation was created with Winmostar's visualization and manual functions based on PDB data. In the process, if steric hindrance occurred in the structure, it was optimized by MM2 method included in Winmostar application.

(D) Eigenvector Following routine was used to optimize the default molecular structure.
MOPAC keyword; PM3 EF PRECISE VECTORS MMOK

(E) Optimization of transition state energy was performed until the gradient norm was achieved below 0.05 kcal / mol / A.
MOPAC keyword; PM3 TS PRECISE MMOK
We did not consider the influence of the solvent, but performed optimization calculation that added the water molecular hydrogen bond network separately.

(F) After TS optimization calculation, confirmation of the transition state was confirmed by the fact that there is only one negative frequency in vibration calculation.

The present invention can be used to design artificial proteins containing amino acid residues of unnatural amino acids.

Claims

A method for estimating the adaptability of an unnatural amino acid to a protein in a ribosomal protein synthesis system, comprising the following steps a and b:
(Step a)
An aminoacyl-tRNA of the unnatural amino acid represented by the general formula (1);
A peptidyl tRNA having any amino acid sequence represented by the general formula (2-1) or (2-2),
Reaction transition state (general formula (3-1), (3-2), (3-3), (3-4) or (3-5)) or its structure reaction in the peptidyl group transfer reaction of A step of performing transition state structure optimization calculation (TS optimization calculation) by the molecular orbital method with a structure omitted as appropriate in order to reduce calculation load from a part far from the center as an initial structure.
(Step b)
A step of judging that the adaptability of the unnatural amino acid to the protein in the protein synthesis system by ribosome is high when it can be confirmed that the structure obtained as a result of the step a is a transition state structure.

(A a1 represents a structural moiety other than an amino group and a carboxyl group involved in the peptide bond in the non-natural amino acid.
B a1 represents an arbitrary group or a group which forms a cyclic structure of B a1 -A a1 -N t1 together with the above-mentioned A a1 .
The tRNA a1 represents a tRNA covalently linked to O a12 at the 3 'end. )

(TRNA P1 represents a tRNA covalently bound to O P1 in the 3 'end.
P P1 represents the peptidyl group. )

(Structural portion of the tRNA P12 -O P12 -H P12 has the general formula and tRNA P1 in (2-1) represents the same structure. Namely, the oxygen O P12 is bound to 2 'carbon of the 3'-terminal sugar of tRNA P1 The atom, HP12 is a hydrogen atom bonded to OP12 , and tRNA P12 is a portion of tRNA P1 from which OP 12 and HP 12 have been removed.
The meaning of the symbols in the general formula (2-2) is the same as in the general formula (2-1). )

(Structure N t1 (H t1 ) -C t1 (O t1 ) moiety is a reaction center in the reaction transition state structure of the first step of stepwise amidification reaction,
The distance between N t1 and C t1 is 1.53 Å to 1.77 Å,
The distance between N t1 and H t1 is 1.10 Å to 1.35 Å.
The distance between H t1 and O t1 is 1.35 Å to 1.45 Å.
The meaning of the symbols in the general formula (3-1) is the same as in the general formulas (1) and (2-1). )

(H t12 is a hydrogen atom which protonated O t1 , which was carbonyl oxygen in the first step of the stepwise amidation reaction.
The moiety C t1 -O p1 -H t12 -O t1 is the reaction center in the reaction transition state structure of the second step of the stepwise amidification reaction,
The distance between C t1 and O p1 is 1.45 Å to 1.70 Å.
Distance of O p1 and H t12 is 1.05Å ~ 1.30Å,
The distance between H t12 and O t1 is 1.10 Å to 1.40 Å,
The distance between O t1 and C t1 is 1.22 Å to 1.42 Å.
The meaning of the symbols in the general formula (3-2) is the same as in the general formulas (1) and (2-1). )

(Structure N t1 (H t1 ) -C t1 (O p1 ) moiety is a reaction center in the transition state structure of a concerted amidation reaction having a four-membered ring structure,
The distance between N t1 and C t1 is 1.40 Å to 1.65 Å,
The distance between N t1 and H t1 is 1.00 Å to 1.24 Å,
The distance between C t1 and O p1 is 1.35 Å to 2.40 Å.
Distance O p1 and H t1 is 1.00Å ~ 1.84Å.
The meaning of the symbols in the general formula (3-3) is the same as in the general formulas (1) and (2-1). )

