JP2518917B2 - Protein structure analysis method - Google Patents
Protein structure analysis methodInfo
- Publication number
- JP2518917B2 JP2518917B2 JP1029261A JP2926189A JP2518917B2 JP 2518917 B2 JP2518917 B2 JP 2518917B2 JP 1029261 A JP1029261 A JP 1029261A JP 2926189 A JP2926189 A JP 2926189A JP 2518917 B2 JP2518917 B2 JP 2518917B2
- Authority
- JP
- Japan
- Prior art keywords
- nmr
- protein
- spectrum
- dimensional
- peaks
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Description
【発明の詳細な説明】 ［産業上の利用分野］ 本発明は、核磁気共鳴（NMR）スペクトルに基づいて
蛋白質の構造解析を行う際に用いられる構造解析方法に
関するものである。TECHNICAL FIELD The present invention relates to a structural analysis method used for structural analysis of a protein based on a nuclear magnetic resonance (NMR) spectrum.
［従来技術］ NMRを利用した蛋白質の構造解析方法が、スイスのＷ
thrich等により開発されている。この方法は、COSYタ
イプ及びNOEタイプの２次元NMRを使用すると共に、蛋白
質の化学構造の特殊性を考慮し、３つのステップからな
る一般的かつ体系的な構造解析方法である。[Prior Art] A protein structure analysis method using NMR is described in W of Switzerland.
Developed by thrich and others. This method is a general and systematic structural analysis method that uses two-dimensional NMR of COSY type and NOE type and takes into account the peculiarities of the chemical structure of protein, and consists of three steps.
例えば、第２図（ａ）に示すような1H−NMRスペクト
ルが、同図（ｂ）に示すような蛋白質について得られた
とする。このスペクトル中の各ピークが具体的に第２図
（ｂ）のペプチドのどの水素核に由来しているのかを確
定するのが帰属問題である。For example, assume that the 1 H-NMR spectrum as shown in FIG. 2 (a) was obtained for the protein as shown in FIG. 2 (b). The assignment problem is to determine which hydrogen nucleus of the peptide in FIG. 2 (b) specifically originates each peak in this spectrum.
２次元NMRを利用した上記従来法では、それを次の３
つのステップで行う。In the above conventional method using two-dimensional NMR,
Do it in one step.
（１）COSY2次元NMRスペクトルもしくはその類似スペク
トルで各アミノ酸に属するスピン系を同定する。その
際、第３図のような２次元パターンが利用される。(1) The spin system belonging to each amino acid is identified by the COZY two-dimensional NMR spectrum or its similar spectrum. At that time, a two-dimensional pattern as shown in FIG. 3 is used.
（２）隣接するアミノ酸のつながりの情報を得るため
に、NOESY2次元NMRスペクトルを測定する。その際、第
４図に示すアミド水素（NH）の隣接基とのNOEが利用さ
れる。(2) NOESY two-dimensional NMR spectrum is measured in order to obtain information on the connection between adjacent amino acids. In that case, NOE with the adjacent group of amido hydrogen (NH) shown in FIG. 4 is utilized.
（３）（１），（２）で得たNMR的アミノ酸のつながり
の情報を既知の一次構造のアミノ酸配列（第２図
（ｂ））と比較し帰属を決定する。(3) The assignment of NMR amino acid linkage information obtained in (1) and (2) is compared with the amino acid sequence of a known primary structure (FIG. 2 (b)) to determine the assignment.
［発明が解決しようとする課題］ しかし、水素核（1H）NMRスペクトルを用いたこの方
法は、分子量１万以下の蛋白質についてはかなり成功し
ているが、分子量がそれ以上のものでは、アミド水素
（NH）の1H−NMRスペクトルにおける化学シフトの重な
りのため、例え２次元NMR上でパターンとして分離でき
ても帰属に曖昧さが残ってしまう。[Problems to be Solved by the Invention] However, this method using the hydrogen nucleus ( 1 H) NMR spectrum has been quite successful for proteins with a molecular weight of 10,000 or less, but for proteins with a molecular weight higher than that, amide Due to the overlap of chemical shifts in the 1 H-NMR spectrum of hydrogen (NH), the ambiguity remains in the assignment even if it can be separated as a pattern on two-dimensional NMR.
例として、分子量6000のBPTI（bovine pancreatic tr
ipsin inhibitor）と分子量11700のチオレドキシン（E.
