NMR supplement The second decade into the third millenium Kurt Wuthrich NMR spectroscopy is one of the principal experimental techniques of structural biology, with abilities to determine atomic resolution structures as well as investigate dynamics and intermolecular interactions of biological macromolecules. There is plenty of room for continued progress of this young branch of science, based on further technical advances as well as innovative funding strategies and project organization In the first Nature Structural Biology spe- number of three-dimensional structures used for quantitative characterization of cial issue on nuclear magnetic resonance of proteins and nucleic acids that were structural and dynamic aspects of protei spectroscopy(NMR), Gerhard Wagner known at the end of 1989, so that we now and nucleic acid hydration in solution. recounted the development of NMr have an extensive data base of three- NMR data on dynamics and solvation from 1946 to the present!. The history of dimensional structures in the Brookhaven have so far in most instances been collect- NMR includes, besides spectacular Protein Data Bank. ed with small proteins and nucleic acid achievements in physics, chemistry, Impressive testimony to the fragments, but this fundamental informa- materials science and medicine2 the recent evolution of structural biol tion applies to any molecular size and can determination of the complete three- numbers in Table I are only a partial be used for the interpretation of crystal dimensional structure of a globular reflection of the increasing prominence of structures with regard to structure-fund protein in solution in 1984 and subse- NMR, since in addition to structure deter- tion correlations in physiological fluids quently the evolution of NMr into a key mination this technique can provide a However, although NMR data on molecu technique for structural biolo lar dynamics have long attracted gy. The achievements of NMR with biological macromole 231 keen interest by theoreticians o the inherent complexities of cules reviewed by Wagner and dynamic macromolecular struc the conditions under which lures have so far limited the use hey have been attained repre of this information in the prac sent the platform for future of analyzing biochemical dvances, which are the prime measurements and designing focus of this article ovel projects. In a certain sense these nmr data - on A remarkable field dynamics and solvation The increasingly important role appear to be ahead of their that NMR plays in structural time: here, then, is an open biology is illustrated by Table 1 avenue for the future. ver which lists the number of novel recent observations also indi NMR structures of proteins and cate that our view of internal nucleic acids published annually Fig. 1 NMR structure of the recombinant murine prion protein In a mobility of proteins may during the period 1990-1996. In first step the structure of the fragment 121-231(which includes a still be largely incomplete and he table these numbers are fur- globular domain from residues 126-2263) olved. In the intact that additional insight on ther placed in perspective by coil, where high mobility of the individual residues is manifested by low frequency motions can sponding data for the other (adapted from ref. 20. mes shorter than 1 ns for the 15N-'H groups be anticipated from novel comparison with the corre- rotation techniques of structure determi reticula Iso from the nation. It is quite obvious that residual anisotrol so far, nearly all atomic resolution struc- wealth of supplementary, unique tions reviewed by Prestegard is in his tures of biological macromolecules have mation that is complementary to contribution to this volume been solved either by X-ray diffraction in structure datas. Wagner presented a sur- NMR spectroscopy is special among the single crystals or by NMR in solution, vey on NMR characterization of molecu- techniques of structural biology in its abil where the number of new crystal struc- lar dynamics of proteins and nucleic acids ity to observe and characterize unfolded tures published exceeds the number of in solution, and the present issue includes polypeptide chains in solution 6. new NMR structures about three-to four- a further thorough review of this area