Use of deuterium labeling in NMR: overcoming a sizeable problem Michael Sattler and Stephen W Fesik*

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1 Ways & Means 1245 Use of deuterium labeling in NMR: overcoming a sizeable problem Michael Sattler and Stephen W Fesik* Address: Abbott Laboratories, 47G AP10,100, Abbott Park Road, Abbott Park, IL , USA. *Corresponding author. fesiks@steves.abbott.com Structure 15 November 1996, 4: Current Biology Ltd ISSN 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 13 C- and 15 N-labeled proteins, which has dramatically improved the applicability of NMR to the study of larger proteins (MW < 20 ka) [1]. owever, two main challenges remain for the structure determination of even larger proteins (MW >20ka) 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 (Fig. 1a) and by decreasing the relaxation rates of 13 C and 15 N spins (Fig. 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 [2,3]. Another use of deuteration is to reduce the dipolar interaction between 13 C or 15 N and the directly bound proton spin which is the main source of relaxation in 13 C- and 15 N-labeled proteins [4,5]. ue to the significantly smaller gyromagnetic ratio ( ) of the deuterium spin ( ~1/6.5 ), the relaxation rates are scaled proportional to ( / ) 2 ~0.02. Therefore, the relaxation times of 13 C and 15 N 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]. Another advantage of the increased relaxation times obtained upon deuteration is that constant-time experiments (which yield poor signalto-noise with fully protonated proteins) can be applied with high sensitivity [7]. 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. Preparation of deuterium labeled proteins Various strategies have been developed for deuterating proteins (for a review see [8]). These strategies include methods for the specific deuteration of selected residue types or of selected positions in aromatic side chains [8 14], random fractional deuteration [2,3,15 17], and the complete deuteration of nonexchangable protons [3,18]. One of the most useful approaches is random fractional deuteration, especially when combined with uniform 13 C- and 15 N- labeling. Fractionally deuterated and uniformly 13 C-, 15 N-labeled proteins are easily prepared. To do this, bacteria that overexpress the protein of interest are grown in a minimal medium containing either uniformly 13 C-labeled sodium acetate [18] or glucose [17] and 15 N-labeled ammonium chloride in water containing the desired amount of Figure 1 N 1 13C 1 γ /γ ~ 1/6.5 Theoretical considerations for the use of deuterium labeling. Suppression of spin diffusion by fractional deuteration of side chains in larger proteins. ue to the elimination of competing relaxation pathways in the network of dipolar coupled protons, relaxation rates of the remaining protons (e.g. N ) are greatly reduced which leads to smaller linewidths and higher signal-to-noise. The dipolar interaction between 13 C or 15 N and an attached proton, which is proportional to the square of the gyromagnetic ratios, is significantly reduced by replacing the proton with deuterium leading to smaller relaxation rates. 2 13C N 0.02

2 1246 Structure 1996, Vol 4 No 11 Figure 2 13 C α N 10.0 N N,C projections from three-dimensional NCA experiments. The projections were obtained from spectra recorded on: uniformly [ 15 N, 13 C]-labeled Shc phosphotyrosine-binding (PTB) domain and 75 % 2, uniformly [ 15 N, 13 C]-labeled Shc PTB domain complexed with an unlabeled tyrosine-phosphorylated 12-mer peptide. 