(Structure N t1 -C t1 -O p1 -H p12 -O p12 -H t1 moiety is the reaction center in the transition state structure of the concerted amidation reaction having a six-membered ring structure,
The distance between N t1 and C t1 is 1.40 Å to 2.10 Å.
The distance between N t1 and H t1 is 1.00 Å to 1.20 Å,
The distance between C t1 and O p1 is 1.35 Å to 2.30 Å.
Distance of O p1 and H p12 is 1.00Å ~ 1.56Å,
The distance between H p12 and O p12 is 1.00 Å to 1.67 Å,
The distance between Op 12 and H t1 is 1.20 Å to 1.96 Å.
The meaning of the symbols in the general formula (3-4) is the same as in the general formulas (1) and (2-2). )

(H s12 -O s1 -H s11 is one of the solvent water molecules, and the structure N t1 -C t1 -O p1 -H s11 -O s1 -H p12 -O p12 -H t1 moiety has an eight-membered ring structure A reaction center in the transition state structure of the concerted amidification reaction,
The distance between N t1 and C t1 is 1.40 Å to 1.79 Å.
The distance between N t1 and H t1 is 1.00 Å to 1.26 Å,
The distance between C t1 and O p1 is 1.40 Å to 2.21 Å,
Distance of O p1 and H s12 is 1.13Å ~ 1.43Å,
The distance between H s12 and O s1 is 1.00 Å to 1.37 Å,
The distance between O s1 and H p12 is 1.30 Å to 1.69 Å,
The distance between H p12 and O p12 is 0.98 Å to 1.24 Å,
The distance between Op 12 and H t1 is 1.17 Å to 1.76 Å.
The meaning of the symbols in the general formula (3-5) is the same as in the general formulas (1) and (2-2). )
The method according to claim 1, wherein the general formula (3-1) is the following general formula (3-1-1).

(The meaning of the symbol in the general formula (3-1-1) is the same as in the general formula (3-1).)
The structure represented by the general formula (1) is a structure represented by the following general formula (4),
The structures represented by the general formulas (3-1), (3-2), (3-3), (3-4), and (3-5) have the following general formulas (5-1) and (5-3), respectively. The method according to claim 1, which is a structure represented by 5-2), (5-3), (5-4), (5-5).

(C α1 is an α carbon of the non-natural amino acid, and R a1 and R a2 each independently represent a side chain structure of the non-natural amino acid (however, R a2 may be a hydrogen atom).
Other symbols are the same as in the general formula (1). )

(The distances between N t1 and C t1 , N t1 and H t1 , and H t1 and O t1 are the same as in the general formula (3-1).
The meaning of the symbols in the general formula (5-1) is the same as in the general formulas (1), (2-1) and (4). )

(The meanings of the symbols R a1 , C α1 and R a2 are the same as in the general formula (4). The meanings of the symbols in the general formula (5-2) and the interatomic distance are the same as in the general formula (3-2). )

(The meanings of the symbols R a1 , C α1 and R a2 are the same as in the general formula (4). The meaning of the symbols in the general formula (5-3) and the interatomic distance are the same as in the general formula (3-3). )

(The meanings of the symbols R a1 , C α1 and R a2 are the same as in the general formula (4). The meanings of the symbols in the general formula (5-4) and the interatomic distance are the same as in the general formula (3-4). )

(The meanings of the symbols R a1 , C α1 and R a2 are the same as in the general formula (4). The meaning of the symbols in the general formula (5-5) and the interatomic distance are the same as in the general formula (3-5). )
A method for estimating the adaptability of an unnatural amino acid to a protein in a ribosomal protein synthesis system, comprising the following steps of c to e.
(Step c)
Prepare three-dimensional structural data obtained by X-ray structural analysis of ribosome in which tRNA or its derivative is bound to A and P sites,
In the three-dimensional structure data, according to the following conditions:
An aminoacyl-tRNA of the unnatural amino acid represented by the general formula (1);
A peptidyl tRNA having any amino acid sequence represented by the general formula (2-1) or (2-2),
A step of incorporating the assumed reaction transition state (general formula (3-1), (3-2), (3-3), (3-4) or (3-5)) in the peptidyl group transfer reaction of
(Condition 1) The general formula (3) is such that the atomic coordinates of the 3 'terminal carbon of tRNA a1 are located at least near the 3' terminal carbon of the tRNA at the position of the tRNA on the A site in the three-dimensional structure data. 1) Incorporate the assumed structure represented by (3-2), (3-3), (3-4) or (3-5).
(Condition 2) The atomic formula of the 3 'terminal carbon of tRNA P1 is located at least near the 3' terminal carbon of the tRNA at the position of the tRNA on the P site in the three-dimensional structure data, 1) Incorporate the assumed structure represented by (3-2), (3-3), (3-4) or (3-5).
(Step d)
Transition state structure optimization calculation (TS optimization calculation) by molecular orbital method with a structure obtained by appropriately removing the structure far from the reaction center of the structure obtained by the step c or the structure, in order to reduce the calculation load as an initial structure Process of
(Step e)
A step of judging that the adaptability of the unnatural amino acid to the protein in the protein synthesis system by ribosome is high when the structure obtained as a result of the step d is confirmed to be a transition state structure.