by Applications of interest to the protein foldThe harvest of new structures in 1996 Kays. In addition, direct observation of folding problem'are discussed in the pre exceeds the corresponding numbers for nuclear Overhauser effects (NOE) sent NMR special issue in articles by 1990 by more than four-fold. From 1994 between water protons and macromolecu- Dyson and Wrights, and by Dobson and onward the total number of new struc- lar hydrogen atomsand measurement of Horel9. In addition, recent structure tures published per year exceeds the total nuclear magnetic relaxation dipersion are determinations revealed the existence of nature structural biology . NMR supplement. july 1998
NMR supplement 492 nature structural biology • NMR supplement • july 1998 used for quantitative characterization of structural and dynamic aspects of protein and nucleic acid hydration in solution. NMR data on dynamics and solvation have so far in most instances been collected with small proteins and nucleic acid fragments, but this fundamental information applies to any molecular size and can be used for the interpretation of crystal structures with regard to structure-function correlations in physiological fluids9. However, although NMR data on molecular dynamics have long attracted keen interest by theoreticians10,11, the inherent complexities of dynamic macromolecular structures have so far limited the use of this information in the practice of analyzing biochemical measurements and designing novel projects. In a certain sense these NMR data — on dynamics and solvation — appear to be ahead of their time: here, then, is an open avenue for the future. Very recent observations also indicate that our view of internal mobility of proteins may still be largely incomplete and that additional insight on low frequency motions can be anticipated from novel NMR approaches12–14, in particular also from the residual anisotropic interactions reviewed by Prestegard15 in his contribution to this volume. NMR spectroscopy is special among the techniques of structural biology in its ability to observe and characterize unfolded polypeptide chains in solution16,17. Applications of interest to the ‘protein folding problem’ are discussed in the present NMR special issue in articles by Dyson and Wright18, and by Dobson and Hore19. In addition, recent structure determinations revealed the existence of In the first Nature Structural Biology special issue on nuclear magnetic resonance spectroscopy (NMR), Gerhard Wagner recounted the development of NMR from 1946 to the present1. The history of NMR includes, besides spectacular achievements in physics, chemistry, materials science and medicine2, the determination of the complete threedimensional structure of a globular protein in solution in 19843 and subsequently the evolution of NMR into a key technique for structural biology. The achievements of NMR with biological macromolecules reviewed by Wagner1 and the conditions under which they have been attained represent the platform for future advances, which are the prime focus of this article. A remarkable field The increasingly important role that NMR plays in structural biology is illustrated by Table 1, which lists the number of novel NMR structures of proteins and nucleic acids published annually during the period 1990–1996. In the table these numbers are further placed in perspective by comparison with the corresponding data for the other techniques of structure determination. It is quite obvious that, so far, nearly all atomic resolution structures of biological macromolecules have been solved either by X-ray diffraction in single crystals or by NMR in solution4, where the number of new crystal structures published exceeds the number of new NMR structures about three- to fourfold. The harvest of new structures in 1996 exceeds the corresponding numbers for 1990 by more than four-fold. From 1994 onward the total number of new structures published per year exceeds the total number of three-dimensional structures of proteins and nucleic acids that were known at the end of 1989, so that we now have an extensive data base of threedimensional structures in the Brookhaven Protein Data Bank. Impressive testimony to the amazing recent evolution of structural biology, the numbers in Table 