JP2009112282A - Miniature protein and crystal thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a miniature protein capable of forming a specific three-dimensional steric structure (hairpin structure) similar to chignolin which is a known miniature protein, having excellent thermal stability compared with chignolin and enabling crystallization and to provide a fusion protein containing the miniature protein. <P>SOLUTION: Provided is a protein containing an amino acid sequence expressed by following formula 1, wherein the region corresponding to the formula 1 has a steric structure defined by a specific atomic coordinate or a steric structure having a root mean square deviation (RMSD) of ≤5 Å from the coordinate of the main chain. Formula 1: XaaTyrAspProGluThrGlyThrTrpXbb. In the formula, Xaa and Xbb are each an arbitrary amino acid residue other than Gly and Ala; and the carboxyl group may be amidated when Xbb is a C-terminal. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a novel ultrafine protein that forms a unique three-dimensional structure and a crystal of the protein.

Protein is one of the most important substances that bear life, and it is known that formation of a unique three-dimensional structure is essential for expression of its function. Until now, the general view was that 30-50 amino acids are the minimum required for a protein to form a stable conformation, but recently only 10 amino acids are stable. A protein that forms a three-dimensional structure, signoline, was artificially designed and synthesized (Non-patent Document 1).
This very small protein named signoline is highly soluble and exists as a monomer in a buffer solution. From the results of structural analysis by two-dimensional ¹ H-NMR method, It was confirmed that the strands showed a certain hairpin structure. Furthermore, from the analysis of the one-dimensional NMR spectrum and circular dichroism (CD) spectrum, signoline was heat-denatured in the same manner as the natural protein, and the denaturation temperature was 39 ° C to 42 ° C. In addition, it was also confirmed that the sample was rapidly regenerated almost quantitatively, and the whole molecule changed cooperatively, indicating a two-state structural transition. In this way, signoline has a “stable intrinsic three-dimensional structure” that is essential for proteins, despite its molecular weight of about 1000, which is far from natural proteins. It has attracted attention as a change in the concept of conventional proteins because it possesses the “denaturation / regeneration ability”, and is the cover of the American scientific journal “Structure” in the August 2004 issue.
Although signoline itself does not have a “function” property, there are many natural proteins having a partial sequence consisting of a similar sequence of signoline (for example, retinoic acid receptor, hyaluronic acid lyase, TFIB transcription initiation factor, etc.) The partial sequence has a partial structure exhibiting a hairpin structure very similar to that of signoline (Non-patent Document 2). That is, it is considered that a segment similar to the signoline sequence always forms a constant hairpin structure independently without being affected by any sequence added before or after the segment.
Therefore, it can be said that the essential attribute of the signoline is this “hairpin structure” itself. In other words, since it can be used as a standard substance as a minimum protein and a recognition substance such as an antibody recognizing this characteristic hairpin structure can be obtained, a pharmaceutical utilizing purification by affinity chromatography and signal transmission of ligand / receptor It can be said that it is a substance that inherently has an effect as a lead compound in development. For this reason, we will develop the technology for signoline in the future, peptide drugs with excellent degradation resistance, protein standards used for calibration of analytical measuring instruments, etc., nanobio devices with excellent molecular recognition ability It is expected to spread to the medical, agricultural and industrial fields, such as application to
However, in order to use signoline as a standard substance, it is important to allow crystallization, but it is important to ensure the purity as a standard substance. However, the denaturation temperature of signoline is around 40 ° C., so that it can be crystallized. Therefore, it cannot be said that the thermal stability is high, and the structural stability is not sufficient. Therefore, it is required to further improve the structural stability without changing the characteristic “signature of hairpin” of signoline.
S. Honda, K. Yamasaki, Y. Sawada, and H. Morii "10-residue folded peptide designed by segment statistics" Structure 12 (8) 1507-1518 (2004). Chemistry and Industry, Vol. 58, No. 8, 940-943 (2005)

  An object of the present invention is to provide a very small protein that forms a unique three-dimensional structure (hairpin structure) similar to that of the known very small protein signoline and is superior in stability to signoline. It is also to provide a crystal of the protein to allow it to be purified with high purity.