(A a1 represents a structural moiety other than an amino group and a carboxyl group involved in the peptide bond in the non-natural amino acid.
B a1 represents an arbitrary group or a group which forms a cyclic structure of B a1 -A a1 -N t1 together with the above-mentioned A a1 .
The tRNA a1 represents a tRNA covalently linked to O a12 at the 3 'end. )

(TRNA P1 represents a tRNA covalently bound to O P1 in the 3 'end.
P P1 represents the peptidyl group. )

(Structural portion of the tRNA P12 -O P12 -HP 12 has the general formula and tRNA P1 in (2-1) represents the same structure. Namely, the oxygen O P12 is bound to 2 'carbon of the 3'-terminal sugar of tRNA P1 The atom, HP12 is a hydrogen atom bonded to OP12 , and tRNA P12 is a portion of tRNA P1 from which OP 12 and HP 12 have been removed.
The meaning of the symbols in the general formula (2-2) is the same as in the general formula (2-1). )

(Structure N t1 (H t1 ) -C t1 (O t1 ) moiety is a reaction center in the reaction transition state structure of the first step of stepwise amidification reaction,
The distance between N t1 and C t1 is 1.53 Å to 1.77 Å,
The distance between N t1 and H t1 is 1.10 Å to 1.35 Å.
The distance between H t1 and O t1 is 1.35 Å to 1.45 Å.
The meaning of the symbols in the general formula (3-1) is the same as in the general formulas (1) and (2-1). )

(H t12 is a hydrogen atom that protonated O t1 , which was carbonyl oxygen in the second step of the stepwise amidation reaction.
The moiety C t1 -O p1 -H t12 -O t1 is the reaction center in the reaction transition state structure of the second step of the stepwise amidification reaction,
The distance between C t1 and O p1 is 1.45 Å to 1.70 Å.
Distance of O p1 and H t12 is 1.05Å ~ 1.30Å,
The distance between H t12 and O t1 is 1.10 Å to 1.40 Å,
The distance between O t1 and C t1 is 1.22 Å to 1.42 Å.
The meaning of the symbols in the general formula (3-2) is the same as in the general formulas (1) and (2-1). )

(Structure N t1 (H t1 ) -C t1 (O p1 ) moiety is a reaction center in the transition state structure of a concerted amidation reaction having a four-membered ring structure,
The distance between N t1 and C t1 is 1.40 Å to 1.65 Å,
The distance between N t1 and H t1 is 1.00 Å to 1.24 Å,
The distance between C t1 and O p1 is 1.35 Å to 2.40 Å.
Distance O p1 and H t1 is 1.00Å ~ 1.84Å.
The meaning of the symbols in the general formula (3-3) is the same as in the general formulas (1) and (2-1). )

(Structure N t1 -C t1 -O p1 -H p12 -O p12 -H t1 moiety is the reaction center in the transition state structure of the concerted amidation reaction having a six-membered ring structure,
The distance between N t1 and C t1 is 1.40 Å to 2.10 Å.
The distance between N t1 and H t1 is 1.00 Å to 1.20 Å,
The distance between C t1 and O p1 is 1.35 Å to 2.30 Å.
Distance of O p1 and H p12 is 1.00Å ~ 1.56Å,
The distance between H p12 and O p12 is 1.00 Å to 1.67 Å,
The distance between Op 12 and H t1 is 1.20 Å to 1.96 Å.
The meaning of the symbols in the general formula (3-4) is the same as in the general formulas (1) and (2-2). )