2 O. In order to optimize the expression of the protein, cell cultures can be adapted to grow in 2 O by increasing the amount of 2 O in the growth medium from zero to the desired percentage with each growth cycle. owever, in practice this is only necessary to obtain deuteration levels of greater than 75%. Thus, deuterated and uniformly 13 C- and 15 N-labeled proteins are readily obtained with only slight modifications of the procedures used to generate uniformly 13 C- and 15 N-labeled proteins. Assignment of the backbone resonances The first step in the structure determination of proteins by NMR is the assignment of the 1, 13 C and 15 N 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]. One of the experiments, for example, is a three-dimensional NCA experiment [19] in which the chemical shifts of the amide proton ( N ) and nitrogen (N) are correlated to the chemical shift of the C spin ( N C ). 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-tonoise, especially for proteins greater than 20ka in molecular weight. An example of the advantages of using fractionally deuterated versus fully protonated 13 C-, 15 N-labeled proteins in the triple resonance experiments is shown in Figure 2. Projections of three-dimensional NCA experiments are shown that were recorded on the 23ka complex of the Shc phosphotyrosine-binding (PTB) domain (190 residues) complexed to a 12-residue tyrosine phosphorylated peptide [20]. In these experiments the 13 C-, 15 N-labeled protein was fully protonated (Fig. 2a) or fractionally deuterated (Fig. 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 N, N and C spins due to the fractional deuteration. The increase in signal-tonoise is especially pronounced for the C spins, which have very fast relaxation rates due to the dipolar coupling with the directly bound proton. The downscaling of this interaction upon substituting the by deuterium, leads to a large increase in the C relaxation times. For example, in NMR studies of a 37ka Trp repressor NA complex, it has recently been demonstrated, that the C relaxation times increase from 16.5ms (100% 1 ) to 130ms (70% 2 ) [7]. Moreover, due to this improvement in sensitivity, NMR experiments that employ constant-time periods for the chemical shift evolution can be used which

3 Ways & Means euterium labeling in NMR Sattler and Fesik 1247 Figure 3 N N N Two-dimensional N, N NOESY planes. Results obtained on an 15 N-labeled RNA methyltransferase, ErmAm, with a mixing time of 80 ms and uniformly 2 -, 15 N-labeled ErmAm with a mixing time of 200 ms. 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 NCA spectrum shown in Figure 2a, C chemical shifts are recorded during a constant-time period of 1/ 1 J(C,C )~28ms in order to refocus the undesired 1 J(C,C ) coupling. 2 decoupling is applied during the constant-time period [4,5,7]. On a fully protonated protein, the very short relaxation times of the C 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 [17 22]. These techniques have allowed the backbone assignments of larger proteins with molecular weights as high as 64ka [21]. Assignment of the side chain resonances After assigning the backbone resonances, the next step in the determination of protein structure by NMR is to assign the side chain 1 and 13 C signals. Usually CC TOCSY ( C C total correlation spectroscopy) experiments [23,24], in which side chain 1 and 13 C signals are correlated with each other [1], are used for this purpose. owever, 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 1 and 13 C chemical shifts to the well dispersed N,N signals of the backbone amides in C(CO)N TOCSY experiments [25 27]. ata analysis of these experiments is straightforward and much faster than for CC TOCSY experiments. This is because side chain chemical shifts can simply be read out at the N and N chemical shifts of the amide of the neighboring residue. owever, as these experiments involve a number of magnetization transfer steps ( C C C N N ), including transfer via the fast relaxing C spin, they yield poor signal-to-noise for proteins with molecular weights greater than20ka. In contrast, when applied to a deuterated protein, these experiments become feasible. For the side chain 13 C assignments, a perdeuterated sample can be used [28]. In this case, magnetization transfer originates from the 13 C spins and is transferred back to the amide protons. Alternatively, side chain 13 C 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

