Stable isotope-labeled aliphatic amino acid and NMR structural analysis of protein using same

Abstract

Provided is a stable isotope-labeled aliphatic amino acid enabling the assignment of the signal of an amino acid residue side chain by increasing to the maximum the observation sensitivity to an NMR signal of the same amino acid residue side chain, and allowing NOE (nuclear Overhauser effect) between protons in the amino acid residue to be observed. The stable isotope-labeled aliphatic amino acid is for constituting a protein and satisfies all of the following conditions (1) to (3): (1) two or more carbon atoms are labeled with .sup.13C; (2) of two or more carbon atoms labeled with .sup.13C, a carbon atom other than a carbon atom of a methyl group, which is capable of bonding to a hydrogen atom, has one .sup.1H directly bonded thereto, while the carbon atom of the methyl group has at least one .sup.1H directly bonded thereto; and (3) other carbon atoms adjacent to all the .sup.13C are all .sup.12C.

Claims

1. A stable isotope-labeled amino acid for constituting a protein, satisfying all of the following conditions (1) to (3): (1) two or more carbon atoms are labeled with .sup.13C; (2) the two or more carbon atoms labeled with .sup.13C include a carbon atom of a methyl group, a carbon atom other than the carbon atom of the methyl group, or a combination thereof, wherein the carbon atom other than the carbon atom of the methyl group has one .sup.1H directly bonded thereto, and the carbon atom of the methyl group has at least one .sup.1H directly bonded thereto; and (3) carbon atoms adjacent to all the .sup.13C are all .sup.12C, wherein the amino acid is glutamic acid (Glu), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), proline (Pro), glutamine (Gln), arginine (Arg), threonine (Thr), or valine (Val); and when the amino acid is leucine (Leu), a carbon atom of the methylene group present at the side chain of the amino acid is labeled with .sup.13C, and one of two hydrogen atoms directly bonded to the .sup.13C is stereo-selectively labeled with .sup.2H.

2. The stable isotope-labeled amino acid according to claim 1, wherein (4) the number of carbon atoms located between the .sup.13C having .sup.1H directly bonded thereto and another carbon atom having .sup.1H directly bonded thereto is three or less.

3. The stable isotope-labeled amino acid according to claim 2, wherein (5) the other carbon atom having .sup.1H directly bonded thereto is labeled with .sup.13C.

4. The stable isotope-labeled amino acid according to claim 1, wherein (6) a carbon atom of at least one methylene chain among methylene chains present at side chains of the amino acid is labeled with .sup.13C, and one of two hydrogen atoms directly bonded to the .sup.13C is stereo-selectively labeled with .sup.2H.

5. The stable isotope-labeled amino acid according to claim 1, wherein (7) one of two hydrogen atoms directly bonded to a .sup.12C carbon atom of a methylene chain present at the side chain of the amino acid is labeled with .sup.2H, or both of the two hydrogen atoms are .sup.1H or .sup.2H.

6. The stable isotope-labeled amino acid according to claim 1, wherein (8) in a case where pro-chiral gem-methyl groups are present, one of the gem-methyl groups is labeled with .sup.12C.sup.2H.sub.3 or .sup.13C.sup.2H.sub.3.

7. A stable isotope-labeled amino acid, which is represented by one of the following ten structural formulas: ##STR00010## wherein R represents .sup.13C.sup.1H.sub.3, .sup.13C.sup.1H.sub.2D, or .sup.13C.sup.1HD.sub.2, H has the same meaning as .sup.1H, D has the same meaning as .sup.2H, C has the same meaning as .sup.12C, * represents a stereogenic center, the amino acids are any one of enantiomers with respect to the stereogenic center, n represents any one of 0 to 2, and in the case where n is 1, the amino acid may be a racemate or any one of enantiomers with respect to a carbon atom to which the corresponding deuterium is bonded.