(H s12 -O s1 -H s11 is one of the solvent water molecules, and the structure N t1 -C t1 -O p1 -H s11 -O s1 -H p12 -O p12 -H t1 moiety has an eight-membered ring structure A reaction center in the transition state structure of the concerted amidification reaction,
The distance between N t1 and C t1 is 1.40 Å to 1.79 Å.
The distance between N t1 and H t1 is 1.00 Å to 1.26 Å,
The distance between C t1 and O p1 is 1.40 Å to 2.21 Å,
Distance of O p1 and H s12 is 1.13Å ~ 1.43Å,
The distance between H s12 and O s1 is 1.00 Å to 1.37 Å,
The distance between O s1 and H p12 is 1.30 Å to 1.69 Å,
The distance between H p12 and O p12 is 0.98 Å to 1.24 Å,
The distance between Op 12 and H t1 is 1.17 Å to 1.76 Å.
The meaning of the symbols in the general formula (3-5) is the same as in the general formulas (1) and (2-2). )
The method according to claim 4, further comprising the following e ′ step after the e step:
(E 'process)
The structure confirmed to be a transition state structure in the step e) is
The cyclic structure containing the reaction center (in a form incorporating the structure represented by the general formula (3-4) or the general formula (3-5), the cyclic structure containing tRNA P12 of the general formula is included ) By adjusting the twist angle of the bond axis of the other part within the range that steric hindrance does not occur, the three-dimensional carbon corresponding atom of tRNA a1 and the three-terminal carbon corresponding atom of tRNA P1 are respectively three-dimensional In the case where the displacement between the terminal 3 'carbon of tRNA on the A site and the terminal 3' carbon of tRNA on the P site is within 0.3 Å in structural data, and fitting to the three-dimensional structural data is possible:
It is judged that the adaptability of the unnatural amino acid to the protein is higher.
When incorporating the assumed structure represented by the general formula (3-1), (3-2), (3-3), (3-4) or (3-5) in the step c,
A part of the structure of a similar structural compound from which a transition state optimization structure is obtained, or a case where it is judged by the method according to claim 1 or 2 that the aptitude for introducing the unnatural amino acid into a protein is high. 6. The method according to claim 4, wherein a part of the structure obtained as a result of the step a), which has been confirmed to be a transition state structure in the step b, is incorporated.
The method according to any one of claims 4 to 6, further comprising the following step f.
(Step f)
It is confirmed that the structure obtained as a result of the step d is a transition state structure, and
When the three-dimensional structural data is an X-ray analysis structure of a Thermus thermophilus ribosome, the side chain of the unnatural amino acid is formed by 2451A, 2452C, 2506U and 2585U in the ribosome When it fits in space without steric hindrance,
Alternatively, when the three-dimensional structural data is an X-ray analysis structure of a ribosome other than Thermus thermophilus, the side chain of the unnatural amino acid is in the ribosome of Thermus thermophilus. In the space formed by the bases corresponding to 2451A, 2452C, 2506U and 2585U of
And a step of judging that the adaptability of the unnatural amino acid to the protein in the protein synthesis system by ribosome is high.
A method for estimating the adaptability of an unnatural amino acid to a protein in a ribosomal protein synthesis system, comprising the following g and h steps.
(Step g)
An aminoacyl-tRNA of any amino acid represented by the general formula (6):
A peptidyl tRNA having as a constituent an amino acid residue of the non-natural amino acid represented by the general formula (7-1) or (7-2);
Reaction transition state (general formula (8-1), (8-2), (8-3), (8-4) or (8-5)) or reaction of its structure in the peptidyl group transfer reaction of A step of performing transition state structure optimization calculation (TS optimization calculation) by the molecular orbital method with a structure omitted as appropriate in order to reduce calculation load from a part far from the center as an initial structure.
(Step h)
A step of judging that the adaptability of the unnatural amino acid to the protein in the protein synthesis system by ribosome is high when it can be confirmed that the structure obtained as a result of the step g is a transition state structure.

(A a2 represents a structural moiety other than an amino group and a carboxyl group involved in peptide bond in any of the amino acids.
B a2 represents an arbitrary group or a group which forms a cyclic structure of B a2 -A a2 -N t2 together with the above-mentioned A a2 .
tRNA a2 represents a tRNA covalently linked to O a22 at the 3 'end. )

(A P represents a structural moiety other than an amino group and a carboxyl group involved in a peptide bond in the non-natural amino acid.
B P represents any group, or the A P and integral with it B P -A P -N group to form a cyclic structure of P.
tRNA P2 represents a tRNA covalently bound to O P2 in the 3 'end.
P P2 represents the peptidyl group. )

(The structural portion of tRNA P22 -OP 22 -HP 22 has the same structure as tRNA P2 in the general formula (7-1). That is, OP22 is an oxygen bonded to the 2 'carbon of the 3' terminal sugar of tRNA P2 The atom, HP22 is a hydrogen atom bonded to OP22 , and tRNA P22 is a portion of tRNA P2 excluding OP 22 and HP 22 .
The meaning of the symbols in the other general formula (7-2) is the same as in the general formula (7-1). )

(Structure N t2 (H t2 ) -C t2 (O t2 ) moiety is a reaction center in the reaction transition state structure of the first step of the stepwise amidation reaction,
The distance between N t2 and C t2 is 1.53 Å to 1.77 Å,
The distance between N t2 and H t2 is 1.10 Å to 1.35 Å.
The distance between H t2 and O t2 is 1.35 Å to 1.45 Å.
The meaning of the symbols in the general formula (8-1) is the same as in the general formulas (6) and (7-1). )

