Use of deuterium labeling in NMR: overcoming a sizeable problem

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is a useful technique for determining the three-dimensional structures of proteins in solution. In the last few years new NMR techniques have been developed and applied to uniformly 13C- and 15N-labeled proteins, which has dramatically improved the applicability of NMR to the study of larger proteins (MW <20kDa) [[1]Clore G.M. Gronenborn A.M. Multidimensional heteronuclear nuclear magnetic resonance of proteins.Methods Enzymol. 1994; 239 (95131769): 349-363Crossref PubMed Scopus (247) Google Scholar]. However, two main challenges remain for the structure determination of even larger proteins (MW >20kDa) by NMR. These are the low signal-to-noise (due to increased relaxation rates caused by the slower overall tumbling of larger proteins in solution) and the lack of spectral resolution due to the large number of signals. One possible approach for extending the use of NMR to larger systems is through the utilization of deuterium labeling. Upon deuteration the signal-to-noise in the NMR spectra is improved by suppressing spin diffusion (Figure 1a) and by decreasing the relaxation rates of 13C and 15N spins (Figure 1b). In larger proteins that are fully protonated, the efficient distribution of magnetization through the spin system of dipolar coupled protons (spin diffusion) leads to large linewidths of the NMR signals and, therefore, low signal-to-noise. If the density of protons is decreased through the use of a deuterated protein, many of these relaxation pathways are eliminated, and the signal-to-noise of the NMR spectra is dramatically improved [2LeMaster D.M. Richards F.M. NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteriation.Biochemistry. 1988; 27 (88163560): 142-150Crossref PubMed Scopus (261) Google Scholar, 3Torchia D.A. Sparks S.W. Bax A. Delineation of α-helical domains in deuteriated staphylococcal nuclease by 2D NOE NMR spectroscopy.J. Am. Chem. Soc. 1988; 110: 2320-2321Crossref Scopus (76) Google Scholar]. Another use of deuteration is to reduce the dipolar interaction between 13C or 15N and the directly bound proton spin which is the main source of relaxation in 13C- and 15N-labeled proteins [4Browne D.T. Kenyon G.L. Packer E.L. Sternlicht H. Wilson D.M. Studies of macromolecular structure by 13C nuclear magnetic resonance. II. A specific labeling approach to the study of histidine residues in proteins.J. Am. Chem. Soc. 1973; 95: 1316-1323Crossref PubMed Scopus (67) Google Scholar, 5Grzesiek S. Anglister J. Ren H. Bax A. 13C line narrowing by 2H decoupling in 2H/13C/15N-enriched proteins. Application to triple resonance 4D J connectivity of sequential amides.J. Am. Chem. Soc. 1993; 115: 4369-4370Crossref Scopus (220) Google Scholar]. Due to the significantly smaller gyromagnetic ratio (γ) of the deuterium spin (γD ∼ 1/6.5 γH), the relaxation rates are scaled proportional to (γD/γH)2 ∼ 0.02. Therefore, the relaxation times of 13C and 15N spins are greatly increased which leads to smaller linewidths and higher signal-to-noise. Smaller linewidths also result from the elimination of passive couplings in deuterated proteins [[6]Kushlan D.M. LeMaster D.M. Resolution and sensitivity enhancement of heteronuclear correlation for methylene resonances via 2H enrichment and decoupling.J. Biomol. NMR. 1993; 3 (94154512): 701-708Crossref PubMed Scopus (50) Google Scholar]. Another advantage of the increased relaxation times obtained upon deuteration is that constant-time experiments (which yield poor signal-to-noise with fully protonated proteins) can be applied with high sensitivity [[7]Yamazaki T. Kay L.E. et al.An HNCA pulse scheme for the backbone assignment of 15N, 13C, 2H-labeled proteins: application to a 37-kDa trp repressor–DNA complex.J. Am. Chem. Soc. 1994; 116: 6464-6465Crossref Scopus (169) Google Scholar]. Constant-time experiments yield spectra with much higher resolution and therefore much less overlap of cross-peaks. Because of these advantages, the use of deuterium labeling is playing an important role in the structure determination of larger proteins by NMR. In the following report, we discuss recent advances in the use of deuterium labeling in heteronuclear multidimensional NMR experiments. Various strategies have been developed for deuterating proteins (for a review see [[8]LeMaster D.M. Isotope labeling in solution protein assignment and structural analysis.Prog. NMR Spectrosc. 