4 1248 Structure 1996, Vol 4 No 11 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], allowing the use of only one sample for obtaining the side chain assignments in larger proteins. istance restraints 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 (C C); amide and nonexchangable protons (N C); and amide protons (N N). 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 1 spins on both the originating and destination proton. For NOE cross-peaks between fully protonated exchangeable amide protons and nonexchangable sidechain 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 1 spins in a fractionally deuterated sample. owever, NOEs with considerably enhanced sensitivity are observed between exchangeable N 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 [2,3,11,12,29,30]. An example of the improvements in NOE spectra of a large protein the RNA methyl transferase ErmAm 28 ka) when deuterated is shown in Figure 3. Compared to the 15 N-edited NOE spectrum, acquired with the fully protonated protein (Fig. 3a), many more NOE cross-peaks are observed with considerably higher signal-to-noise with the uniformly 2 - and 15 N- labeled protein (Fig. 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 [2,3,29,30]. Conclusions and perspectives In summary, the use of deuteration in combination with uniformly 13 C- and 15 N-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, threedimensional structures of proteins up to 30 ka have been determined [20,31,32; SWF, unpublished data]. Further developments of NMR methods and variation of labeling techniques promise to push the molecular weight limit even further. References 1. Clore, G.M. & Gronenborn, A.M. (1994). Multidimensional heteronuclear nuclear magnetic resonance of proteins. Methods Enzymol. 239, LeMaster,.M. & Richards, F.M. (1988). NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteriation. Biochemistry 27, Torchia,.A., Sparks, S.W. & Bax, A. (1988). elineation of -helical domains in deuteriated staphylococcal nuclease by 2 NOE NMR spectroscopy. J. Am. Chem. Soc. 110, Browne,.T., Kenyon, G.L., Packer, E.L., Sternlicht,. & Wilson,.M. (1973). Studies of macromolecular structure by 13 C nuclear magnetic resonance. II. A specific labeling approach to the study of histidine residues in proteins. J. Am. Chem. Soc. 95, Grzesiek, S., Anglister, J., Ren,. & Bax, A. (1993). 13 C line narrowing by 2 decoupling in 2 / 13 C/ 15 N-enriched proteins. Application to triple resonance 4 J connectivity of sequential amides. J. Am. Chem. Soc. 115, Kushlan,.M. & LeMaster,.M. (1993). Resolution and sensitivity enhancement of heteronuclear correlation for methylene resonances via 2 enrichment and decoupling. J. Biomol. NMR 3, Yamazaki, T., et al., & Kay, L.E. (1994). An NCA pulse scheme for the backbone assignment of 15 N, 13 C, 2 -labeled proteins: application to a 37-ka trp repressor NA complex. J. Am. Chem. Soc. 116, LeMaster,.M. (1994). Isotope labeling in solution protein assignment and structural analysis. Prog. NMR Spectrosc. 26, Crespi,.L., Rosenberg, R.M. & Katz, J.J. (1968). Proton magnetic resonance of proteins fully deuterated except for 1 -leucine side chains. Science 161, Markley, J.L., Putter, I. & Jardetzky, O. (1968). igh-resolution nuclear magnetic resonance spectra of selectively deuterated staphylococcal nuclease. Science 161, Tsang, P., Wright, P.E. & Rance, M. (1990). Specific deuteration strategy for enhancing direct nuclear Overhauser effects in high molecular weight complexes. J. Am. Chem. Soc. 112, Reisman, J., Jariel-Encontre, I., su, V.L., Parello, J., Geiduschek, E.P. & Kearns,.R. (1991). Improving two-dimensional 1 NMR NOESY spectra of a large protein by selective deuteriation. J. Am. Chem. Soc. 113, Arrowsmith, C.., Pachter, R., Altman, R.B., Iyer, S.B. & Jardetzky, O. (1990). Sequence-specific 1 NMR assignments and secondary structure in solution of Escherichia coli trp repressor. Biochemistry 29, Metzler, W.J., Wittekind, M., Goldfarb, V., Mueller, L. & Farmer II, B.T. (1996). Incorporation of 1 / 13 C/ 15 N-[Ile, Leu, Val] into a perdeuterated, 15 N-labeled protein: potential in structure determination of large proteins by NMR. J. Am. Chem. Soc. 118, Shon, K.