The present inventors first considered the modification of these functional groups, considering that the unique structure is maintained by the strong hydrogen bonding between the functional groups of each amino acid constituting signoline. The modification that contributed to the above was not found. For example, to neutralize the C-terminal charge, the terminal carboxylic acid was amidated, but rather the structural stability decreased (comparison of CLN001 and CLN002 in Table 2).
Therefore, the present inventors decided to create a 10-amino acid signoline mutant, and paid attention to Gly at positions 1 and 10 corresponding to both ends of the hairpin structure in the 10 amino acid sequence. Substitution of amino acid residues at both positions with small amino acids, branched amino acids, charged amino acids, aromatic amino acids, and further amidation of each 10th amino acid, systematically synthesized, to improve its thermal stability Analysis (Tables 1 and 2).
As a result, it was surprisingly confirmed that all amino acids other than Gly and Ala have improved thermal stability compared to signoline, regardless of positive charge, negative charge, polarity, degree of branching, aromaticity, etc. It was. Specifically, the enthalpy change (ΔH _m ) was 27.5 kJ mol ⁻¹ , but all increased to 20.7 or higher up to 50.7 and denaturation temperature (Tm) was 314.9 K at 316.5 or higher and 343.2 at maximum. Rose to. When amidated, the effect was slightly reduced, but the thermal stability improvement effect for signoline was almost the same as that for the free carboxylic acid terminal.
Of the mutants having improved thermal stability, the Gly1Tyr / Gly10Tyr mutant of SEQ ID NO: 2 (Formula 2) was crystallized as a typical one, and the crystallization was also successful. This means that each of the mutants of the present invention guarantees high purity as a standard substance as well as structural stability.
In addition, the crystals obtained were of good quality, and when they were used to determine atomic coordinates by X-ray diffraction (Table 3), it was confirmed that the hairpin structure almost the same as that of signoline was maintained. . That is, the mutant of the present invention has a root mean square residual with the three-dimensional structure defined by the atomic coordinates described in Table 3 or the main chain coordinates described in Table 3, in which the “hairpin structure” is quantified. The difference (RMSD) can be specified by the property of “stereostructure having a thickness of 5 angstroms or less”.
Further, since the signoline mutant of the present invention has a structurally higher stability than signoline, the fusion protein having a partial sequence consisting of the amino acid sequence of the mutant (formula 1: SEQ ID NO: 1) is also included in the fusion protein. It is almost certain that the hairpin structural properties will remain stable.

The present invention has been completed with the above knowledge, and is specifically as follows.
[1] A protein comprising an amino acid sequence represented by the following formula 1, wherein the region corresponding to formula 1 is a three-dimensional structure defined by atomic coordinates described in Table 3, or a main chain described in Table 3. A protein having a three-dimensional structure with a root mean square residual (RMSD) within 5 angstroms.
XaaTyrAspProGluThrGlyThrTrpXbb (Formula 1)
(Wherein Xaa and Xbb represent any amino acid residue other than Gly and Ala, and the carboxyl group when Xbb is the C-terminus may be amidated.)
[2] The protein according to [1], comprising the amino acid sequence represented by Formula 1.
[3] The protein according to [1] above, which is a fusion protein of a protein consisting of the amino acid sequence represented by Formula 1 and an arbitrary protein.
[4] In Formula 1, Xaa is selected from the group of Thr, Val, Ile, Glu, Phe, Tyr and Trp, and Xbb is from the group of Thr, Val, Ile, Lys, Arg, Phe, Tyr and Trp. The protein according to any one of [1] to [3], wherein the protein is a selected amino acid residue, and the carboxyl group when Xbb is C-terminal may be amidated.
[5] In Formula 1, any one of the above [1] to [4], wherein Xaa and Xbb are both Tyr, and the carboxyl group when Xbb is C-terminal may be amidated. protein.
[6] A protein comprising an amino acid sequence represented by Formula 2 (SEQ ID NO: 2).
TyrTyrAspProGluThrGlyThrTrpTyr (Formula 2: SEQ ID NO: 2)
[7] A nucleic acid encoding the protein according to any one of [1] to [5], including a nucleic acid encoding the amino acid sequence represented by Formula 1.
[8] A nucleic acid encoding a protein consisting of the amino acid sequence represented by Formula 2 (SEQ ID NO: 2).
[9] A recombinant vector containing the nucleic acid according to [7] or [8].
[10] A transformant comprising the recombinant vector according to [9].
[11] A crystal of the protein according to [2] or [6], wherein the crystallographic objectivity is P2 ₁ 2 ₁ 2, the unit cell constant is a = 1.9 ± 0.5 nm, b = 3.4 ± Crystals with 0.5 nm and c = 1.2 ± 0.5 nm.