1994; 26: 371-419Abstract Full Text PDF Scopus (95) Google Scholar]). These strategies include methods for the specific deuteration of selected residue types or of selected positions in aromatic side chains [8LeMaster D.M. Isotope labeling in solution protein assignment and structural analysis.Prog. NMR Spectrosc. 1994; 26: 371-419Abstract Full Text PDF Scopus (95) Google Scholar, 9Crespi H.L. Rosenberg R.M. Katz J.J. Proton magnetic resonance of proteins fully deuterated except for 1H-leucine side chains.Science. 1968; 161: 795-796Crossref PubMed Scopus (76) Google Scholar, 10Markley J.L. Putter I. Jardetzky O. High-resolution nuclear magnetic resonance spectra of selectively deuterated staphylococcal nuclease.Science. 1968; 161: 1249-1251Crossref PubMed Scopus (152) Google Scholar, 11Tsang P. Wright P.E. Rance M. Specific deuteration strategy for enhancing direct nuclear Overhauser effects in high molecular weight complexes.J. Am. Chem. Soc. 1990; 112: 8183-8185Crossref Scopus (22) Google Scholar, 12Reisman J. Jariel-Encontre I. Hsu V.L. Parello J. Geiduschek E.P. Kearns D.R. Improving two-dimensional 1H NMR NOESY spectra of a large protein by selective deuteriation.J. Am. Chem. Soc. 1991; 113: 2787-2789Crossref Scopus (22) Google Scholar, 13Arrowsmith C.H. Pachter R. Altman R.B. Iyer S.B. Jardetzky O. Sequence-specific 1H NMR assignments and secondary structure in solution of Escherichia coli trp repressor.Biochemistry. 1990; 29 (91002525): 6332-6341Crossref PubMed Scopus (113) Google Scholar, 14Metzler W.J. Wittekind M. Goldfarb V. Mueller L. Farmer II, B.T. Incorporation of 1H/13C/15N-[Ile, Leu, Val] into a perdeuterated, 15N-labeled protein: potential in structure determination of large proteins by NMR.J. Am. Chem. Soc. 1996; 118: 6800-6801Crossref Scopus (71) Google Scholar], random fractional deuteration [2LeMaster D.M. Richards F.M. NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteriation.Biochemistry. 1988; 27 (88163560): 142-150Crossref PubMed Scopus (261) Google Scholar, 3Torchia D.A. Sparks S.W. Bax A. Delineation of α-helical domains in deuteriated staphylococcal nuclease by 2D NOE NMR spectroscopy.J. Am. Chem. Soc. 1988; 110: 2320-2321Crossref Scopus (76) Google Scholar, 15Shon K.-J. Kim Y. Colnago L.A. Opella S.J. NMR studies of the structure and dynamics of membrane-bound bacteriophage Pf1 coat protein.Science. 1991; 252 (92022520): 1303-1305Crossref PubMed Scopus (155) Google Scholar, 17Yamazaki T. Lee W.L. Arrowsmith C.H. Muhandiram D.R. Kay L.E. A suite of triple resonance NMR experiments for the backbone assignment of 15N, 13C, 2H labeled proteins with high sensitivity.J. Am. Chem. Soc. 1994; 116: 11655-11666Crossref Scopus (479) Google Scholar], and the complete deuteration of nonexchangable protons [3Torchia D.A. Sparks S.W. Bax A. Delineation of α-helical domains in deuteriated staphylococcal nuclease by 2D NOE NMR spectroscopy.J. Am. Chem. Soc. 1988; 110: 2320-2321Crossref Scopus (76) Google Scholar, 18Venters R.A. Huang C.-C. Farmer II, B.T. Trolard R. Spicer L.D. Fierke C.A. High-level 2H/13C/15N labeling of proteins for NMR studies.J. Biomol. NMR. 1995; 5 (95375527): 339-344Crossref PubMed Scopus (101) Google Scholar]. One of the most useful approaches is random fractional deuteration, especially when combined with uniform 13C- and 15N-labeling. Fractionally deuterated and uniformly 13C-, 15N-labeled proteins are easily prepared. To do this, bacteria that overexpress the protein of interest are grown in a minimal medium containing either uniformly 13C- labeled sodium acetate [[18]Venters R.A. Huang C.