8. A stable isotope-labeled amino acid, which is represented by one of the following ten structural formulas: ##STR00011## wherein R represents .sup.13C.sup.1H.sub.3, .sup.13C.sup.1H.sub.2D, or .sup.13C.sup.1HD.sub.2, R.sub.2 represents .sup.1H or .sup.2H, R.sub.3 represents .sup.12C.sup.1H.sub.3, .sup.12C.sup.1H.sub.2D, or .sup.12C.sup.1HD.sub.2, H has the same meaning as .sup.1H, D has the same meaning as .sup.2H, C has the same meaning as .sup.12C, * represents a stereogenic center, the amino acids are any one of enantiomers with respect to the stereogenic center, n represents any one of 0 to 2, and in the case where n is 1, the amino acid may be a racemate or any one of enantiomers with respect to a carbon atom to which the corresponding deuterium is bonded.

9. A composition comprising at least one of the stable isotope-labeled amino acids according to claim 1.

10. A composition comprising at least one of the stable isotope-labeled amino acids according to claim 7, which further comprises at least one of stable isotope-labeled amino acids represented by one of the following nine structural formulas: ##STR00012## wherein R represents .sup.13C.sup.1H.sub.3, .sup.13C.sup.1H.sub.2D, or .sup.13C.sup.1HD.sub.2, H has the same meaning as .sup.1H, D has the same meaning as .sup.2H, C has the same meaning as .sup.12C, * represents a stereogenic center, and the amino acids are any one of enantiomers with respect to the stereogenic center.

11. A method for incorporating a stable isotope-labeled amino acid into a protein, comprising the step of synthesizing a protein using cultured cells, microorganisms, or a cell-free protein synthesis system in presence of the stable isotope-labeled amino acid according to claim 1.

12. An NMR structural analysis method for a protein, comprising the step of measuring an NMR spectrum of a solution of a purified protein obtained by the method for incorporating a stable isotope-labeled amino acid into a protein according to claim 11.

13. A composition comprising at least one of the stable isotope-labeled amino acids according to claim 8, which further comprises at least one of stable isotope-labeled amino acids represented by one of the following nine structural formulas: ##STR00013## wherein R represents .sup.13C.sup.1H.sub.3, .sup.13C.sup.1H.sub.2D, or .sup.13C.sup.1HD.sub.2, H has the same meaning as .sup.1H, D has the same meaning as .sup.2H, C has the same meaning as .sup.12C, * represents a stereogenic center, and the amino acids are any one of enantiomers with respect to the stereogenic center.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows an NMR spectrum at the β-position of a leucine residue of [.sup.2H; .sup.15N]MSG incorporating [ul-.sup.13C; .sup.15N]Leu.

(2) FIG. 2 shows an NMR spectrum at the β-position of a leucine residue of [.sup.2H; .sup.15N]MSG incorporating SAIL-Leu.

(3) FIG. 3 shows an NMR spectrum at the β-position of leucine of [.sup.2H; .sup.15N]MSG incorporating oro-SAIL-Leu.

(4) FIG. 4 shows an NMR spectrum at the β-position of a leucine residue of [.sup.2H; .sup.15N]MSG incorporating Leu (1) that is stable isotope-labeled leucine of the present invention.

(5) FIG. 5 shows an NMR spectrum at the β-position of a leucine residue of [.sup.2H; .sup.15N]MSG incorporating Leu (2) that is stable isotope-labeled leucine of the present invention.

(6) FIG. 6 shows a sequence-specific assignment of Hβ3 in the leucine residue of the [.sup.2H; .sup.15N]MSG incorporating Leu (1) that is the stable isotope-labeled leucine of the present invention.

(7) FIG. 7 shows a sequence-specific assignment of Hβ2 in the leucine residue of the [.sup.2H; .sup.15N]MSG incorporating Leu (2) that is the stable isotope-labeled leucine of the present invention.

(8) FIG. 8 shows NOE spectra between β2, β3 protons and a δ1 methyl proton (a), a δ2 methyl proton (b), or an amide proton (c) of L180 in the MSG.

(9) FIG. 9 shows the conformation of side chains of the L180 in the MSG.

(10) FIG. 10 shows an NMR spectrum at the α-position of a proline residue of [.sup.2H; .sup.15N]MSG incorporating [ul-.sup.13C; .sup.15N]Pro.