  According to the present invention, a very small protein that retains the characteristic “hairpin structure” similar to that of the known very small protein signoline, has a greatly improved thermal stability and can be easily crystallized compared to signoline, and a crystal thereof. I was able to provide it. This makes it possible to provide a reference material with higher accuracy, and is useful for searching for immunoreactive substances and information-transmitting substances based on its characteristic and stable hairpin structure, and is expected to be an excellent lead compound. Is also big.

Hereinafter, the present invention will be described in detail.
The present invention includes a very small protein having a specific “hairpin structure” consisting of the amino acid sequence represented by Formula 1 (SEQ ID NO: 1), and a fusion comprising Formula 1 as a partial sequence, wherein the partial sequence exhibits a “hairpin structure” It is an invention related to proteins.
Here, for a specific “hairpin structure”, the root mean square residual (RMSD) with respect to the three-dimensional structure defined by the atomic coordinates described in Table 3 or the main chain coordinates described in Table 3 is 5 It can be quantified as “a three-dimensional structure within angstrom”.
The ultrafine protein of the present invention is a bulky protein other than Ala, which is a small amino acid, in the signoline, a known ultrafine protein consisting of 10 amino acids, in which glycine (Gly), which is the 1st and 10th amino acids, is used. It can be said that it is a signoline mutant converted to a new amino acid, or a terminal amidated product thereof, and thus all related techniques such as a production method applicable to signoline, a purification method, and a production method of a fusion protein can be applied.
Therefore, embodiments of the present invention are shown below, but are not limited thereto.

1. Method for Producing the Protein of the Present Invention A signoline mutant can be produced by an organic chemical method such as a solid phase peptide synthesis method. It can also be produced as a recombinant protein using genetic engineering techniques. Protein production methods using such techniques are well known in the art and are briefly described below.
(1) Method for producing a signoline mutant by an organic chemical method When a protein is chemically produced by a solid-phase peptide synthesis method, a polycondensation reaction of an activated amino acid derivative is preferably performed using an automatic synthesizer. By repeating, a protected polypeptide having the amino acid sequence of the signoline mutant of the present invention is synthesized on a resin. At this time, by appropriately selecting the resin to be used, the C-terminus of the final product can be converted to a carboxyl group or amidated. Next, the protective polypeptide is cleaved from the resin and the side chain protecting group is cleaved simultaneously. This cleaving reaction is known to have an appropriate cocktail depending on the type of resin, protecting group, and amino acid composition (supervised by Shigeo Ohno, Yoshifumi Nishimura (1997) Protein Experiment Protocol 2-Structure Analysis, Hide Junsha). Thereafter, the crude protein is transferred from the organic solvent layer to the aqueous layer, and the target mutant protein is purified. As a purification method, reverse phase chromatography can be used (supervised by Shigeo Ohno and Yoshifumi Nishimura).
(1997) Protein Experiment Protocol 2-Structural Analysis, Shujunsha).

(2) Method for producing a signoline mutant by a genetic engineering technique When producing a recombinant protein, the signoline mutant of the present invention is a transformant into which a recombinant vector into which a nucleic acid encoding the amino acid sequence is inserted is introduced. Can be obtained by culturing and harvesting from the culture. Any host / vector system for transformation may be used as long as it is a system used in a normal genetic engineering technique, such as prokaryotic cells, yeast, insect cells, animal and plant cells, and plants.
The method of culturing the transformant is performed according to a usual method used for culturing a host. When the signoline mutant of the present invention is produced in cells or cells after culturing, the protein is obtained by crushing the cells or cells by sonication, repeated freeze-thawing, homogenizer treatment, etc. Collect. When the protein is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, by using general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination, Thus, the signoline mutant of the present invention can be isolated and purified.
The C-terminal amidation reaction when produced as a recombinant protein can be accomplished, for example, by treating the product isolated and purified with peptidylglycine monooxygenase (Kenzaku Mizuno, Toshiyuki Matsuo (1988) C-terminal amidating enzyme of active peptide, Biochemistry, 60, 551-557).
In addition, when a so-called cell-free synthesis system in which only factors (enzymes, nucleic acids, ATP, amino acids, etc.) involved in protein biosynthesis reactions are mixed is used, the signoline mutant of the present invention can be transformed from a vector without using living cells. Can be synthesized in vitro (Masato Okada, Kaori Miyazaki (2004) Protein Experiment Notes (top), Yodosha). Thereafter, using the same purification method as described above, the signoline mutant of the present invention can be isolated and purified from the mixed solution after the reaction.