-C. Farmer II, B.T. Trolard R. Spicer L.D. Fierke C.A. High-level 2H/13C/15N labeling of proteins for NMR studies.J. Biomol. NMR. 1995; 5 (95375527): 339-344Crossref PubMed Scopus (101) Google Scholar] or glucose [[17]Yamazaki T. Lee W.L. Arrowsmith C.H. Muhandiram D.R. Kay L.E. A suite of triple resonance NMR experiments for the backbone assignment of 15N, 13C, 2H labeled proteins with high sensitivity.J. Am. Chem. Soc. 1994; 116: 11655-11666Crossref Scopus (479) Google Scholar] and 15N-labeled ammonium chloride in water containing the desired amount of D2O. In order to optimize the expression of the protein, cell cultures can be adapted to grow in D2O by increasing the amount of D2O in the growth medium from zero to the desired percentage with each growth cycle. However, in practice this is only necessary to obtain deuteration levels of greater than 75 % . Thus, deuterated and uniformly 13C- and 15N-labeled proteins are readily obtained with only slight modifications of the procedures used to generate uniformly 13C- and 15N-labeled proteins. The first step in the structure determination of proteins by NMR is the assignment of the 1H, 13C and 15N chemical shifts. This is accomplished by correlating the chemical shifts of backbone nuclei via 1J-couplings using a suite of triple resonance NMR experiments [[1]Clore G.M. Gronenborn A.M. Multidimensional heteronuclear nuclear magnetic resonance of proteins.Methods Enzymol. 1994; 239 (95131769): 349-363Crossref PubMed Scopus (247) Google Scholar]. One of the experiments, for example, is a three-dimensional HNCA experiment [[19]Kay L.E. Ikura M. Tschudin R. Bax A. Three-dimensional triple-resonance NMR spectroscopy of isotopically enriched proteins.J. Magn. Reson. 1990; 89: 496-514Google Scholar] in which the chemical shifts of the amide proton (HN) and nitrogen (N) are correlated to the chemical shift of the Cα spin (H→N →Cα). The efficiency of these triple resonance experiments relies on the relaxation properties of the spins involved in the magnetization transfer, as the decay of magnetization during the delays in the pulse sequence decreases the signal-to-noise, especially for proteins greater than 20kDa in molecular weight. An example of the advantages of using fractionally deuterated versus fully protonated 13C-, 15N-labeled proteins in the triple resonance experiments is shown in Figure 2. Projections of three-dimensional HNCA experiments are shown that were recorded on the 23kDA complex of the Shc phosphotyrosine-binding (PTB) domain (190 residues) complexed to a 12-residue tyrosine phosphorylated peptide [[20]Zhou M.-M. Fesik S.W. et al.Structure and ligand recognition of the phosphotyrosine binding domain of Shc.Nature. 1995; 378 (96097066): 584-592Crossref PubMed Scopus (323) Google Scholar]. In these experiments the 13C-, 15N-labeled protein was fully protonated (Figure 2a) or fractionally deuterated (Figure 2b). The number of cross-peaks visible in the spectrum of the 75 % fractionally deuterated sample is much higher than in the spectrum recorded on fully protonated Shc PTB domain. This gain in signal-to-noise results from the increased relaxation times of HN, N and Cα spins due to the fractional deuteration. The increase in signal-to-noise is especially pronounced for the Cα spins, which have very fast relaxation rates due to the dipolar coupling with the directly bound Hα proton. The downscaling of this interaction upon substituting the Hα by deuterium, leads to a large increase in the Cα relaxation times. For example, in NMR studies of a 37kDa Trp repressor–DNA complex, it has recently been demonstrated, that the Cα relaxation times increase from 16.5 ms (100 % 1H) to 130ms (70 % 2H) [[7]Yamazaki T. Kay L.E. et al.An HNCA pulse scheme for the backbone assignment of 15N, 13C, 2H-labeled proteins: application to a 37-kDa trp repressor–DNA complex.J. Am. Chem. Soc. 1994; 116: 6464-6465Crossref Scopus (169) Google Scholar]. Moreover, due to this improvement in sensitivity, NMR experiments that employ constant-time periods for the chemical shift evolution can be used which greatly improves the resolution in the spectra as illustrated in Figure 2. The improved resolution is especially useful in studies of larger proteins, in which the large number of cross-peaks overlap even in heteronuclear, multidimensional NMR experiments. For the projection of the high resolution three-dimensional HNCA spectrum shown in Figure 2a, Cα chemical shifts are recorded during a constant-time period of 1/1J(Cα, Cβ) ∼ 28ms in order to refocus the undesired 1J(Cα, Cβ) coupling. 