1 are only a partial reflection of the increasing prominence of NMR, since in addition to structure determination this technique can provide a wealth of supplementary, unique information that is complementary to crystal structure data5. Wagner1 presented a survey on NMR characterization of molecular dynamics of proteins and nucleic acids in solution, and the present issue includes a further thorough review of this area by Kay6. In addition, direct observation of nuclear Overhauser effects (NOE) between water protons and macromolecular hydrogen atoms7 and measurement of nuclear magnetic relaxation dipersion8 are The second decade — into the third millenium Kurt Wüthrich NMR spectroscopy is one of the principal experimental techniques of structural biology, with abilities to determine atomic resolution structures as well as investigate dynamics and intermolecular interactions of biological macromolecules. There is plenty of room for continued progress of this young branch of science, based on further technical advances as well as innovative funding strategies and project organization. Fig. 1 NMR structure of the recombinant murine prion protein. In a first step the structure of the fragment 121–231 (which includes a globular domain from residues 126–22638) was solved. In the intact protein with residues 23–231, the segment 23–126 forms an extended coil, where high mobility of the individual residues is manifested by rotational correlation times shorter than 1 ns for the 15N-1H groups (adapted from ref. 20)
NMR supplement △v(15N) mes of t- 20, 60 and 320 ns [HzI 320 320 and Ad('H)- 15 p.p. m. axial Tc(ns T(ns ymmetry was assumed for assumed to be15°for 60 10 20 side of the 15N-H moiety were not considered 0 50010001500 50010001500 VCH MHz] V(H)[MHz native, folded proteins that contain long tures in the healthy cell. Flexible polypep- on dissection of their modular architec flexible coils attached to well structured tide coils attached to globular domains ture, which is an approach used in a wide the prion protein(Fig. 1), with a globular future NMR studies, considering that their well as X-ray diffraction. Finally, the solid domain containing a-helical and B-sheet characterization by alternative methods is state NMR techniques presented by secondary structure, and an N-terminal very limited and NMR has the potential Griffin I in this issue carry the promise of domain of nearly equal size that forms a for further refinement of this class of information on very large systemS TROSY this extended coil exceeds the diameter structures mobile extended coiled The lens a new NMR (transverse relaxation-optimized spec of the globular domain by almost ten-fold. Large molecules oscopy)32, promises a further several- A similar structure consisting of a globular The title"NMRstructures.beyond 20,000 fold increase of the molecular size domain and a flexibly extended coil has Mr"(M,=relative molecular mass)used by accessible with solution NMR. TROSY been observed for a yeast heat-shock tran- Clore and gronenborn" in the first Nature makes use of the fact that at ' H frequen scription factor2l. Considering the practi- Structural Biology special issue on NMR cies in the range 900-1,000 MHz nearly cal difficulties in preparing proteins with reflects on a stigma that has traditionally complete cancellation of transverse relax long extended coils for structural studies, been attached to solution NMR, that is"its ation effects can be achieved for one of the one is tempted to speculate that this struc- use is limited to small molecular sizes". four multiplet components observed for ure type has so far largely escaped detailed Actually, during the last few years the size N-H moieties 2), and TROSY characterization and may be quite com- limit for de novo protein structure determin- exclusively observes this narrow comp mon in nature. In this context it is interest- ation has been moved to about 30,000 M, by nent. Fig. 3 shows that already at 750 ing that the crystal structure of the the use of stable isotope labeling with 13C, MHz, TROSY yields significantly narrow nucleosome core particle contains 15N and 2H2425 combined with the use of er spectral linewidths and improved sensi numerous extended polypeptide segments triple-resonance experiments2 z7 and het- tivity