(H t22 is a hydrogen atom which protonated O t2 , which was carbonyl oxygen in the second step of the stepwise amidation reaction.
The moiety C t2 -O p2 -H t22 -O t2 moiety is the reaction center in the reaction transition state structure of the second stage of the stepwise amidation reaction,
The distance between C t2 and O p2 is 1.45 Å to 1.70 Å.
Distance of O p2 and H t22 is 1.05Å ~ 1.30Å,
The distance between H t22 and O t2 is 1.10 Å to 1.40 Å,
The distance between O t2 and C t2 is 1.22 Å to 1.42 Å.
The meaning of the symbols in the general formula (8-2) is the same as in the general formulas (6) and (7-1). )

(Structure N t2 (H t2 ) -C t2 (O p2 ) moiety is a reaction center in the transition state structure of a concerted amidation reaction having a four-membered ring structure,
The distance between N t2 and C t2 is 1.40 Å to 1.65 Å,
The distance between N t2 and H t2 is 1.00 Å to 1.24 Å,
The distance between C t2 and O p2 is 1.35 Å to 2.40 Å,
Distance O p2 and H t2 is 1.00Å ~ 1.84Å.
The meaning of the symbols in the general formula (8-3) is the same as in the general formulas (6) and (7-1). )

(Structure N t2 -C t2 -O p2 -H p22 -O p22 -H t2 moiety is the reaction center in the transition state structure of the concerted amidation reaction having a six-membered ring structure,
The distance between N t2 and C t2 is 1.40 Å to 2.10 Å,
The distance between N t2 and H t2 is 1.00 Å to 1.20 Å,
The distance between C t2 and O p2 is 1.35 Å to 2.30 Å.
Distance of O p2 and H p22 is 1.00Å ~ 1.56Å,
The distance between H p22 and O p22 is 1.00 Å to 1.67 Å,
The distance between Op 22 and H t2 is 1.20 Å to 1.96 Å.
The meaning of the symbol in the general formula (8-4) is the same as in the general formulas (6) and (7-2). )

(H s21 -O s2 -H s22 is one of the solvent water molecules, and the structure N t2 -C t2 -O p2 -H s21 -O s2 -H p22 -O p22 -H t2 moiety has an eight-membered ring structure A reaction center in the transition state structure of the concerted amidification reaction,
The distance between N t2 and C t2 is 1.40 Å to 1.79 Å.
The distance between N t2 and H t2 is 1.00 Å to 1.26 Å,
The distance between C t2 and O p2 is 1.40 Å to 2.21 Å,
Distance of O p2 and H s21 is 1.13Å ~ 1.43Å,
The distance between H s21 and O s2 is 1.00 Å to 1.37 Å,
The distance between O s2 and H p22 is 1.30 Å to 1.69 Å,
The distance between H p22 and O p22 is 0.98 Å to 1.24 Å,
The distance between Op 22 and H t2 is 1.17 Å to 1.76 Å.
The meaning of the symbol in the general formula (8-5) is the same as in the general formulas (6) and (7-2). )
The method according to claim 8, wherein the general formula (8-1) is the following general formula (8-1-1).

(The meaning of the symbol in the general formula (8-1-1) is the same as in the general formula (8-1).)
The structure represented by the general formula (7-1) or (7-2) is a structure represented by the following general formula (9-1) or (9-2),
The structures represented by the general formulas (8-1), (8-2), (8-3), (8-4), and (8-5) have the following general formulas (10-1) and (10), respectively. The method according to claim 8, characterized in that it has a structure represented by -2), (10-3), (10-4) or (10-5).

(C α2 is the α carbon of the non-natural amino acid, and R P1 and R P2 each independently represent the side chain structure of the non-natural amino acid (however, R P2 may be a hydrogen atom).
The meaning of the other symbols is the same as in the general formula (7-1). )

(TRNA p22 , OP 22 , and HP 22 have the same meaning as in general formula (7-2). The meanings of other symbols are the same as in general formula (9-1).)

((Structure N t2 (H t2 ) -C t2 (O t2 ) part, the distances between N t2 and C t2 , N t2 and H t2 , and H t2 and O t2 are the same as in the general formula (8-1).
The meaning of the symbols in the general formula (10-1) is the same as in the general formulas (6) and (7-1). )

(The meanings of the symbols R P1 , C α2 and R P2 are the same as in the general formula (9-1). The meaning of the symbols in the general formula (10-2) and the interatomic distance are the same as in the general formula (8-2) the same.)

(The meanings of the symbols R P1 , C α2 and R P2 are the same as in the general formula (9-1). The meaning of the symbols in the general formula (10-3) and the interatomic distance are the same as in the general formula (8-3) the same.)