2H decoupling is applied during the constant-time period [4Browne D.T. Kenyon G.L. Packer E.L. Sternlicht H. Wilson D.M. Studies of macromolecular structure by 13C nuclear magnetic resonance. II. A specific labeling approach to the study of histidine residues in proteins.J. Am. Chem. Soc. 1973; 95: 1316-1323Crossref PubMed Scopus (67) Google Scholar, 5Grzesiek S. Anglister J. Ren H. Bax A. 13C line narrowing by 2H decoupling in 2H/13C/15N-enriched proteins. Application to triple resonance 4D J connectivity of sequential amides.J. Am. Chem. Soc. 1993; 115: 4369-4370Crossref Scopus (220) Google Scholar, 7Yamazaki T. Kay L.E. et al.An HNCA pulse scheme for the backbone assignment of 15N, 13C, 2H-labeled proteins: application to a 37-kDa trp repressor–DNA complex.J. Am. Chem. Soc. 1994; 116: 6464-6465Crossref Scopus (169) Google Scholar]. On a fully protonated protein, the very short relaxation times of the Cα spins (<20ms) would lead to a rapid decay of magnetization during the constant-time period making these experiments impractical. Similar gains in sensitivity and resolution have been achieved for other triple resonance experiments used in assigning the backbone resonances of proteins [17Yamazaki T. Lee W.L. Arrowsmith C.H. Muhandiram D.R. Kay L.E. A suite of triple resonance NMR experiments for the backbone assignment of 15N, 13C, 2H labeled proteins with high sensitivity.J. Am. Chem. Soc. 1994; 116: 11655-11666Crossref Scopus (479) Google Scholar, 18Venters R.A. Huang C.-C. Farmer II, B.T. Trolard R. Spicer L.D. Fierke C.A. High-level 2H/13C/15N labeling of proteins for NMR studies.J. Biomol. NMR. 1995; 5 (95375527): 339-344Crossref PubMed Scopus (101) Google Scholar, 19Kay L.E. Ikura M. Tschudin R. Bax A. Three-dimensional triple-resonance NMR spectroscopy of isotopically enriched proteins.J. Magn. Reson. 1990; 89: 496-514Google Scholar, 20Zhou M.-M. Fesik S.W. et al.Structure and ligand recognition of the phosphotyrosine binding domain of Shc.Nature. 1995; 378 (96097066): 584-592Crossref PubMed Scopus (323) Google Scholar, 21Shan X. Gardner K.H. Muhandiram D.R. Rao N.S. Arrowsmith C.H. Kay L.E. Assignment of 15N, 13Cα, 13Cβ, and HN resonances in an 15N, 13C, 2H labeled 64kDa trp repressor–operator complex using triple resonance NMR spectroscopy and 2H-decoupling.J. Am. Chem. Soc. 1996; 118: 6570-6579Crossref Scopus (129) Google Scholar, 22Nietlispach D. Laue E.D. et al.An approach to the structure determination of larger proteins using triple resonance NMR experiments in conjunction with random fractional deuteration.J. Am. Chem. Soc. 1996; 118: 407-415Crossref Scopus (95) Google Scholar]. These techniques have allowed the backbone assignments of larger proteins with molecular weights as high as 64kDa [[21]Shan X. Gardner K.H. Muhandiram D.R. Rao N.S. Arrowsmith C.H. Kay L.E. Assignment of 15N, 13Cα, 13Cβ, and HN resonances in an 15N, 13C, 2H labeled 64kDa trp repressor–operator complex using triple resonance NMR spectroscopy and 2H-decoupling.J. Am. Chem. Soc. 1996; 118: 6570-6579Crossref Scopus (129) Google Scholar]. After assigning the backbone resonances, the next step in the determination of protein structure by NMR is to assign the side chain 1H and 13C signals. Usually HCCH–TOCSY (H →C →C →H total correlation spectroscopy) experiments [23Fesik S.W. Eaton H.L. Olejniczak E.T. Zuiderweg E.R.P. 2D and 3D NMR spectroscopy employing 13C–13C magnetization transfer via isotropic mixing. Spin system identification in large proteins.J. Am. Chem. Soc. 1990; 112: 886-888Crossref Scopus (172) Google Scholar, 24Bax A. Clore G.M. Gronenborn A.M. 1H–1H correlation via isotropic mixing of 13C magnetization, a new three-dimensional approach for assigning 1H and 13C spectra of 13C- enriched proteins.J. Magn. Reson. 1990; 88: 425-431Google Scholar], in which side chain 1H and 13C signals are correlated with each other [[1]Clore G.M. Gronenborn A.M. Multidimensional heteronuclear nuclear magnetic resonance of proteins.Methods Enzymol. 