for observation of 5N-H groups in in the multimolecular aggregate, indicat- eronuclear-resolved2s and -edited 29 NMR. an oligomeric protein of 110,000 M, than ing that formation of the oligomeric struc- More important ture may start with subunits that contain there is no Table 1 Annual publications of novel atomic resolution sizeable extended coils. The existence of a priori rigid size structures of proteins and nucleic acids(ref. 4) extended polypeptide coils under physio- barrier: As men- logical conditions is expected to depend on tioned above, much Method protective chaperoning by other of the NMr data X-ray crystallography NMR other methods macromolecules. The assembly of internal mobility (single crystals (solution) (crystals, fibers formation of the nucleosome core particle, to any molecular 1991 or processes such as those leading to dis- size. In the article 207 994 thus be governed in subtle ways by chaper- Downing NMR 1995 one systems that ensure maintenance and studies with very big 1996 112 controlled release of the flexible coil struc proteins are based nature structural biology. NMR supplement. july 1998
NMR supplement nature structural biology • NMR supplement • july 1998 493 on dissection of their modular architecture, which is an approach used in a wide variety of structural studies by NMR as well as X-ray diffraction. Finally, the solid state NMR techniques presented by Griffin31 in this issue carry the promise of information on very large systems. A new NMR experiment, TROSY (transverse relaxation-optimized spectroscopy)32, promises a further severalfold increase of the molecular size accessible with solution NMR. TROSY makes use of the fact that at 1H frequencies in the range 900–1,000 MHz nearly complete cancellation of transverse relaxation effects can be achieved for one of the four multiplet components observed for 15N-1H moieties (Fig. 2), and TROSY exclusively observes this narrow component. Fig. 3 shows that already at 750 MHz, TROSY yields significantly narrower spectral linewidths and improved sensitivity for observation of 15N-1H groups in an oligomeric protein of 110,000 Mr than native, folded proteins that contain long flexible coils attached to well structured globular domains. A striking example is the prion protein (Fig. 1), with a globular domain containing α-helical and β-sheet secondary structure, and an N-terminal domain of nearly equal size that forms a highly mobile extended coil20. The length of this extended coil exceeds the diameter of the globular domain by almost ten-fold. A similar structure consisting of a globular domain and a flexibly extended coil has been observed for a yeast heat-shock transcription factor21. Considering the practical difficulties in preparing proteins with long extended coils for structural studies, one is tempted to speculate that this structure type has so far largely escaped detailed characterization and may be quite common in nature. In this context it is interesting that the crystal structure of the nucleosome core particle22 contains numerous extended polypeptide segments in the multimolecular aggregate, indicating that formation of the oligomeric structure may start with subunits that contain sizeable extended coils. The existence of extended polypeptide coils under physiological conditions is expected to depend on protective chaperoning by other macromolecules. The assembly of oligomeric structures — for example, the formation of the nucleosome core particle, or processes such as those leading to disease-related amyloid formation — could thus be governed in subtle ways by chaperone systems that ensure maintenance and controlled release of the flexible coil structures in the healthy cell. Flexible polypeptide coils attached to globular domains could well become yet another focus for future NMR studies, considering that their characterization by alternative methods is very limited and NMR has the potential for further refinement of this class of structures. Large molecules The title “NMR structures ... beyond 20,000 Mr” (Mr = relative molecular mass) used by Clore and Gronenborn23 in the first Nature Structural Biology special issue on NMR reflects on a stigma that has traditionally been attached to solution NMR, that is “its use is limited to small