(3) Fusion protein In the case of a fusion protein containing a region corresponding to the amino acid sequence represented by Formula 1 (SEQ ID NO: 1) in the present invention, the region has a “hairpin structure” similar to that of the signoline mutant. Therefore, purification, detection, etc. can be easily performed using the structural features.
The protein fused with the signoline mutant is preferably a protein that itself is useful, such as a physiologically active protein. In the case of a fusion protein with a membrane protein, a transformant expressed so as to display a plurality of “hairpin structures” by a signoline mutant on the membrane surface of the transformed cell can be obtained.
The fusion protein of the present invention can be chemically conjugated with any natural protein or synthetic or recombinant protein after the synoline mutant has been synthesized. Moreover, the nucleic acid which codes each protein can be connected, and it can obtain as a recombinant fusion protein using a genetic engineering method.
At that time, after purifying the recombinant fusion protein, the amino acid sequence between the protein fused with the signoline mutant is divided by, for example, limited hydrolysis using a protease, so that the signoline mutant and the fused protein are separated. Each can also be isolated and purified.

2. Method for Measuring Thermal Stability of Protein of the Present Invention The thermal stability of the signoline mutant of the present invention is evaluated using circular dichroism (CD) spectrum, fluorescence spectrum, infrared spectroscopy, differential scanning calorimetry, etc. can do. In particular, the CD spectrum is a spectroscopic analysis method that sharply reflects changes in the secondary structure of the protein, and thus provides an indication that the signoline mutant has a unique three-dimensional structure, and Changes in the three-dimensional structure with respect to temperature can be observed, and the structural stability can be evaluated thermodynamically and quantitatively.

3. The protein crystallization method of the present invention Crystallization utilizes the property that protein precipitates as a crystal under a specific condition by adding a precipitant to the target protein solution or reducing the amount of solvent by evaporation or the like. is doing. Crystallization requires highly purified protein. In addition, physical and chemical factors such as protein concentration, salt concentration, hydrogen ion concentration (pH), type and concentration of precipitating agent, type and concentration of other additives, and temperature are involved in the crystallization conditions. ing. In addition, there are many crystallization methods such as batch method, dialysis method, vapor diffusion method, etc. for adding precipitant and adjusting the amount of solvent (Blundell, TL & Johnson, LN (1976) Protein Crystallography, Academic Press , New York, 59-82). Thus, in order to obtain a protein crystal suitable for X-ray crystal analysis, it is necessary to obtain a highly pure protein and to optimize various conditions and methods in crystallization.
The signoline mutant used in the crystal of the present invention is a protein consisting of the amino acid sequence represented by SEQ ID NO: 2, and is referred to herein as CLN025. As the crystallization method, a batch method, a dialysis method, a vapor diffusion method, or the like can be used. Regarding physical and chemical factors such as protein concentration, salt concentration, pH, type and concentration of precipitating agent, type and concentration of other additives, and temperature, protein concentration is 1 to 20 mg / ml, preferably 5 mg / mL , 35-100 mM, preferably 72 mM citrate buffer (pH 3-6, preferably pH 5.0) as buffer, 15-40%, preferably 29% saturated ammonium sulfate as precipitant, temperature 0-30 ° C., Preferably, a crystal belonging to the space group P2 ₁ 2 ₁ 2 of CLN025 can be obtained by allowing to stand at 10 ° C. for 2 to 10 weeks. However, since it is a well-known fact that those skilled in the art can crystallize the same protein under different conditions, the present invention is not limited to the conditions described here, and is substantially the same as the present invention. Crystals of the signoline mutants that give the crystallographic constant of are within the scope of the present invention.