1994; 239 (95131769): 349-363Crossref PubMed Scopus (247) Google Scholar], are used for this purpose. However, although these experiments are quite sensitive even for proteins with higher molecular weight, analyzing the spectra is complicated due to the extensive signal overlap obtained with larger proteins. In principle, side chain assignments are most easily achieved by correlating the side chain 1H and 13C chemical shifts to the well dispersed HN, N signals of the backbone amides in HC(CO)NH–TOCSY experiments [25Logan T.M. Olejniczak E.T. Xu R.X. Fesik S.W. Side chain and backbone assignments in isotopically labeled proteins from two heteronuclear triple resonance experiments.FEBS Lett. 1992; 314 (93106196): 413-418Abstract Full Text PDF PubMed Scopus (118) Google Scholar, 26Montelione G.T. Lyons B.A. Emerson S.D. Tashiro M. An efficient triple resonance experiment using carbon-13 isotropic mixing for determining sequence-specific resonance assignments of isotopically-enriched proteins.J. Am. Chem. Soc. 1992; 114: 10974-10975Crossref Scopus (268) Google Scholar, 27Grzesiek S. Anglister J. Bax A. Correlation of backbone amide and aliphatic side-chain resonances in 13C/15N-enriched proteins by isotropic mixing of 13C magnetization.J. Magn. Reson. B. 1993; 101: 114-119Crossref Scopus (569) Google Scholar]. Data analysis of these experiments is straightforward and much faster than for HCCH–TOCSY experiments. This is because side chain chemical shifts can simply be read out at the HN and N chemical shifts of the amide of the neighboring residue. However, as these experiments involve a number of magnetization transfer steps (H →C →Cα→C′ →N →HN), including transfer via the fast relaxing Cα spin, they yield poor signal-to-noise for proteins with molecular weights greater than 20kDa. In contrast, when applied to a deuterated protein, these experiments become feasible. For the side chain 13C assignments, a perdeuterated sample can be used [[28]Farmer II, B.T. Venters R.A. Assignment of side-chain 13C resonances in perdeuterated proteins.J. Am. Chem. Soc. 1995; 117: 4187-4188Crossref Scopus (62) Google Scholar]. In this case, magnetization transfer originates from the 13C spins and is transferred back to the amide protons. Alternatively, side chain 13C assignments can be obtained on a fractionally deuterated protein. For this purpose, as for the assignments of side chain proton signals, a compromise has to be found for the deuteration level with respect to the dilution of side-chain protons that determine the observable magnetization and the improvement of relaxation times by the deuteration level. In a recent study, 50 % fractional deuteration was found to optimize the sensitivity for experiments that correlate side-chain resonances with the amide protons [[22]Nietlispach D. Laue E.D. et al.An approach to the structure determination of larger proteins using triple resonance NMR experiments in conjunction with random fractional deuteration.J. Am. Chem. Soc. 1996; 118: 407-415Crossref Scopus (95) Google Scholar], allowing the use of only one sample for obtaining the side chain assignments in larger proteins. The primary parameters used to derive three-dimensional structures from NMR are the interatomic distances that are measured by nuclear Overhauser effects (NOEs). Three types of cross-peaks have to be considered in NOE experiments. These include NOEs between: nonexchangable protons (CH↔CH); amide and nonexchangable protons (NH↔CH); and amide protons (NH↔NH). In a fractionally deuterated protein, cross-peaks between nonexchangable protons are expected to have less signal-to-noise compared to a fully protonated sample, due to the dilution of the 1H spins on both the originating and destination proton. For NOE cross-peaks between fully protonated exchangeable amide protons and nonexchangable side-chain protons, the overall sensitivity observed with a fully protonated and a fractionally deuterated protein is about the same. This is because the gain in signal-to-noise from the increase of relaxation times is compensated for by the dilution of available 1H spins in a fractionally deuterated sample. However, NOEs with considerably enhanced sensitivity are observed between exchangeable HN protons on a protein with 100 % deuteration of the side-chain spin systems. Furthermore, as spin diffusion into the side chains is eliminated, longer NOE mixing times can be used, allowing NOEs corresponding to longer distances to be detected [2LeMaster D.M. Richards F.M. NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteriation.Biochemistry. 