molecular sizes”. Actually, during the last few years the size limit for de novo protein structure determination has been moved to about 30,000 Mr by the use of stable isotope labeling with 13C, 15N and 2H24,25 combined with the use of triple-resonance experiments26,27 and heteronuclear-resolved28 and -edited29 NMR. More importantly, there is no a priori rigid size barrier: As mentioned above, much of the NMR data on internal mobility and hydration apply to any molecular size. In the article by Campbell and Downing30 NMR studies with very big proteins are based Fig. 2 Frequency dependence from 100–1800 MHz of the full resonance line width at half height for amide groups in TROSY experiments calculated for three correlation times of τc = 20, 60 and 320 ns, which represent spherical proteins with molecular weights of 50,000, 150,000 and 800,000 Mr. a, 1HN linewidth. b, 15N linewidth. (The calculation used ∆σ(15N) = 155 p.p.m. and ∆σ(1HN) = 15 p.p.m.; axial symmetry was assumed for both tensors; the angle between the principal tensor axis and the N–H bond was assumed to be 15° for 15N and 10° for 1HN; dN–H = 0.104 nm; effects of long-range dipoledipole couplings with spins outside of the 15N-1H moiety were not considered. Table 1 Annual publications of novel atomic resolution structures of proteins and nucleic acids (ref. 4) Method X-ray crystallography NMR Other methods Year (single crystals) (solution) (crystals, fibers) 1990 109 23 2 1991 123 36 - 1992 168 61 - 1993 207 59 - 1994 352 100 2 1995 394 102 - 1996 461 112 - a b
NMR supplement a TRoSY onN)/b Cosy Ippm] 115 襄B B ◆C C 125 O2(H) [ppm] 2(H Ippm C a"(15N) w MW wOwa b'(H) 938.97.874 115112125123125122 CO2(H) Ippm cO2(5N)Ippm eopterin aldolase from Staphylococcus aureus in H2O solution. This protein is a homo-octamer with subunits of 121 amino acid residues. a, TRO HSoC. b Conventional HSoC. monomer concentration 0. 4 mM, PH 5.5, T 20C, ' H frequency 750 MHz, recording time per spectrum 1 h). In both dimensions the positions of esponding peaks in(a) and (b) differ by J(5N, 'H) the corresponding, conventional NMR thus extend the use of SAr by NMr33 number of NMr groups, and that X-ray experiments (Fig. 2)predicts (where SAR stands for'structure-activity crystallography has already made the ster that TROSY experiments with suitably relationships) to larger systems. The tobig science: in 1996 the majority of X isotope-labeled systems can yield infor- TROSY principle is readily applicable for ray structure determinations, including mative data on proteins in particles with improvement of a wide variety of more essentially all structures with M, above molecular weights of several hundred complex NMR experiments, also with 100,000, made use of high intensity syn- thousand M, such as membrane proteins aromatic 13C-H groups34 hrotron X-ray sources in addition to lubilized in micelles or lipid vesicles, local X-ray equipment. In contrast, the proteins attached to nucleic acid frag- Big science NMR ents or homo-oligomer ric proteins. For When comparing the contributions from small science, with the groups being example, direct correlation experiments X-ray crystallography and NMR in Table equipped with commercial spectrom- of the kind shown in Fig 3 enable chemi- 1, one has to consider that the number of eters and working individually on the cal shift mapping of intermolecular con- research groups in macromolecular crys- preparation of isotope-labeled proteins tacts in very large aggregates, and will tallography exceeds by a large margin the and nucleic acids nature structural biology . NMR supplement. july 1998
NMR supplement 494 nature structural biology • NMR supplement • july 1998 number of NMR groups, and that X-ray crystallography has already made the step to ‘big science’: in 1996 the majority of Xray structure determinations, including essentially all structures with Mr above 100,000, made use of high intensity synchrotron X-ray sources in addition to local X-ray equipment4. In contrast, the NMR community is oriented to doing ‘small science’, with the groups being equipped with commercial spectrometers and working individually on the preparation of isotope-labeled proteins and nucleic acids. the corresponding, conventional NMR experiments. Theory (Fig. 2) predicts that TROSY