(11) FIG. 11 shows an NMR spectrum at the γ-position of the proline residue of the [.sup.2 H; .sup.15 N]MSG incorporating [ul-.sup.13C; .sup.15N]Pro.

(12) FIG. 12 shows an NMR spectrum at the α-position of a proline residue of [.sup.2H; .sup.15N]MSG incorporating SAIL-Pro.

(13) FIG. 13 shows an NMR spectrum at the γ-position of the proline residue of the [.sup.2H; .sup.15N]MSG incorporating SAIL-Pro.

(14) FIG. 14 shows an NMR spectrum at the α-position of a proline residue of [.sup.2H; .sup.15N]MSG incorporating Pro (3) that is stable isotope-labeled proline of the present invention.

(15) FIG. 15 shows an NMR spectrum at the γ-position of the leucine residue of the [.sup.2H; .sup.15N]MSG incorporating Pro (3) that is the stable isotope-labeled proline of the present invention.

(16) FIG. 16 shows sequence-specific assignment of Ha in the proline residue of the [.sup.2H; .sup.15N]MSG incorporating Pro (3) that is the stable isotope-labeled proline of the present invention.

DESCRIPTION OF EMBODIMENTS

(17) Hereinafter, the present invention will be described in detail with reference to the drawings.

(18) <Stable Isotope-Labeled Amino Acid>

(19) A stable isotope-labeled aliphatic amino acid of the present invention satisfies all of the following conditions (1) to (3).

(20) (1) two or more carbon atoms are labeled with .sup.13C;

(21) (2) of two or more carbon atoms labeled with .sup.13C, a carbon atom other than a carbon atom of a methyl group, which is capable of bonding to a hydrogen atom, has one .sup.1H directly bonded thereto, while the carbon atom of the methyl group has at least one .sup.1H directly bonded thereto; and

(22) (3) other carbon atoms adjacent to all the .sup.13C are all .sup.12C.

(23) When the structure of a protein is analyzed according to an NMR technique, the observation sensitivity to NMR signals of amino acid residues is sufficiently increased by using the stable isotope-labeled aliphatic amino acid of the present invention, and NOE between protons in the amino acid residue is allowed to be observed. Thereby, it is made possible to assign the signal of amino acid residue side chains. This also makes it possible to analyze the three-dimensional structure of a methylene proton on a side chain of an over 80 kDa protein, which has been impossible heretofore, and allows detailed information on the structure of the high-molecular-weight protein to be obtained.

(24) To be more specific, the stable isotope-labeled aliphatic amino acid of the present invention satisfying the conditions (1) and (3) makes it possible to avoid a complication of .sup.13C signals due to spin couplings. Conventionally known methods including methods described in International Publication No. WO2003/053910 and 2 utilize .sup.13C—.sup.13C spin couplings for assignment of .sup.13C signals derived from a stable isotope-labeled aliphatic amino acid. Accordingly, it has been necessary to avoid a complication in a spectrum by employing a constant time evolution technique that is an approach sacrificing the sensitivity and resolution of an NMR spectrum. In contrast, the stable isotope-labeled aliphatic amino acid satisfying the conditions (1) and (3) makes it possible to avoid a complication of .sup.13C signals derived from the stable isotope-labeled aliphatic amino acid without employing the constant time evolution technique. Thus, an NMR structural analysis method can be performed with high sensitivity and high resolution even on a protein having a higher molecular weight.

(25) Moreover, since the stable isotope-labeled aliphatic amino acid of the present invention satisfies the conditions (2), the information on the three-dimensional structure determined by the assignment of .sup.1H signals utilizing NOE is maintained, and also .sup.1H does not excessively remain in a side chain of the stable isotope-labeled aliphatic amino acid. Thus, it is possible to avoid the broadening of a .sup.1H-derived signal due to a dipole interaction. Hence, an NMR structural analysis can be performed with high sensitivity and high resolution even on a protein having a higher molecular weight.

(26) In other words, in the present invention, .sup.1H bonding to .sup.13C is isolated from the other .sup.13C—.sup.1H in the amino acid. This makes it possible to prevent a complication of the .sup.1H NMR signal, and to observe a signal derived from .sup.13C—.sup.1H with high sensitivity and high resolution.