Coli TRX）について、アミド水素のピークの重なりのヒ
ストグラムを第５図に示した。図から分るように、BPTI
ではよく分離したアミド水素（縮退度１）が最も多く、
縮退度3,4は急速になくなっている。As an example, BPTI (bovine pancreatic tr
ipsin inhibitor) and thioredoxin (E.
FIG. 5 shows a histogram of overlapping peaks of amide hydrogen for Coli TRX). As you can see from the figure, BPTI
Well separated most often amido hydrogen (degeneracy 1),
Degeneration degree 3, 4 is disappearing rapidly.
一方、チオレドキシンは、分子量に比例したピークの
広がり0.02ppmをとった場合、縮退度２が最も多く、し
かも、縮退度６に及ぶ激しいピークの重なりを持つ。On the other hand, in the case of thioredoxin, when the spread of the peak in proportion to the molecular weight is 0.02 ppm, the degree of degeneration 2 is the largest, and further, the degree of degeneracy 6 is severe.
このように、従来法では、蛋白質の分子量が大きくな
ると、スペクトル中のピークの重なりが激しくなって構
造解析が事実上不可能になってしまう。As described above, according to the conventional method, when the molecular weight of the protein is increased, the peaks in the spectrum are heavily overlapped with each other, which makes structural analysis practically impossible.
本発明は、上述した点に鑑みてなされたものであり、
大きな分子量の蛋白質であっても解析が可能な構造解析
方を提供することを目的としている。The present invention has been made in view of the above points,
It is an object of the present invention to provide a structural analysis method capable of analyzing even a protein having a large molecular weight.
［課題を解決するための手段］ この目的を達成するため、本発明の蛋白質の構造解析
方法は、2Hラベル及び15Nラベルされた蛋白質であって2
Hのラベル化率LH（％）が０＜LH＜100である蛋白質を含
む被検試料について15N−1H異核種３次元NMR測定を行
い、得られたデータに基づいて構造解析を行うことを特
徴としている。[Means for Solving the Problems] To achieve this object, the structural analysis method of a protein of the present invention is a 2 H label and 15 N labeled protein 2
Perform 15 N- 1 H heteronuclear three-dimensional NMR measurement on a test sample containing a protein with a labeling rate LH (%) of 0 <LH <100, and perform structural analysis based on the obtained data. Is characterized by.
［作用］ 以下、本発明を詳説する。[Operation] Hereinafter, the present invention will be described in detail.
上述のような激しいピークの重なりを減らすには、
ピークを狭小化し、本来の分離を向上させるとともに、
1H−NMRに新しい次元即ち15N−NMRを導入し、両者の
２次元相関スペクトルを測定し、その新次元軸上で水素
核のピークを分離すれば良い。To reduce the overlap of intense peaks as described above,
While narrowing the peak and improving the original separation,
It suffices to introduce a new dimension into 1 H-NMR, that is, 15 N-NMR, measure the two-dimensional correlation spectrum of both, and separate the hydrogen nucleus peaks on the new dimension axis.
上記のピークの狭小化は、重水素核（2H）の一様部
分ラベル法で解決できることがLeMaster等により示され
た（Biochemistry 24,7263（1985））。その原理は重水
素核の導入により、スピン系をAX化し、ピークの微細構
造を簡単化することにある。第６図にその例を示す。2H
ラベルしていないもののスペクトルＡに比べ、2Hラベル
したもののスペクトルＢは、各ピークが狭小化されると
共に、微細構造が簡易化されていることが分かる。尚、
この場合、試料中の水素核を75％の割合で重水素に置換
している。It was shown by LeMaster et al. That the above-mentioned narrowing of the peak can be solved by the uniform partial labeling method of the deuterium nucleus ( 2 H) (Biochemistry 24,7263 (1985)). The principle is to introduce a deuterium nucleus to make the spin system AX and simplify the fine structure of the peaks. An example is shown in FIG. 2 H
It can be seen that the spectrum B of the 2 H-labeled product has each peak narrowed and the fine structure is simplified as compared with the spectrum A of the unlabeled product. still,
In this case, 75% of the hydrogen nuclei in the sample are replaced with deuterium.