experiments with suitably isotope-labeled systems can yield informative data on proteins in particles with molecular weights of several hundred thousand Mr, such as membrane proteins solubilized in micelles or lipid vesicles, proteins attached to nucleic acid fragments, or homo-oligomeric proteins. For example, direct correlation experiments of the kind shown in Fig. 3 enable chemical shift mapping of intermolecular contacts in very large aggregates, and will thus extend the use of SAR by NMR33 (where SAR stands for ‘structure-activity relationships’) to larger systems. The TROSY principle is readily applicable for improvement of a wide variety of more complex NMR experiments, also with aromatic 13C-1H groups34. Big science NMR When comparing the contributions from X-ray crystallography and NMR in Table 1, one has to consider that the number of research groups in macromolecular crystallography exceeds by a large margin the Fig. 3 Comparison of conventional and TROSY-type 15N-1H correlation spectra of the uniformly 15N- and 2H-labeled 110,000 Mr protein 7,8-dihydroneopterin aldolase from Staphylococcus aureus in H2O solution. This protein is a homo-octamer with subunits of 121 amino acid residues. a, TROSYHSQC. b, Conventional HSQC. c, a’(1H) and b’(1H) show cross sections along the 1H frequency axis ω2 through three peaks identified by upper case letters in (a) and (b) respectively. d, a”(15N) and b”(15N) show cross sections along the 15N frequency axis ω1 through the same three peaks. (Protein monomer concentration 0.4 mM, pH 5.5, T 20 °C, 1H frequency 750 MHz, recording time per spectrum 1 h). In both dimensions the positions of corresponding peaks in (a) and (b) differ by 1J(15N,1H)/2. a b c d
NMR supplement Introduction of the traits of big science to high-throughput, high-quality structure 1. Wagner, G Nature Struct. Biol. 4. 841-844(1997) biomacromolecular NMR would mean the determination. Facilities operating at nuclear magnetic resonar loss of the opportunity to perform all the highest fields would, however, also initiate steps of a structure determination in the further development of spectroscopic.Bio. 182.295-315(1985) individual laboratories, a comfort that most techniques for the benefit of continued of us presently enjoy. In return we would get progress of biomolecular NMR romolecular structure access to NMR centers with more powerful 5. Wuthrich, K. MMR in structural biology (World equipment than the individual researcher The future Struct Bio. 5, 514-517(1 could afford, as well as support from Considering that the current use of NMR 7 otting G, Liepinsh, E. Wuthrich, K Science 254 specialized in efficient preparation of signs of a young, emerging field chemistry and microbiology laboratories in structural biology still shows typical 8.venu isotope-labeled precursor molecules and research, the results obtained (Table 1) are 9. Wuthrich, K.Acta Crystallogr. D,249-270 production of NMR quantities of labeled truly remarkable and are an encourage- 10. Gel in, B. karplus M. Proc. Nat. Acad. Sci. USA macromolecules In a successful big science scenario the macromolecules could experience an Wu to be comparable in function(if not in size) tralized high-performance facilities for prestegard, .H Nature Struct. Biol. 4. 292-297 with the high intensity light sources of the data collection, which have recently been 13 Tjandra, N.& Bax, A. Science 278. 1111-111 tion that significantly outperforms the USA 35. These projects are intimately relat- Pale r, A: G. Nature stranct, Bio. 5. 55-59 1998). equipment likely to be available to individ- genome sequencing projects, and 15 Prestegard, J.H. Nature Struct. Biol. 5.518-52 al research groups. (This demand con- envisage determination of a comprehen-16.Wuthrich, K.Curr.Opin.Struct.Biol.4.93-99 trasts with the present situation, where the sive set of all naturally occurring protein 1-no06l" D.R. Curr.Opin. Struct.Biol.6,24-30 uipment of regional and national nmr folds 5, 37. NMR will have an indispensable centers consists of spectrometers identical role in any such ambitious venture since 18 99-503(199 to those available in the laboratories of most experience indicates that a sizeable frac- 19. Dobson,C& Hore. PJ. Nature Struct. Biol. 5 of the leading individual research groups. tion of all proteins will not be amenable to an 504-507(1998) and efficiency of NMR structure determi- information. Considering the current rate D. Richmond, T. Nature 389, 251-260 ation, including structure refinement of progress in structural biology (Table 1)23. Clore, G.M.