4). X-ray diffraction atomic coordinate determination method The most commonly used method for clarifying the three-dimensional structure of proteins is the X-ray crystal structure analysis method. That is, the target protein is crystallized, the monochromatic X-ray is applied to the crystal, and the three-dimensional structure of the protein is clarified based on the obtained X-ray diffraction image. Using the crystal of the signoline mutant obtained in the previous section, the three-dimensional structure is clarified by this X-ray crystal structure analysis method (translated by Noriaki Hirayama (2004) Introduction to X-ray Analysis for Brow Life Systems, Chemistry) be able to.
The crystal of the signoline mutant CLN025 belongs to the orthorhombic space group P2 ₁ 2 ₁ 2 and has a unit cell of 1.9 ± 0.5 nm in the a-axis direction, 3.4 ± 0.5 nm in the b-axis direction, c-axis In the direction of 1.2 ± 0.5 nm. By the X-ray crystal structure analysis technique using this crystal, the three-dimensional structure coordinates (values indicating the spatial positional relationship between each atom) of the signoline mutant CLN025 of the present invention can be obtained for the first time. The obtained structure coordinates are shown in accordance with the notation method of protein data bank (PDB) generally used by those skilled in the art (Table 3).

The present invention will be specifically described with reference to examples. However, the technical scope of the present invention is not limited to these examples.
(Example 1)
In this example, based on the amino acid sequence of a known very small protein, signoline, a mutant in which Gly1 and Gly10 of signoline were substituted with different amino acids was systematically analyzed.
Specifically, novel amino acids shown in Table 1 were designed, and each peptide was synthesized. The peptide was synthesized organically by a known Fmoc solid phase synthesis method. Specifically, synthesis was performed using a solid phase method automatic peptide synthesizer PSSM-8 system (Shimadzu Corporation) according to this synthesis manual. As the solid support, 2-chlorotrityl resin was used. The synthetic scale was 74 mg (50.0 μmol) of resin per reaction vessel, and Fmoc-amino acid was added 2.5 times the amount of resin. After completion of the synthesis, dimethylformamide and dichloromethane were added to the peptide resin in the reaction vessel and washed sequentially, and then the peptide resin was dried under reduced pressure for 1 hour. After drying, transfer to a centrifuge tube, and add a cleavage cocktail (trifluoroacetic acid / ethanedithiol / triisopropylsilane / water = 4.2 ml / 0.4 ml / 0.2 ml / 0.2 to 74 mg scale resin) Stir for hours. Then, the cooled diethyl ether was added, centrifugation was performed, the depositing deposit was collect | recovered, and it dried under reduced pressure after air-drying. A 5% aqueous acetic acid solution was added to this and dissolved, and insoluble matters were removed with a filter to obtain a crude peptide solution.

Table 1. Synoline mutants synthesized

(Example 2)
In this example, the crude peptide solution of the signoline mutant synthesized in Example 1 was purified by liquid chromatography, and its identification and purity were confirmed by mass spectrometry.
The crude peptide solution was reverse phase liquid chromatography (HPLC; eluent 0.1% trifluoroacetic acid aqueous solution (A solution) and 100% acetonitrile aqueous solution (B solution) equipped with C18 column (YMC-R & D-ODS). , Gradient A solution 100-0% / B solution 0-100%, flow rate 7.0 ml / min, analysis wavelength 220 nm). The gradient was set to change 5% in 30 min depending on the HPLC analysis. The peptide solution fractionated and purified by HPLC was concentrated under reduced pressure under centrifugation, dissolved in water, and then lyophilized to obtain a purified signoline mutant.
Mass spectrometry was performed with an electrospray ionization mass spectrometer QP8000α (Shimadzu Corporation) after diluting the purified sample with solution A and adjusting the concentration to 50 ug / 100 uL. As a result of comparing the molecular weight of the detected peak with the theoretical molecular weight (Table 1) calculated from the amino acid sequence of the synthesized sample, both samples matched within the measurement error, and the desired signoline mutant was synthesized. It was confirmed that Further, no significant peak other than the target protein was found in the mass spectrum.