上記の新次元の導入によるピークの分離は、15N−1
HCOSY２次元NMR（15NHMQC）により可能となる。そのス
ペクトルの例を第７図に示した。この図は、横軸が1H−
NMR、縦軸が15N−NMRで、分子量16000の蛋白質ペプチド
アミドNHの２次元相関スペクトルである。この図から、
もし、15N核による縦方向の分離を行わなければ、±0.0
2ppmの広がりでは〜10個、±0.01ppmを取っても〜５個
の化学シフトの重なりがあることが理解される。The separation of peaks due to the introduction of the above new dimension is 15 N- 1
It becomes possible by HCOSY two-dimensional NMR ( 15 NHMQC). An example of the spectrum is shown in FIG. In this figure, the horizontal axis is 1 H-
NMR, vertical axis is 15 N-NMR, and is a two-dimensional correlation spectrum of a protein peptide amide NH having a molecular weight of 16000. From this figure,
If vertical separation by 15 N nucleus is not performed, ± 0.0
It is understood that there is an overlap of ~ 10 chemical shifts with a spread of 2 ppm, and ~ 5 chemical shifts even if ± 0.01 ppm is taken.
従って、1H−NMRにおけるピークの分離を基礎とした
従来の帰属方法は、この蛋白質分子には適用不可能とい
うことになる。Therefore, the conventional attribution method based on the separation of peaks in 1 H-NMR cannot be applied to this protein molecule.
以上の考察から、本発明者は、との方法を取り入
れた３次元NMRにより分子量が大きくても帰属を決定で
きることを見出した。From the above consideration, the present inventor has found that the attribution can be determined even by a large molecular weight by three-dimensional NMR incorporating the above method.
［実施例］ 第１図は、本発明の一例を示す流れ図である。第１図
に示すように、本発明の構造解析方法においては、 （０）先ず、2Hラベル及び15Nラベルされた蛋白質を含
む被検試料が用意される。Embodiments FIG. 1 is a flow chart showing an example of the present invention. As shown in FIG. 1, in the structure analysis method of the present invention, (0) first, a test sample containing 2 H-labeled and 15 N-labeled proteins is prepared.
（１）次に、15N−1H３次元NMR例えばHMQC−COSYが実施
され、分離されたピークに対しアミノ酸スピン系の同定
が行われる。第８図に示すような２次元NMRを用いた従
来法では重なり合って分離が困難なピークも、第８図
（ｂ）に示すような３次元NMRを用いた本発明では、新
たに導入された15Nの軸方向に水素核のピークが分離さ
れているため、曖昧さのない同定が可能である。(1) Next, 15 N- 1 H three-dimensional NMR, such as HMQC-COSY, is performed to identify the amino acid spin system for the separated peaks. Peaks that are difficult to separate due to the conventional method using two-dimensional NMR as shown in FIG. 8 have been newly introduced in the present invention using three-dimensional NMR as shown in FIG. 8 (b). Since the hydrogen nucleus peaks are separated in the 15 N axial direction, unambiguous identification is possible.
（２）次に、15N−1H３次元NMR例えばNOESY−HMQCが実
施され、得られたスペクトルに基づいて隣接するアミノ
酸のつながりの情報を得る。(2) Next, 15 N- 1 H three-dimensional NMR, such as NOESY-HMQC, is performed to obtain information on the connection of adjacent amino acids based on the obtained spectrum.
（３）（１），（２）で得たNMR的アミノ酸のつながり
の情報を既知の一次構造のアミノ酸配列と比較し、帰属
を決定する。(3) The assignment of NMR amino acid linkages obtained in (1) and (2) is compared with the amino acid sequence of known primary structure to determine the assignment.
尚、2Hのラベル化率LH（％）は、０＜LH＜100で、ラ
ンダムな部分又は全ラベル、15Nのラベル化率LN（％）
は、80≦LN≦100で、ランダムな部分又は全ラベルが好
ましい。In addition, the labeling rate LH (%) of 2 H is 0 <LH <100, random part or all labels, and the labeling rate LN (%) of 15 N
Is 80 ≦ LN ≦ 100, and a random part or whole label is preferable.
また、上記（１），（２）の測定は、試料を軽水と混
合して実施される。特に、100％2Hラベルした場合、そ
のままでは15N−1H３次元NMRの1Hの信号が出てこない
が、軽水と混合すると、15Nと結合している2Hだけが1H
に置換されるため、アミド水素のピークだけが出現し、
ピークが一層簡単化され、帰属の決定が容易になる。The measurements of (1) and (2) above are carried out by mixing the sample with light water. In particular, when labeled with 100% 2 H, the 1 H signal of 15 N- 1 H three-dimensional NMR does not come out as it is, but when mixed with light water, only 2 H bound to 15 N is 1 H.