(The meanings of the symbols R P1 , C α2 and R P2 are the same as in the general formula (9-2). The meaning of the symbols in the general formula (10-4) and the interatomic distance are the same as in the general formula (8-4) the same.)

(The meanings of the symbols R P1 , C α2 and R P2 are the same as in the general formula (9-2). The meanings of the symbols in the general formula (10-5) and the interatomic distance are the same as in the general formula (8-5) the same.)
A method for estimating the adaptability of an unnatural amino acid to a protein in a ribosomal protein synthesis system, comprising the following ik steps:
(Step i)
Prepare three-dimensional structural data obtained by X-ray structural analysis of ribosome in which tRNA or its derivative is bound to A and P sites,
In the three-dimensional structure data, according to the following conditions:
An aminoacyl-tRNA of any amino acid represented by the general formula (6):
A peptidyl tRNA having as a constituent an amino acid residue of the non-natural amino acid represented by the general formula (7-1) or (7-2);
A step of incorporating an assumed reaction transition state (general formula (8-1), (8-2), (8-3), (8-4) or (8-5)) in the peptidyl group transfer reaction of
(Condition 3) The general formula (8) is such that the atomic coordinates of the 3 'terminal carbon of tRNA a2 are located at least near the 3' terminal carbon of the tRNA at the position of the tRNA on the A site in the three-dimensional structure data. Incorporate the assumed structure represented by -1), (8-2), (8-3), (8-4) or (8-5)).
(Condition 4) The general formula (8) is such that atomic coordinates of the 3 'terminal carbon of tRNA P2 are located at least near the 3' terminal carbon of the tRNA at the position of the tRNA on the P site in the three-dimensional structure data. Incorporate the assumed structure represented by -1), (8-2), (8-3), (8-4) or (8-5)).
(Step j)
Transition state structure optimization calculation (TS optimization calculation) by molecular orbital method with the structure obtained by appropriately removing the structure far from the reaction center of the structure obtained by the step i or the structure omitted to reduce the calculation load as an initial structure Process of
(Step k)
A step of judging that the adaptability of the unnatural amino acid to the protein in the protein synthesis system by ribosome is high when the structure obtained as a result of the step j is confirmed to be a transition state structure.

(A a2 represents a structural moiety other than an amino group and a carboxyl group involved in peptide bond in any of the amino acids.
B a2 represents an arbitrary group or a group which forms a cyclic structure of B a2 -A a2 -N t2 together with the above-mentioned A a2 .
tRNA a2 represents a tRNA covalently linked to O a22 at the 3 'end. )

(A P represents a structural moiety other than an amino group and a carboxyl group involved in a peptide bond in the non-natural amino acid.
B P represents any group, or the A P and integral with it B P -A P -N group to form a cyclic structure of P.
tRNA P2 represents a tRNA covalently bound to O P2 in the 3 'end.
P P2 represents the peptidyl group. )

(The structural portion of tRNA P22 -OP 22 -HP 22 has the same structure as tRNA P2 in the general formula (7-1). That is, OP22 is an oxygen bonded to the 2 'carbon of the 3' terminal sugar of tRNA P2 The atom, HP22 is a hydrogen atom bonded to OP22 , and tRNA P22 is a portion of tRNA P2 excluding OP 22 and HP 22 .
The meaning of the symbols in the other general formula (7-2) is the same as in the general formula (7-1). )

(Structure N t2 (H t2 ) -C t2 (O t2 ) moiety is a reaction center in the reaction transition state structure of the first step of the stepwise amidation reaction,
The distance between N t2 and C t2 is 1.53 Å to 1.77 Å,
The distance between N t2 and H t2 is 1.10 Å to 1.35 Å.
The distance between H t2 and O t2 is 1.35 Å to 1.45 Å.
The meaning of the symbols in the general formula (8-1) is the same as in the general formulas (6) and (7-1). )

(H t22 is a hydrogen atom which protonated O t2 , which was carbonyl oxygen in the second step of the stepwise amidation reaction.
The moiety C t2 -O p2 -H t22 -O t2 moiety is the reaction center in the reaction transition state structure of the second stage of the stepwise amidation reaction,
The distance between C t2 and O p2 is 1.45 Å to 1.70 Å.
Distance of O p2 and H t22 is 1.05Å ~ 1.30Å,
The distance between H t22 and O t2 is 1.10 Å to 1.40 Å,
The distance between O t2 and C t2 is 1.22 Å to 1.42 Å.
The meaning of the symbols in the general formula (8-2) is the same as in the general formulas (6) and (7-1). )