(Example 3)
In this example, the three-dimensional structure and thermal stability of the signoline mutant were evaluated. Circular dichroism (CD) spectra are known to be spectroscopic analytical methods that sensitively reflect changes in protein secondary structure. Each signoline mutant is given at what temperature by giving a suggestion that the protein has a unique three-dimensional structure and observing the molar ellipticity corresponding to the intensity of the CD spectrum while changing the temperature of the sample. Can be clarified.
Each of the isolated and purified signoline mutants was prepared in an aqueous solution (20 mM potassium phosphate buffer, pH 7.0) containing a concentration of about 1.0 mM. The concentration of the signoline mutant was determined using the molar extinction coefficient in Table 1-2. This sample solution was poured into a cylindrical cell (cell length: 0.1 cm), and the CD805 was moved from 260 nm to 195 nm at a temperature of 20 ° C. using a J805 circular dichroism spectrophotometer (JASCO). A spectrum was obtained. In all the mutants synthesized, a positive peak at 229 nm characteristic of signoline was detected (FIG. 1). From this, it is strongly inferred that all mutants form a three-dimensional structure equivalent to signoline. The same sample was then heated to 100 ° C. and further cooled to 100 ° C. to 20 ° C. to obtain a circular dichroism spectrum from 260 nm to 195 nm. The molar ellipticity of the spectrum re-cooled after heating recovered 98% or more, confirming the reversibility of the three-dimensional structure of the signoline mutant. In addition, the measurement wavelength was fixed at 229 nm, the temperature was increased from 0 ° C. to 100 ° C. at a rate of 1 ° C./min, and the change in molar ellipticity with time was measured. The resulting theoretical equation of the two-state phase transition model for thermal melting curve (Fumio Arisaka (2004) Protein Science Primer for Bioscience, Mohanabo) and analyzed using denaturing at denaturation temperature T _m, and T _m The enthalpy change ΔH _{m of} was determined.
As a result, in the measured signoline mutant, when substituted with amino acids other than small amino acids such as Gly and Ala, the thermal stability is higher than that of the original signoline, regardless of whether the terminal carboxylic acid is amidated or not. It has become clear that it has improved (Table 2). For example, CLN025 exhibits a denaturation temperature of 28 ° C. or higher compared to signoline.

Table 2 Thermostability of signoline mutants

(Example 4)
In this example, a single crystal of CLN025 among the synthesized signoline mutants was prepared. CLN025 did not show the highest value in either enthalpy change or denaturation temperature, and was selected as a typical example.
The obtained protein sample was crystallized using the hanging drop method shown below. In order to obtain crystals belonging to the space group P21212, this protein sample was dissolved in 50 mM sodium citrate buffer pH 5.0 to a concentration of 10 mg / ml, and 1 μl of this protein sample solution was equivalent to the crystallization agent solution. (Mixed 0.1M sodium citrate buffer pH 5.0 and saturated ammonium sulfate solution in a ratio of 5: 2) was dropped and mixed on a cover glass (Hampton) using a pipetman to obtain a crystallization solution. The above-mentioned crystallization agent solution was poured into a 24-well plate manufactured by Hampton, covered with a cover glass to which the crystallization solution was dropped, and sealed with high vacuum grease. The plate was stored in an incubator kept at 10 ° C. After about 3 weeks, crystals having a size of about 40 μm × 40 μm × 50 μm were obtained in the crystallization solution.

(Example 5)
In this example, the three-dimensional structure of CLN025 was determined by X-ray diffraction analysis.
The single crystal obtained in Example 4 was sealed in a glass capillary for crystal analysis (inner diameter 0.5 mm, wall thickness 10 μm) together with a small amount of mother liquor and subjected to an X-ray diffraction experiment. As the X-ray, the copper Kα ray obtained from the rotating anti-cathode X-ray generator M06-XHF (Mac Science) was used, and the diffraction data measuring device was SMART6000 (Bruker AXS), with a resolution of 1.11 mm. The diffraction data up to were collected. Using the program SMART (Bruker AXS Co., Ltd.), the diffraction pattern was indexed and the integrated intensity was measured and digitized to obtain 20393 intensity data. At this stage, the crystal parameters of the single crystal used were determined. That is, the crystal space group was orthorhombic P2 ₁ 2 ₁ 2, and the lattice constants were a = 19.246 (4) Å, b = 33.597 (7) Å, and c = 1.11.551 (2) Å. Furthermore, they were merged and scaled using the program SAINT + (Bruker AXS), and 3094 unique intensity data were obtained. _Their R _merge value was 5.7%.