(27) The stable isotope-labeled aliphatic amino acid of the present invention preferably further satisfies at least one or more conditions selected from the group consisting of the following conditions (4) to (8):

(28) (4) the number of carbon atoms located between the .sup.13C having .sup.1H directly bonded thereto and another carbon atom having .sup.1H directly bonded thereto is three or less;

(29) (5) under the condition (4), the other carbon atom having .sup.1H directly bonded thereto is labeled with .sup.13C;

(30) (6) a carbon atom of at least one methylene chain among methylene chains present at side chains of the amino acid is labeled with .sup.13C, and one of two hydrogen atoms directly bonded to the .sup.13C is stereo-selectively labeled with .sup.2H;

(31) (7) one of two hydrogen atoms directly bonded to a .sup.12C carbon atom of a methylene chain present at the side chain of the amino acid is labeled with .sup.2H, or both of the two hydrogen atoms are concurrently .sup.1H or .sup.2H; and

(32) (8) in a case where pro-chiral gem-methyl groups are present, one of the gem-methyl groups is labeled with .sup.12C.sup.2H.sub.3 or .sup.13C.sup.2H.sub.3.

(33) Preferably, the stable isotope-labeled aliphatic amino acid of the present invention simultaneously satisfies the conditions (4) to (6) among any of these conditions.

(34) The stable isotope-labeled aliphatic amino acid of the present invention satisfying the condition (4) facilitates assignment of .sup.1H signals by utilizing NOE observed between multiple .sup.1Hs, and facilitates assignment of structures of amino acid residues in a protein without utilizing a sequential assignment technique having been conventionally performed.

(35) Moreover, the stable isotope-labeled aliphatic amino acid of the present invention satisfying the condition (5) further facilitates assignment of .sup.1H signals by utilizing NOE observed between multiple .sup.1Hs, and further facilitates assignment of structures of amino acid residues in a protein without utilizing the sequential assignment technique having been conventionally performed.

(36) The stable isotope-labeled aliphatic amino acid of the present invention satisfying the condition (6) makes it possible to simplify a signal derived from .sup.13C—.sup.1H, and also enables stereospecific assignment of the entire —.sup.13C.sup.1H.sup.2H— because .sup.1H is stereo-selectively bonded to the .sup.13C.

(37) Furthermore, the stable isotope-labeled aliphatic amino acid of the present invention satisfying the condition (7) makes it possible, in the case where the two hydrogen atoms bonded to the .sup.12C are labeled with .sup.2H, to simplify a signal of the .sup.1H bonded to the other carbon atom; meanwhile, in the case where at least one of the hydrogen atoms bonded to the .sup.12C is .sup.1H, NOE observed between this .sup.1H and the .sup.1H bonded to the other carbon atom enables assignment of structures of amino acid residues in a protein.

(38) Furthermore, the stable isotope-labeled aliphatic amino acid of the present invention satisfying the condition (8) makes it possible to reduce the number of signals from methyl groups, accordingly making it possible to avoid a complication of the signals.

(39) The stable isotope aliphatic amino acid is not particularly limited, as long as the aliphatic amino acid satisfies the conditions (1) to (3) and constitutes a protein. Nevertheless, the aliphatic amino acid is preferably glutamic acid (Glu), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), proline (Pro), glutamine (Gln), arginine (Arg), threonine (Thr), or valine (Val).

(40) More specifically, the stable isotope-labeled aliphatic amino acid of the present invention is preferably represented by one of the following ten structural formulas:

(41) ##STR00004##
where R represents .sup.13C.sup.1H.sub.3, .sup.13C.sup.1H.sub.2D, or .sup.13C.sup.1HD.sub.2, H has the same meaning as .sup.1H, D has the same meaning as .sup.2H, C has the same meaning as .sup.12C, * represents a stereogenic center, the amino acids are any one of enantiomers with respect to the stereogenic center, n represents any one of 0 to 2, and in the case where n is 1, the amino acid may be a racemate or anyone of enantiomers with respect to a carbon atom to which the corresponding deuterium is bonded.