(Structure N t2 (H t2 ) -C t2 (O p2 ) moiety is a reaction center in the transition state structure of a concerted amidation reaction having a four-membered ring structure,
The distance between N t2 and C t2 is 1.40 Å to 1.65 Å,
The distance between N t2 and H t2 is 1.00 Å to 1.24 Å,
The distance between C t2 and O p2 is 1.35 Å to 2.40 Å,
Distance O p2 and H t2 is 1.00Å ~ 1.84Å.
The meaning of the symbols in the general formula (8-3) is the same as in the general formulas (6) and (7-1). )

(Structure N t2 -C t2 -O p2 -H p22 -O p22 -H t2 moiety is the reaction center in the transition state structure of the concerted amidation reaction having a six-membered ring structure,
The distance between N t2 and C t2 is 1.40 Å to 2.10 Å,
The distance between N t2 and H t2 is 1.00 Å to 1.20 Å,
The distance between C t2 and O p2 is 1.35 Å to 2.30 Å.
Distance of O p2 and H p22 is 1.00Å ~ 1.56Å,
The distance between H p22 and O p22 is 1.00 Å to 1.67 Å,
The distance between Op 22 and H t2 is 1.20 Å to 1.96 Å.
The meaning of the symbol in the general formula (8-4) is the same as in the general formulas (6) and (7-2). )

(H s21 -O s2 -H s22 is one of the solvent water molecules, and the structure N t2 -C t2 -O p2 -H s21 -O s2 -H p22 -O p22 -H t2 moiety has an eight-membered ring structure A reaction center in the transition state structure of the concerted amidification reaction,
The distance between N t2 and C t2 is 1.40 Å to 1.79 Å.
The distance between N t2 and H t2 is 1.00 Å to 1.26 Å,
The distance between C t2 and O p2 is 1.40 Å to 2.21 Å,
Distance of O p2 and H s21 is 1.13Å ~ 1.43Å,
The distance between H s21 and O s2 is 1.00 Å to 1.37 Å,
The distance between O s2 and H p22 is 1.30 Å to 1.69 Å,
The distance between H p22 and O p22 is 0.98 Å to 1.24 Å,
The distance between Op 22 and H t2 is 1.17 Å to 1.76 Å.
The meaning of the symbol in the general formula (8-5) is the same as in the general formulas (6) and (7-2). )
The method according to claim 11, wherein after the k step, the following k 'step is included.
(K 'process)
The structure confirmed to be a transition state structure in the k step is
The cyclic structure including the reaction center (in a form incorporating the structure represented by the general formula (8-4) or the general formula (8-5), includes a cyclic structure containing the tRNA P22 of the general formula ) By adjusting the twist angle of the bond axis of the other part within the range where steric hindrance does not occur, the three-dimensional carbon corresponding atom of tRNA a2 and the three-terminal carbon corresponding atom of tRNA P2 are respectively three-dimensional In the case where the displacement between the terminal 3 'carbon of tRNA on the A site and the terminal 3' carbon of tRNA on the P site is within 0.3 Å in structural data, and fitting to the three-dimensional structural data is possible:
It is judged that the adaptability of the unnatural amino acid to the protein is higher.
When incorporating the assumed structure represented by the general formula (8-1), (8-2), (8-3), (8-4), or (8-5) in the step i,
A part of the structure of a similar structural compound for which a transition state optimization structure is obtained, or a case where it is judged by the method according to claim 8 or 9 that the aptitude for introducing the unnatural amino acid into a protein is high. The method according to claim 11 or 12, characterized in that a part of the structure obtained as a result of the g step is incorporated, which has been confirmed to be a transition state structure in the h step.
The method according to any one of claims 11 to 13, further comprising the following 1 step:
(L process)
It is assumed that the structure obtained as a result of the j step is a transition state structure, and
When the three-dimensional structural data is an X-ray analysis structure of a Thermus thermophilus ribosome, the side chain of the unnatural amino acid is formed by 2451A, 2452C, 2506U and 2585U in the ribosome When it fits in space without steric hindrance,
Alternatively, when the three-dimensional structural data is an X-ray analysis structure of a ribosome other than Thermus thermophilus, the side chain of the unnatural amino acid is in the ribosome of Thermus thermophilus. In the space formed by the bases corresponding to 2451A, 2452C, 2506U and 2585U of
And a step of judging that the adaptability of the unnatural amino acid to the protein in the protein synthesis system by ribosome is high.
The structure represented by the general formula (7-1) is a structure represented by the following general formula (9-1),
The structure represented by the general formula (8-1) is a structure represented by the following general formula (10-1),
The method according to any one of claims 11 to 14, characterized in that it comprises the following m steps:
(M process)
In the i step, the structure represented by the general formula (10-1) is incorporated, and in the structure obtained as a result of the j step, the twist angle ∠N t2 -C t2 -C α2 -R P1 is 0 ° to 108 ° Determining that the adaptability of the unnatural amino acid to the protein in the protein synthesis system by ribosome is high.