Therefore, only the peak of amide hydrogen appears,
The peaks are further simplified, making it easier to determine attribution.
前記（１）における15N−1H３次元NMR測定は、HMQC−
COSYに限らず、これと同等の情報が得られれば、例えば
COSY−HMQC,HMQC−RELAY,RELAY−HMQC,HMQC−HOHAHA,HO
HAHA−HMQC等の各種測定法が使用できる。The 15 N- 1 H three-dimensional NMR measurement in (1) above is HMQC-
Not only COSY, but if equivalent information can be obtained, for example
COSY-HMQC, HMQC-RELAY, RELAY-HMQC, HMQC-HOHAHA, HO
Various measurement methods such as HAHA-HMQC can be used.
また、上記（２）における15N−1H３次元NMR測定は、
NOESY−HMQCに限らず、これと同等の情報が得られれ
ば、例えばHMQC−NOESY等の測定法も使用できる。In addition, the 15 N- 1 H three-dimensional NMR measurement in (2) above
Not limited to NOESY-HMQC, measurement methods such as HMQC-NOESY can also be used if equivalent information can be obtained.
［効果］ 以上詳述した如く、本発明によれば、分子量の大きな
蛋白質試料であっても、正確に構造解析を行うことの出
来る方法が実現される。[Effects] As described in detail above, according to the present invention, a method capable of accurately performing structural analysis even with a protein sample having a large molecular weight is realized.
第１図は本発明の一実施例を示す流れ図、第２図は蛋白
質の一次構造及びその1H−NMRスペクトルを示す図、第
３図は２次元パターンを示す図、第４図はアミド水素
（NH）の隣接基とのNOEを説明する図、第５図はアミド
水素のピークの重なりのヒストグラムを示す図、第６図
は2Hラベルの有無によるスペクトルの違いを示す図、第
７図は15N−1HCOSY２次元NMR（15NHMQC）スペクトルの
例を示す図、第８図は２次元NMRと３次元NMRを比較した
図である。FIG. 1 is a flow chart showing an embodiment of the present invention, FIG. 2 is a diagram showing a primary structure of a protein and its 1 H-NMR spectrum, FIG. 3 is a diagram showing a two-dimensional pattern, and FIG. 4 is an amidohydrogen. FIG. 5 is a diagram for explaining NOE with an adjacent group of (NH), FIG. 5 is a diagram showing a histogram of overlapping of amido hydrogen peaks, and FIG. 6 is a diagram showing differences in spectra depending on the presence or absence of a 2 H label. Is a diagram showing an example of 15 N- 1 HCOSY two-dimensional NMR ( 15 NHMQC) spectrum, and FIG. 8 is a diagram comparing two-dimensional NMR and three-dimensional NMR.
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 特開 昭63−175754（ＪＰ，Ａ) Ｂｉｏｃｈｅｍｉｓｔｒｙ1985，24, ｐ．7263−7268 ─────────────────────────────────────────────────── ─── Continuation of front page (56) References JP-A-63-175754 (JP, A) Biochemistry 1985, 24, p. 7263-7268
Claims (1)
って2Hのラベル化率LH（％）が０＜LH＜100である蛋白
質を含む被検試料について15N−1H異核種３次元NMR測定
を行い、得られたデータに基づいて構造解析を行うこと
を特徴とする蛋白質の構造解析方法。1. A 2 H label and 15 N labeled a protein 2 H label rate LH (%) is 0 <LH <For a test sample containing protein is 100 15 N-1 H different species A method for structural analysis of protein, which comprises performing three-dimensional NMR measurement and performing structural analysis based on the obtained data.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1029261A JP2518917B2 (en) | 1989-02-07 | 1989-02-07 | Protein structure analysis method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1029261A JP2518917B2 (en) | 1989-02-07 | 1989-02-07 | Protein structure analysis method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02208579A JPH02208579A (en) | 1990-08-20 |
| JP2518917B2 true JP2518917B2 (en) | 1996-07-31 |
Family
ID=12271339
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1029261A Expired - Fee Related JP2518917B2 (en) | 1989-02-07 | 1989-02-07 | Protein structure analysis method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2518917B2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6111066A (en) | 1997-09-02 | 2000-08-29 | Martek Biosciences Corporation | Peptidic molecules which have been isotopically substituted with 13 C, 15 N and 2 H in the backbone but not in the sidechains |
| JP4340538B2 (en) * | 2001-12-19 | 2009-10-07 | 独立行政法人科学技術振興機構 | Stable isotope-labeled amino acids and their incorporation into target proteins, protein NMR structure analysis methods, and regioselective stable isotope-labeled fumaric and tartaric acid production methods |
| US20080293150A1 (en) * | 2004-02-02 | 2008-11-27 | Toshiyuki Kohno | Nmr Signal Assignment Method |