& Gronenborn, A.M. Nature Struc Much of the aforementioned demands performance Nmr centers should be 24 toaster, D.M. rog: MR Specr. 26, 371-419 for improved equipment in big science encouraged by the necessary innovations 25. Kainosho, M. Nature Struct. Bio. 4. 858-86 NMR centres could probably be met by in funding 26. Bax, A& Grzesiek, s AcC. Chem. Res. 26, 131-138 improved performance at the presently available field strengths corresponding to 27. Kay, L.E.& Gardner, K.H. Curr Opin Struct Biol. 'H resonance frequencies up to 800 MHz, Acknowledgments omzerischer Nationalfonds is uiderweg, E.R.P. J. Magn. Reson. 78 which can be anticipated from on-site con- gratefully acknowledged. The author thanks 588-593(198 29. Otting. G.& Wuthrich, K. Q. Rev. Biophys. 23. struction of specialized NMR equipment K Pervushin, R Riek and G Wider for the and from operation in a suitably shielded preparation of the figures and for discussions ning. A.K. Nature Struct. experience in general and more specifically Kurt Wuthrich is at the Institut Proc. Nali k rier and stable environment nonetheless 5.508-513(1998). the TrosY experiment2 (Fig. 2) indicate fur, Molekularbiologie und Biophysik, 33. Sh%er, S.B. Hajduk, Pl. Meadows. R. P. Fesik additional advantages of higher fields, in Eidgenossische Technische Hochschule s W Science 274. 1531-1534(1996) particular in extending the upper size range Honggerberg, CH-8093 Zurich, Switzerland 34. Per. chem. Soc. in the press. 1702(1997) er G.& wuthrich, K.J. enable to anal Politically aceptable big science NMR Correspondence should be addressed to K W. 37. Pennis E Science 219: 2. 180-182(1996) centers would be laid out primarily for Fax: +4116331151 nature structural biology. NMR supplement. july 1998
NMR supplement nature structural biology • NMR supplement • july 1998 495 1. Wagner, G. Nature Struct. Biol. 4, 841–844 (1997). 2. Grant, D.M. & Harris. R.K. (eds) Encyclopedia of nuclear magnetic resonance (Wiley, New York; 1996). 3. Williamson, M.P., Havel, T.F. & Wüthrich, K. J. Mol. Biol. 182, 295–315 (1985). 4. Hendrickson, W.A. & Wüthrich, K. (eds) Macromolecular structures. (Current Biology, London; 1991–1997). 5. Wüthrich, K. NMR in structural biology (World Scientific, Singapore; 1995). 6. Kay, L.E. Nature Struct. Biol. 5, 514–517 (1998). 7. Otting, G., Liepinsh, E. & Wüthrich, K. Science 254, 974–980 (1991). 8. Venu, K., Denisov, V.P. & Halle, B. J. Am. Chem. Soc. 119, 3122–3134 (1997). 9. Wüthrich, K. Acta Crystallogr. D 51, 249–270 (1995). 10. Gelin, B.R. & Karplus, M. Proc. Natl. Acad. Sci. USA 72, 2002–2006 (1975). 11. Smith, P.E., van Schaik, R.C., Szyperski, T., Wüthrich, K. & van Gunsteren, W.F. J. Mol. Biol. 246, 356–365 (1995). 12. Tolman, J.R., Flanagan, J.M., Kennedy, M.A. & Prestegard, J.H. Nature Struct. Biol. 4, 292–297 (1997). 13. Tjandra, N. & Bax, A. Science 278, 1111–1114 (1997). 14. Akke, M., Liu, J., Cavanagh, J., Erickson, H.P. & Palmer, A.G. Nature Struct. Biol. 5, 55–59 (1998). 15. Prestegard, J.H. Nature Struct. Biol. 5, 518–523 (1998) 16. Wüthrich, K. Curr. Opin. Struct. Biol. 4, 93–99 (1994). 17. Shortle, D.R. Curr. Opin. Struct. Biol. 6, 24–30 (1996). 18. Dyson, H.J. & Wright, P.E. Nature Struct. Biol. 5, 499–503 (1998). 19. Dobson, C. & Hore, P.J. Nature Struct. Biol. 5,. 504–507 (1998). 20. Riek, R., Hornemann, S., Wider, G., Glockshuber, R. & Wüthrich, K. FEBS Lett. 413, 282–288 (1997). 21. Cho, H.S. et al. Prot. Sci. 5, 262–269 (1996). 22. Luger, K., Mäder, A.W., Richmond, R.K., Sargent, D.F. & Richmond, T.J. Nature 389, 251–260 (1997). 23. Clore, G.M. & Gronenborn, A.M. Nature Struct. Biol. 4, 849–853 (1997). 24. LeMaster, D.M. Progr. NMR Spectr. 26, 371–419 (1994). 25. Kainosho, M. Nature Struct. Biol. 4, 858–861 (1997). 26. Bax, A. & Grzesiek, S. Acc. Chem. Res. 26, 131–138 (1993). 27. Kay, L.E. & Gardner, K.H. Curr. Opin. Struct. Biol. 7, 722–731 (1997). 28. Fesik, S. & Zuiderweg, E.R.P. J. Magn. Reson. 78, 588–593 (1988). 29. Otting, G. & Wüthrich, K. Q. Rev. Biophys. 23, 39–96 (1990). 