-J., Kim, Y., Colnago, L.A. & Opella, S.J. (1991). NMR studies of the structure and dynamics of membrane-bound bacteriophage Pf1 coat protein. Science 252, Kalbitzer,.R., Leberman, R. & Wittinghofer, A. (1985). 1 -NMR spectroscopy on elongation factor Tu from Escherichia coli. FEBS Lett. 180, Yamazaki, T., Lee, W.L., Arrowsmith, C.., Muhandiram,.R. & Kay, L.E. (1994). A suite of triple resonance NMR experiments for the backbone assignment of 15 N, 13 C, 2 labeled proteins with high sensitivity. J. Am. Chem. Soc. 116, Venters, R.A., uang, C.-C., Farmer II, B.T., Trolard, R., Spicer, L.. & Fierke, C.A. (1995). igh-level 2 / 13 C/ 15 N labeling of proteins for NMR studies. J. Biomol. NMR 5, Kay, L.E., Ikura, M., Tschudin, R. & Bax, A. (1990). Three-dimensional triple-resonance NMR spectroscopy of isotopically enriched proteins. J. Magn. Reson. 89, Zhou, M.-M., et al., & Fesik, S.W. (1995). Structure and ligand recognition of the phosphotyrosine binding domain of Shc. Nature 378, Shan, X., Gardner, K.., Muhandiram,.R., Rao, N.S., Arrowsmith, C.. & Kay, L.E. (1996). Assignment of 15 N, 13 C, 13 C, and N resonances in an 15 N, 13 C, 2 labeled 64 ka trp repressor operator complex using triple resonance NMR spectroscopy and 2 -decoupling. J. Am. Chem. Soc. 118,

5 22. Nietlispach,., et al., & Laue, E.. (1996). An approach to the structure determination of larger proteins using triple resonance NMR experiments in conjunction with random fractional deuteration. J. Am. Chem. Soc. 118, Fesik, S.W., Eaton,.L., Olejniczak, E.T. & Zuiderweg, E.R.P. (1990). 2 and 3 NMR spectroscopy employing 13 C 13 C magnetization transfer via isotropic mixing. Spin system identification in large proteins. J. Am. Chem. Soc. 112, Bax, A., Clore, G.M. & Gronenborn, A.M. (1990). 1 1 correlation via isotropic mixing of 13 C magnetization, a new three-dimensional approach for assigning 1 and 13 C spectra of 13 C-enriched proteins. J. Magn. Reson. 88, Logan, T.M., Olejniczak, E.T., Xu, R.X. & Fesik, S.W. (1992). Side chain and backbone assignments in isotopically labeled proteins from two heteronuclear triple resonance experiments. FEBS Lett. 314, Montelione, G.T., Lyons, B.A., Emerson, S.. & Tashiro, M. (1992). An efficient triple resonance experiment using carbon-13 isotropic mixing for determining sequence-specific resonance assignments of isotopically-enriched proteins. J. Am. Chem. Soc. 114, Grzesiek, S., Anglister, J. & Bax, A. (1993). Correlation of backbone amide and aliphatic side-chain resonances in 13 C/ 15 N-enriched proteins by isotropic mixing of 13 C magnetization. J. Magn. Reson. B 101, Farmer II, B.T. & Venters, R.A. (1995). Assignment of side-chain 13 C resonances in perdeuterated proteins. J. Am. Chem. Soc. 117, Grzesiek, S., Wingfield, P., Stahl, S., Kaufman, J.. & Bax, A. (1995). Four-dimensional 15N-separated NOESY of slowly tumbling perdeuterated 15 N-enriched proteins. Application to IV-1 Nef. J. Am. Chem. Soc. 117, Venters, R.A., Metzler, W.J., Spicer, L.., Mueller, L. & Farmer II, B.T. (1995). Use of 1 N 1 N NOEs to determine protein global folds in perdeuterated proteins. J. Am. Chem. Soc. 117, Muchmore, S.W., et al., & Fesik, S.W. (1996). X-ray and NMR structure of human Bcl-x L, an inhibitor of programmed cell death. Nature 381, Sattler, M., et al., & Fesik, S.W. (1996). Structure of Bcl-x L Bak peptide complex reveals how regulators of apoptosis interact. Science, in press. Ways & Means euterium labeling in NMR Sattler and Fesik 1249

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