| JP6111387B2 (en) * | 2012-04-13 | 2017-04-12 | 日本電子株式会社 | NMR measurement method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6224688U (en) * | 1985-07-26 | 1987-02-14 |
-
1989
- 1989-02-07 JP JP1029261A patent/JP2518917B2/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| Ｂｉｏｃｈｅｍｉｓｔｒｙ１９８５，２４，ｐ．７２６３−７２６８ |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH02208579A (en) | 1990-08-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Thevenon-Emeric et al. | Determination of amide hydrogen exchange rates in peptides by mass spectrometry | |
| Fesik et al. | Heteronuclear three-dimensional NMR spectroscopy of isotopically labelled biological macromolecules | |
| Vuister et al. | 2D and 3D NMR study of phenylalanine residues in proteins by reverse isotopic labeling | |
| Meyer et al. | Screening mixtures for biological activity by NMR | |
| Zuiderweg et al. | Three-dimensional 13C-resolved proton NOE spectroscopy of uniformly 13C-labeled proteins for the NMR assignment and structure determination of larger molecules | |
| Wagner | NMR investigations of protein structure | |
| Montelione et al. | An efficient triple resonance experiment using carbon-13 isotropic mixing for determining sequence-specific resonance assignments of isotopically-enriched proteins | |
| Neuhaus et al. | Systematic application of high‐resolution, phase‐sensitive two‐dimensional 1H‐NMR techniques for the identification of the amino‐acid‐proton spin systems in proteins: Rabbit metallothionein‐2 | |
| Dumy et al. | Solution structure of regioselectively addressable functionalized templates: An NMR and restrained molecular dynamics investigation | |
| Zhang et al. | Rapid amide proton exchange rates in peptides and proteins measured by solvent quenching and two‐dimensional NMR | |
| Sklenáǩ et al. | Two-dimensional heteronuclear chemical-shift correlation in proteins at natural abundance 15N and 13C levels | |
| US20090115414A1 (en) | Phase-sensitively detected reduced dimensionality nuclear magnetic resonance spectroscopy for rapid chemical shift assignment and secondary structure determination of proteins | |
| Mitsuhiko et al. | Heteronuclear 3D NMR and isotopic labeling of calmodulin: towards the complete assignment of the 1h NMR spectrum | |
| Driscoll et al. | Determination of the secondary structure and molecular topology of interleukin-1. beta. by use of two-and three-dimensional heteronuclear nitrogen-15-proton NMR spectroscopy | |
| Madsen et al. | Improved pulse sequences for measuring coupling constants in 13 C, 15 N-labeled proteins | |
| Deng et al. | Rate and equilibrium constants for protein unfolding and refolding determined by hydrogen exchange-mass spectrometry | |
| Wagner et al. | Toward the complete assignment of the carbon nuclear magnetic resonance spectrum of the basic pancreatic trypsin inhibitor | |
| JP2518917B2 (en) | Protein structure analysis method | |
| Wemmer et al. | [26] Use of nuclear magnetic resonance in probing ligand-macromolecule interactions | |
| US7141432B2 (en) | Method of using reduced dimensionality nuclear magnetic resonance spectroscopy for rapid chemical shift assignment and secondary structure determination of proteins | |
| Bradbury et al. | High resolution nuclear magnetic resonance studies of biopolymers | |
| Chatterjee et al. | A novel protocol based on HN (C) N for rapid resonance assignment in (15N, 13C) labeled proteins: implications to structural genomics | |
| Fesik et al. | Heteronuclear three-dimensional NMR spectroscopy applied to CMP-KDO synthetase (27.5 kD) | |
| Forrest et al. | Characterization of cross-linking of cell walls of Bacillus subtilis by a combination of magic-angle spinning NMR and gas chromatography-mass spectrometry of both intact and hydrolyzed 13C-and 15N-labeled cell-wall peptidoglycan. | |
| Zhang et al. | Selectively 2H‐labeledGlu/Asp: application to pKa measurements in Aβ amyloid peptides |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| LAPS | Cancellation because of no payment of annual fees |