30. Campbell, I.D. & Downing, A.K. Nature Struct. Biol. 5, 496–499 (1998). 31. Griffin, R.G. Nature Struct. Biol. 5, 508–513 (1998). 32. Pervushin, K., Riek, R., Wider, G. & Wüthrich, K. Proc. Natl. Acad. Sci. USA 94, 12366–12371 (1997). 33. Shuker, S.B., Hajduk, P.J., Meadows, R.P. & Fesik, S.W. Science 274, 1531–1534 (1996). 34. Pervushin, K., Riek, R., Wider, G. & Wüthrich, K. J. Am. Chem. Soc., in the press. 35. Normile, D. Science 278, 1700–1702 (1997). 36. Service, R.F. Science 279, 1127–1128 (1998). 37. Pennisi, E. Science 279, 978–979 (1998). 38. Riek R. et al. Nature 382, 180–182 (1996). Introduction of the traits of big science to biomacromolecular NMR would mean the loss of the opportunity to perform all the steps of a structure determination in the individual laboratories, a comfort that most of us presently enjoy. In return we would get access to NMR centers with more powerful equipment than the individual researcher could afford, as well as support from chemistry and microbiology laboratories specialized in efficient preparation of isotope-labeled precursor molecules and production of NMR quantities of labeled macromolecules. In a successful big science scenario the newly established NMR centers would need to be comparable in function (if not in size) with the high intensity light sources of the X-ray crystallographers, with instrumentation that significantly outperforms the equipment likely to be available to individual research groups. (This demand contrasts with the present situation, where the equipment of regional and national NMR centers consists of spectrometers identical to those available in the laboratories of most of the leading individual research groups.) The centralized equipment should provide high quality data for automated spectral analysis, to enable improvement of quality and efficiency of NMR structure determination, including structure refinement against raw NMR data. Much of the aforementioned demands for improved equipment in big science NMR centres could probably be met by improved performance at the presently available field strengths corresponding to 1H resonance frequencies up to 800 MHz, which can be anticipated from on-site construction of specialized NMR equipment and from operation in a suitably shielded and stable environment. Nonetheless, past experience in general and more specifically the TROSY experiment32 (Fig. 2) indicate additional advantages of higher fields, in particular in extending the upper size range amenable to analysis by NMR. Politically acceptable big science NMR centers would be laid out primarily for high-throughput, high-quality structure determination. Facilities operating at highest fields would, however, also initiate further development of spectroscopic techniques for the benefit of continued progress of biomolecular NMR. The future Considering that the current use of NMR in structural biology still shows typical signs of a young, emerging field of research, the results obtained (Table 1) are truly remarkable and are an encouragement for the future. NMR with biological macromolecules could experience an important boost with the creation of centralized high-performance facilities for data collection, which have recently been extensively discussed in Japan35 and in the USA36. These projects are intimately related to genome sequencing projects, and envisage determination of a comprehensive set of all naturally occurring protein folds35,37. NMR will have an indispensable role in any such ambitious venture, since experience indicates that a sizeable fraction of all proteins will not be amenable to structure determination in crystalline form, and increasingly there will also be demands for additional, NMR-specific information. Considering the current rate of progress in structural biology (Table 1) an immediate start of construction of high performance NMR centers should be encouraged by the necessary innovations in funding. Acknowledgments Support by the Schweizerischer Nationalfonds is gratefully acknowledged. The author thanks K. Pervushin, R. Riek and G. Wider for the preparation of the figures and for discussions. Kurt Wüthrich is at the Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg,CH-8093 Zürich, Switzerland. Correspondence should be addressed to K.W. Fax: +41 1 633 11 51