1988; 27 (88163560): 142-150Crossref PubMed Scopus (261) Google Scholar, 3Torchia D.A. Sparks S.W. Bax A. Delineation of α-helical domains in deuteriated staphylococcal nuclease by 2D NOE NMR spectroscopy.J. Am. Chem. Soc. 1988; 110: 2320-2321Crossref Scopus (76) Google Scholar, 11Tsang P. Wright P.E. Rance M. Specific deuteration strategy for enhancing direct nuclear Overhauser effects in high molecular weight complexes.J. Am. Chem. Soc. 1990; 112: 8183-8185Crossref Scopus (22) Google Scholar, 12Reisman J. Jariel-Encontre I. Hsu V.L. Parello J. Geiduschek E.P. Kearns D.R. Improving two-dimensional 1H NMR NOESY spectra of a large protein by selective deuteriation.J. Am. Chem. Soc. 1991; 113: 2787-2789Crossref Scopus (22) Google Scholar, 29Grzesiek S. Wingfield P. Stahl S. Kaufman J.D. Bax A. Four-dimensional 15N-separated NOESY of slowly tumbling perdeuterated 15N-enriched proteins. Application to HIV-1.Nef. J. Am. Chem. Soc. 1995; 117: 9594-9595Crossref Scopus (94) Google Scholar, 30Venters R.A. Metzler W.J. Spicer L.D. Mueller L. Farmer II, B.T. Use of 1HN–1HN NOEs to determine protein global folds in perdeuterated proteins.J. Am. Chem. Soc. 1995; 117: 9592-9593Crossref Scopus (99) Google Scholar]. An example of the improvements in NOE spectra of a large protein the RNA methyl transferase ErmAm 28kDa) when deuterated is shown in Figure 3. Compared to the 15N-edited NOE spectrum, acquired with the fully protonated protein (Figure 3a), many more NOE cross-peaks are observed with considerably higher signal-to-noise with the uniformly 2H- and 15N-labeled protein (Figure 3b). The increased number of NOEs between the amide protons obtained from a perdeuterated sample of ErmAm was very helpful in determining the overall fold of this protein (SWF, unpublished data). Thus, as demonstrated previously, deuteration also promises to be useful for the extraction of distance restraints for larger proteins [2LeMaster D.M. Richards F.M. NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteriation.Biochemistry. 1988; 27 (88163560): 142-150Crossref PubMed Scopus (261) Google Scholar, 3Torchia D.A. Sparks S.W. Bax A. Delineation of α-helical domains in deuteriated staphylococcal nuclease by 2D NOE NMR spectroscopy.J. Am. Chem. Soc. 1988; 110: 2320-2321Crossref Scopus (76) Google Scholar, 29Grzesiek S. Wingfield P. Stahl S. Kaufman J.D. Bax A. Four-dimensional 15N-separated NOESY of slowly tumbling perdeuterated 15N-enriched proteins. Application to HIV-1.Nef. J. Am. Chem. Soc. 1995; 117: 9594-9595Crossref Scopus (94) Google Scholar, 30Venters R.A. Metzler W.J. Spicer L.D. Mueller L. Farmer II, B.T. Use of 1HN–1HN NOEs to determine protein global folds in perdeuterated proteins.J. Am. Chem. Soc. 1995; 117: 9592-9593Crossref Scopus (99) Google Scholar]. In summary, the use of deuteration in combination with uniformly 13C- and 15N-labeling dramatically improves the quality of NMR spectra of larger proteins. These improvements allow the backbone and side-chain signals of larger proteins to be assigned and aids in the acquisition and analysis of NOE data. Using these methods, three-dimensional structures of proteins up to 30kDa have been determined [[20Zhou M.-M. Fesik S.W. et al.Structure and ligand recognition of the phosphotyrosine binding domain of Shc.Nature. 1995; 378 (96097066): 584-592Crossref PubMed Scopus (323) Google Scholar, 31Muchmore S.W. Fesik S.W. et al.X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death.Nature. 1996; 381: 335-341Crossref PubMed Scopus (1266) Google Scholar, 32Sattler M. Fesik S.W. et al.Structure of Bcl-xL–Bak peptide complex reveals how regulators of apoptosis interact.Science. 1996; : in pressGoogle Scholar] SWF, unpublished data]. Further developments of NMR methods and variation of labeling techniques promise to push the molecular weight limit even further.