(42) Further, the stable isotope-labeled aliphatic amino acid of the present invention is more preferably represented by one of the following ten structural formulas:

(43) ##STR00005##
where R represents .sup.13C.sup.1H.sub.3, .sup.13C.sup.1H.sub.2D, or .sup.13C.sup.1HD.sub.2, R.sub.2 represents .sup.1H or .sup.2H, R.sub.3 represents .sup.12C.sup.1H.sub.3, .sup.12C.sup.1H.sub.2D, or .sup.12C.sup.1HD.sub.2, H has the same meaning as .sup.1H, D has the same meaning as .sup.2H, C has the same meaning as .sup.12C, * represents a stereogenic center, the amino acids are any one of enantiomers with respect to the stereogenic center, n represents any one of 0 to 2, and in the case where n is 1, the amino acid may be a racemate or any one of enantiomers with respect to a carbon atom to which the corresponding deuterium is bonded.

(44) These stable isotope-labeled aliphatic amino acids can be prepared by employing a conventionally known preparation method and using reaction raw materials specifically labeled with .sup.13C and .sup.2H in accordance with isotope-labeled sites in the aliphatic amino acid. Specifically, the stable isotope-labeled aliphatic amino acid of the present invention can be prepared, for example, according to the method for preparing an amino acid described in International Publication No. WO2003/053910. Regarding the stable isotope-labeled reaction raw materials to be used, reaction raw materials and so forth (for example, “Acetic acid-2-13C”, “Acetic acid-1-13C”, “deuterium oxide”, “Deuterium”, “Glycine-2-13C,15N”) marketed from Taiyo Nippon Sanso Corporation or the like may be used. Furthermore, the stable isotope-labeled aliphatic amino acid of the present invention may be prepared according to methods for preparing an amino acid described in the following Literatures 1 to 4. Note that the content of International Publication No. WO2003/053910 is incorporated into the description of this specification by reference. [Literature 1] Oba, M.; Ueno, R.; Fukuoka (nee Yoshida), M.; Kainosho, M.; Nishiyama, K., Synthesis of L-threo- and L-erythro-[1-.sup.13C, 2,3-.sup.2H.sub.2]amino acids: novel probes for conformational analysis of peptide side chains., J. Chem. Soc., Perkin Trans. 1 1995, 1603. [Literature 2] Terauchi, T.; Kamikawai, T.; Vinogradov, M. G.; Starodubtseva, E. V.; Takeda, M.; Kainosho, M. Synthesis of stereoarray isotope labeled (SAIL) lysine via the “head-to-tail” conversion of SAIL glutamic acid. Org. Lett., 2011, 13, 161-163. [Literature 3] Okuma, K.; Ono, A. M.; Tsuchiya, S.; Oba, M.; Nishiyama, K.; Kainosho, M.; Terauchi, T. Assymetric synthesis of (2S,3R)- and (2S,3S)-[2-.sup.13C; 3-.sup.2H] glutamic acid. Tetrahedron Lett., 2009, 50, 1482-1484. [Literature 4] Terauchi, T.; Kobayashi, K.; Okuma, K.; Oba, M.; Nishiyama, K.; Kainosho, M. Stereoselective synthesis of triply isotope-labeled Ser, Cys, and Ala: amino acids for stereoarray isotope labeling technology. Org. lett., 2008, 10, 2785-7.

(45) <Composition Containing Stable Isotope-Labeled Amino Acid>

(46) The present invention relates also to a composition containing the above-described stable isotope-labeled aliphatic amino acid. The composition of the present invention contains: at least one selected from the group consisting of the above-described stable isotope-labeled aliphatic amino acids; and a carrier. The combination and concentrations of the stable isotope-labeled aliphatic amino acids in the composition of the present invention may be adjusted appropriately depending on the purpose of use of the composition of the present invention.