(C α2 is the α carbon of the non-natural amino acid, and R P1 and R P2 each independently represent the side chain structure of the non-natural amino acid (however, R P2 may be a hydrogen atom)).

(Structure N t2 (H t2 ) -C t2 (O t2 ) portion, the distances of N t2 and C t2 , N t2 and H t2 , and H t2 and O t2 are the same as in the general formula (8-1).
The meaning of the symbols in the general formula (10-1) is the same as in the general formulas (6) and (7-1). )
When the A P of the general formula (7-1) or (7-2) in the presence hydrophilic atoms, characterized in that it comprises the following n steps, to any one of claims 11 to 13 Method described.
(N steps)
In the structure obtained as a result of the j step, the hydrophilic atom is a group represented by the general formula (8-1), (8-2), (8-3), (8-4) or (8-5). A step of judging that the adaptability of the unnatural amino acid to a protein is high in a ribosomal protein synthesis system, when it is presumed that a hydrogen bond can be formed with Ht2 and / or Ot2 .
The method according to any one of claims 11 to 16, comprising the following steps o to q:
(Step o)
In the structure obtained by the i step,
Furthermore, the reaction of the peptidyl group transfer reaction in the ribosome is a substance which is present in the reaction system of the biological synthesis method by the ribosome and which is confirmed or predicted to promote or not inhibit the peptidyl group transfer reaction by the ribosome A process of setting as a initial structure a structure disposed at the center or its periphery and having a structure to which this is added or a portion far from the reaction center of the structure is omitted in order to reduce the calculation load
A step of performing TS optimization calculation by a molecular orbital method on the initial structure obtained by the step o.
(Step q)
It is confirmed that the structure obtained as a result of the p step is a transition state structure, and ribosomal transfer reaction which proceeds by taking the transition state structure is not inhibited by the steric hindrance by the substance, the ribosome by the ribosome A step of judging that the adaptability of the unnatural amino acid to the protein in the protein synthesis system is high.
The method according to claim 17, further comprising the following r step.
(R process)
It is confirmed that the structure obtained as a result of the p step is a transition state structure, and the general formula (8-1), (8-2), (8-3), (8-4), or 8-4), when it is possible to form a hydrogen bond between the molecule of the transition state optimization structure and the substance, the ribosome synthesis system of ribosomes has the adaptability to introduce the unnatural amino acid into a protein A step of determining high.
The atomic coordinates registered in the Protein Data Bank (PDB) as Accession number 4V5C or 4V5D, the three-dimensional structure data is characterized in that any one of claims 4 to 6 and 11 to 17 Method described.
A method according to any one of the preceding claims, characterized in that the molecular orbital method is a semi-empirical molecular orbital method.
21. An estimation step of estimating adaptability of a non-natural amino acid to a protein in a ribosomal protein synthesis system by the method according to any one of claims 1 to 20;
A process for producing an artificial protein, comprising the step of producing a protein by a ribosomal protein synthesis system using as a raw material a non-natural amino acid determined to have high introduction suitability in the estimation step.
A method of improving the performance of a protein or peptide, comprising
21. An estimation step of estimating adaptability of a non-natural amino acid to a protein in a ribosomal protein synthesis system by the method according to any one of claims 1 to 20;
A production process for producing a protein or a peptide by a protein synthesis system by ribosome using as a raw material a non-natural amino acid determined to have high introduction suitability in the estimation step;
Assessing the performance of the protein or peptide produced in the production step.
A method of improving the productivity or production efficiency of a protein or peptide by a ribosomal protein synthesis system, comprising:
21. An estimation step of estimating adaptability of a non-natural amino acid to a protein in a ribosomal protein synthesis system by the method according to any one of claims 1 to 20;
A production process for producing a protein or a peptide by a protein synthesis system by ribosome using as a raw material a non-natural amino acid determined to have high introduction suitability in the estimation step;
And e) evaluating the productivity or production efficiency of the protein or peptide in the production step.
A drug design method for a drug comprising an artificial protein or artificial peptide into which a non-natural amino acid is introduced,
Selecting a non-naturally occurring amino acid that improves efficacy or reduces side effects by introducing it into a lead compound protein or peptide according to Ligand Based Drug Design (LBDD) and / or Structure-Base Drug Design (SBDD);
An estimation step of estimating adaptability of the unnatural amino acid to the protein or peptide in a ribosomal protein synthesis system by the method according to any one of claims 1 to 18;
A drug design method characterized in that