Table 3 Three-dimensional structural coordinates obtained from crystals of the signoline mutant CLN025
In Table 3, the first line is a description of crystallographic constants. From the left, the unit cell size (a-axis, b-axis, and c-axis in Å units), and the angles formed by each axis (α, β, γ ), Space group, and Z value (number of molecules per unit cell). From the second line onward, the three-dimensional coordinates of each atom are described by lines other than the lines starting with TER and END. ATOM in the first column represents atomic coordinates of the polypeptide portion, and HETATM represents atomic coordinates of a portion other than the polypeptide. The second column of the ATOM or HETATM row is the identification number of the atom, the third column is the name of the atom, the fourth column is the residue name to which the atom belongs, and the fifth column is the residue number to which the atom belongs. The 6th, 7th, and 8th columns are the coordinates of the atom (in units of x, y, and z), the 9th column is the occupancy of the atom, the 10th column is the temperature factor of the atom, The eleventh column shows atomic symbols. The TER line indicates the break of the polypeptide chain, and the END line indicates the final line of the three-dimensional structure coordinates.

  Very small proteins that form a unique three-dimensional structure defined by atomic coordinates have many industrial applications due to their remarkable structural properties and thermal stability. For example, it can be expected to be used as a standard substance for proteins as described above or as a lead compound for molecular target drugs, and as a calibration substance for analytical instruments for measuring the size of molecules, membranes and fibers having nano-sized pores As a test substance for pores of structures, etc., it can be expected to be used as a molecular spacer capable of arranging other molecules with high accuracy. In particular, this ultra-fine protein crystal makes it possible to provide the protein with high purity and to store it in a stable state for a long time. In addition, the atomic coordinates found thereby can be used for molecular simulation, etc. It can be expected to be used as a target for pharmaceutical design based on performance evaluation and structure of large-scale high-precision computers.

CD spectrum of signoline mutant

Claims (11)

A protein comprising an amino acid sequence represented by the following formula 1, wherein the region corresponding to the formula 1 is a three-dimensional structure defined by atomic coordinates described in Table 3, or a main chain coordinate described in Table 3. A protein having a three-dimensional structure with a root mean square residual (RMSD) of 5 angstroms or less.
XaaTyrAspProGluThrGlyThrTrpXbb (Formula 1)
(Wherein Xaa and Xbb represent any amino acid residue other than Gly and Ala, and the carboxyl group when Xbb is the C-terminus may be amidated.)   The protein according to claim 1, comprising the amino acid sequence represented by Formula 1.   The protein according to claim 1, which is a fusion protein of a protein consisting of the amino acid sequence represented by Formula 1 and an arbitrary protein.   In Formula 1, Xaa is selected from the group of Thr, Val, Ile, Glu, Phe, Tyr, and Trp, and Xbb is selected from the group of Thr, Val, Ile, Lys, Arg, Phe, Tyr, and Trp. The protein according to any one of claims 1 to 3, which is an amino acid residue, and the carboxyl group when Xbb is C-terminal may be amidated.   The protein according to any one of claims 1 to 4, wherein in formula 1, Xaa and Xbb are both Tyr, and the carboxyl group when Xbb is C-terminal may be amidated. A protein comprising the amino acid sequence represented by Formula 2 (SEQ ID NO: 2).
TyrTyrAspProGluThrGlyThrTrpTyr (Formula 2: SEQ ID NO: 2)   A nucleic acid encoding a protein according to any one of claims 1 to 5, comprising a nucleic acid encoding the amino acid sequence represented by Formula 1.   A nucleic acid encoding a protein consisting of the amino acid sequence represented by Formula 2 (SEQ ID NO: 2).   A recombinant vector containing the nucleic acid according to claim 7 or 8.   A transformant comprising the recombinant vector according to claim 9. 7. A protein crystal according to claim 2 or 6, wherein the crystallographic objectivity is P2 ₁ 2 ₁ 2, unit cell constants are a = 1.9 ± 0.5 nm, b = 3.4 ± 0.5 nm, and c = A crystal that is 1.2 ± 0.5 nm.