(47) Note that in a case where the composition of the present invention contains at least one selected from Arg, Gln, Glu, Ile, Leu, Lys, Met, Pro, Thr, and Val represented by the above-described 20 specific structural formulas as the stable isotope-labeled aliphatic amino acids, the composition of the present invention preferably further contains at least one of stable isotope-labeled amino acids represented by one of the following nine structural formulas:

(48) ##STR00006##
where R represents .sup.13C.sup.1H.sub.3, .sup.13C.sup.1H.sub.2D, or .sup.13C.sup.1HD.sub.2, H has the same meaning as .sup.1H, D has the same meaning as .sup.2H, C has the same meaning as .sup.12C, * represents a stereogenic center, and the amino acids are any one of enantiomers with respect to the stereogenic center.

(49) <Method for Incorporating Stable Isotope-Labeled Amino Acid into Protein>

(50) The present invention relates also to a method for incorporating the above-described stable isotope-labeled aliphatic amino acid into a protein. Here, the method for incorporating a stable isotope-labeled aliphatic amino acid into a protein of the present invention is a method including the step of synthesizing a protein using cultured cells, microorganisms, or a cell-free protein synthesis system in the presence of the above-described stable isotope-labeled aliphatic amino acid.

(51) To be more specific, in the present invention, introduction of a gene fragment encoding a target protein whose structure is to be analyzed by an NMR technique into a system of the cultured cells, the microorganisms, the cell-free protein synthesis, or the like in such a manner that the gene fragment can be excessively expressed in these systems, and excessive expression of the protein in the presence of the stable isotope-labeled aliphatic amino acid are conducted to synthesize the protein, where a particular amino acid residue is replaced with an amino acid residue derived from the stable isotope-labeled aliphatic amino acid of the present invention.

(52) The detailed conditions in performing such a method for incorporating a stable isotope-labeled aliphatic amino acid into a protein may be conditions designed in accordance with conventional methods for excessively expressing a protein using cultured cells, microorganisms, or a cell-free protein synthesis system.

(53) <NMR Structural Analysis Method for Protein>

(54) The present invention relates also to a method for an NMR structural analysis of a protein, including the step of measuring an NMR spectrum of a solution of a purified protein obtained by the above-described method for incorporating a stable isotope-labeled aliphatic amino acid into a protein. As the method for measuring an NMR spectrum, conventionally known approaches may be employed. In the NMR spectrum measurement, the protein may be bound to a conventionally known ligand and a different protein to measure the NMR spectrum in the state of a complex formed of the protein, the ligand, and the different protein.

(55) The detailed conditions in performing these NMR structural analysis methods may be designed in accordance with conventional NMR structural analysis methods.

EXAMPLES

(56) Hereinafter, the present invention will be described in detail based on Examples. Note that the present invention is not limited at all to Examples shown below.

(57) <Synthesis of Stable Isotope-Labeled Leucine>

(58) Two types of stable isotope-labeled leucines represented by chemical formulas 1 and 2 (hereinafter may be referred to as “Leu (1)” and “Leu (2)”, respectively) were synthesized by a known synthesis method according to approaches described in the above [Literature 1] to [Literature 3] and the following [Literature 5]. The method for synthesizing each stable isotope-labeled leucine is shown below. [Literature 5] Nahm, S.; Weinreb, S. M., N-Methoxy-N-methylamides as effective acylating agents. Tetrahedron Lett., 1981, 22, 38153818.

(59) ##STR00007## ##STR00008##

(60) <Preparation of Proteins Incorporating Stable Isotope-Labeled Leucines>

(61) Malate synthase G (molecular weight 82 kDa; hereinafter maybe referred to as “MSG”) incorporating Leu (1) was prepared by a known approach according to the method in the following Literature 6. However, the leucine addition and culturing methods were modified as follows. After a plasmid MSG-pET28b having a DNA sequence encoding malate synthase G (MSG) was transformed into Escherichia coli BL21(DE3)pLysS strain, the transformed Escherichia coli was grown in an LB medium (2 ml) having been prepared using light water. The grown bacterial cells were collected and cultured at 37° C. for 20 hours in an M9 medium (3 ml) having been prepared using heavy water, in the presence of various vitamins. Then, the resultant was inoculated into a medium (100 ml) for main culturing in which Leu (1) (0.67 mg) had been dissolved, and cultured at 37° C. until the OD.sub.600 reached approximately 0.3. To the resulting culture liquid, Leu (1) (1.33 mg) and IPTG (final concentration of 1 mM) were further added for MSG synthesis induction. After culturing at 37° C. for 8 hours, the bacterial cells were collected by centrifugation. By purifying a protein from the obtained bacterial cells with a known method according to the method in Literature 6, [Leu (1); .sup.2H; .sup.15N]MSG was obtained. [Leu (2); .sup.2H; .sup.15N]MSG was also prepared by employing the same approach as that for Leu (1). [Literature 6] Tugarinov V, Muhandiram R, Ayed A, Kay L E, Four-dimensional NMR spectroscopy of a 723-residue protein: Chemical shift assignments and secondary structure of malate synthase G. J Am Chem Soc, 2002, 124: 10025-10035.

(62) <Structural Analysis by NMR Technique>

(63) To compare the NMR structural analysis method of the present invention with conventional methods, CT TROSY-HSQC of the [.sup.2H; .sup.15N]MSGs, which were prepared by using [ul-.sup.13C; .sup.15N] Leu whose structure is shown in FIG. 1, SAIL-Leu whose structure is shown in FIG. 2, oro-SAIL-Leu whose structure is shown in FIG. 3, and Leu (1) and Leu (2), was measured (FIGS. 1 to 5). Hydrogen Hα in the samples was labeled with a deuterium atom through metabolism because of the culturing in heavy water. MSG is a high-molecular-weight protein consisting of 723 amino acid residues including 70 residues in respect of even leucine residues. In MSG incorporating [ul-.sup.13C; .sup.15N]Leu, 140 signals of the β-position were supposed to be observed, but in fact the signals were significantly broadened due to the influence of a relaxation phenomenon between adjacent .sup.13C and .sup.1H nuclei, so that the signals of the β-position were hardly observed (FIG. 1). Moreover, in SAIL-Leu, since one of β protons was deuterated, the measurement sensitivity was enhanced slightly in comparison with [ul-.sup.13C; .sup.15N]Leu. Nevertheless, signals of only 33 residues were observed out of the 70 residues (FIG. 2). Further, in oro-SAIL-Leu, because of the effect that all the hydrogens present at the vicinal positions of the β proton are deuterated, the number of observed signals of the β-position was increased in comparison with SAIL-Leu. However, still a sufficient sensitivity was not obtained due to the influence of a relaxation phenomenon between .sup.13C nuclei, so that only 38 residues were observed out of the 70 residues (FIG. 3). On the other hand, in Leu (1) and Leu (2) that are the examples of the stable isotope-labeled aliphatic amino acid of the present invention, the carbon atom and the hydrogen atoms at the β-position and the δ-position were specifically labeled with .sup.13C and .sup.2H, and the relaxation influence has been eliminated as much as possible. As a result, it was possible to observe β proton-derived signals corresponding to 66 residues and 67 residues in the MSGs incorporating Leu (1) and Leu (2), respectively (FIGS. 4 and 5).

(64) It was found that utilizing Leu (1) and Leu (2) makes it possible to observe β protons stereospecifically with high sensitivity even in a high-molecular-weight protein (FIGS. 6 and 7). Further, analyzing NOE between a β proton and an amide proton or δ methyl proton in the amino acid residue makes it possible to easily determine sequence-specific sequential assignment and the conformation of Leu side chain. As an example, FIG. 8 shows NOE spectra in the 180th leucine (L180) residue of the MSG. It can be seen that although signals of β2 and β3 protons of L180 were observed from a δ1 methyl proton (FIG. 10a), a δ2 methyl proton (FIG. 10b) and an amide proton (FIG. 10c) of the L180, the relation of the signal intensity varied from one another. Specifically, it can be seen from this result that the distances from the δ1 methyl proton to the β2, β3 protons are approximately the same, but the β3 proton is located closer to the δ2 methyl proton than the β2 proton is. On the other hand, it can be seen that the β2 proton is located closer to the amide proton than the β3 proton is.

(65) These results revealed the relative arrangement of atomic groups of the L180, so that the conformation can be determined accurately (FIG. 9).

(66) In this respect, Leu residues are generally present at an inner side of a protein, and form a core structure through a hydrophobic interaction with other amino acid residues in many cases. Thus, Leu residue-derived information is very useful in analyses of three-dimensional structures of proteins. Leu (1) and Leu (2) that are the examples of the stable isotope-labeled aliphatic amino acid of the present invention are capable of providing signals with high sensitivity even in a high-molecular-weight protein, and also providing information on conformation, accordingly showing a new way of a method for three-dimensional-structural analysis of high-molecular-weight proteins according to a solution NMR technique.

(67) <Synthesis of Stable Isotope-Labeled Proline>

(68) Stable isotope-labeled proline represented by chemical formula 3 (hereinafter may be referred to as Pro (3)) was synthesized by a known synthesis method according to approaches described in the following [Literature 7] to [Literature 9]. The method for synthesizing a stable isotope-labeled proline is shown below.

(69) ##STR00009## [Literature 7] Iida, K.; Kajiwara, M. J. Label. Compd. Radiopharm. 1991, 29, 201. [Literature 8] Kajiwara, M.; Lee, S.-F.; Scott, A. I.; Akhtar, M.; Jones, C. R.; Jordan, P. M. Chem. Commun. 1978, 967. [Literature 9] Reddy, L. R.; Reddy, B. V. S.; Corey, E. J. Org. lett., 2006, 8, 2819.

(70) <Preparation of Protein Incorporating Stable Isotope-Labeled Proline>

(71) Malate synthase G (MSG) using the stable isotope-labeled proline Pro (3) was prepared in accordance with the method in Literature 6. However, the methods for adding and culturing the stable isotope-labeled proline Pro (3) were modified as follows. After a plasmid MSG-pET28b was introduced into E. coli BL21 (DE3) pLysS strain, the resultant was grown in an LB medium (2 ml) having been prepared using light water. The grown bacterial cells were cultured at 37° C. for 20 hours in an M9 medium (3 ml) having been prepared using heavy water, in the presence of various vitamins. Then, the resultant was inoculated into a medium (100 ml) for main culturing in which the stable isotope-labeled proline (Pro (3), 1 mg) had been dissolved, and cultured at 37° C. until OD600=approximately 0.3. To the resulting medium, the stable isotope-labeled proline (Pro (3), 2 mg) and IPTG (final concentration of 1 mM) were added. After culturing at 37° C. for 8 hours, the bacterial cells were collected by centrifugation. By purifying a protein from the obtained bacterial cells in accordance with Literature 6, [Pro; .sup.2H; .sup.15N]MSG was obtained.

(72) <Structural Analysis by NMR Technique>

(73) For comparison with conventional methods, CT TROSY-HSQC of the [.sup.2H; .sup.15N]MSGs, which were prepared by using [ul-.sup.13C; .sup.15N]Pro whose structure is shown in FIGS. 10 and 11, SAIL-Pro whose structure is shown in FIGS. 12 and 13, and Pro (3) whose structure is shown in FIGS. 14 to 16. MSG is a high-molecular-weight protein consisting of 723 amino acid residues including 31 residues in respect of even proline residues. In MSG incorporating [ul-.sup.13C; .sup.15N]Pro, and 31 signals of the α-position and 62 signals of the γ-position were supposed to be observed, but actually the signals were significantly broadened due to the influence of a relaxation phenomenon between adjacent .sup.13C and .sup.1H nuclei, so that the signals were hardly observed (FIGS. 10 and 11). Moreover, in SAIL-Pro also, although one of protons on a side chain was deuterated as described above, a sufficient sensitivity was not obtained due to the influence of a relaxation phenomenon between .sup.13C nuclei, so that most of the 31 residues were not observed (FIGS. 12 and 13). On the other hand, in Pro (3) illustrated in the present invention, the α-position and the γ-position were specifically labeled with .sup.13C and .sup.1H, and the relaxation influence has been eliminated as much as possible. As a result, it became possible to observe proton-derived signals of the α-position corresponding to 31 residues in the MSG incorporating Pro (3) (FIGS. 14 to 16).