清华大学：《生物核磁共振波谱学》课程教学资源（文献资料，英文版）Kinetic studies of protein folding using NMR spectroscopy

NMR supplement Kinetic studies of protein folding using NMR spectroscopy Christopher M. Dobson and Peter J Hore Recent progress has advanced our abilities to use NMR spectroscopy to follow- in real time -the structural and dynamic changes taking place during protein folding In a cell, the starting point of protein rant). This allows the use of biophysical polypeptide than there are molecules in on the ribosome. The process of protein in real time. Two aspects of folding, how- of folding can involve extremely diverse folding continues in a crowded molecu- ever, make this task challenging. The structural ensembles until the very last lar environment, in the presence of a first is that folding is usually fast; many stages of the reaction. This complicates ariety of helper molecules, the most small proteins fold in milliseconds or substantially the analysis of the results of famous of which are the molecular chap- less, although others may take consider- structural studies erones whose major functions include ably longer. The second, and perhaps In order to combat these problems, the control of protein aggregation. Many most significant, is that the initial state one approach has been to utilize a wide small proteins, however, will refold effi- from which the folding reaction is initi- range of spectroscopic techniques, each ciently in dilute aqueous solutions fol- ated is extremely heterogeneous!. The able to monitor the formation of specific lowing transfer from a denaturing ultimate starting point of folding is a aspects of protein structure, in stopped environment(such as 6 M guanidinium random coil, and proteins in strong and quenched flow mode 3. NMR spe chloride) into one where the native state denaturants approach this rather close- troscopy has played a significant role is thermodynamically stable (such as ly?. In the random coil state there are here through its ability to analyze the hat produced by dilution of the denatu- more accessible conformations for a distributions of hydrogen and deuterium in labile sites in proteins, and through pulse labeling to follow in a site-specific manner the formation of structure that Native resonances example as a result of the formation of hydrogen bonds involving amide hydro gens. Much has been learned through to use nmr directly to follow folding In principle such experiments could allow he detailed analysis of the structural ensembles populated at different stages ng reaction, a fundamentally the level of detail in which we are able to define the folding The use of NMR to study reactions of time(sec)1.5 A proteins in real time' started nearly 30 years ago with the objective of studying enzymatic mechanisms. 6. Not long afterwards efforts were made to stud 30& Unfolded resonances protein folding and unfolding, and a variety of experimental strategies were developed for this purpose(reviewed in Fig. 1 Stopped-flow F NMR spectra of the refolding of 6.19F-tryptophan labeled Escherichia coli dihy. ref. 7). Particular emphasis was placed on slow reactions to overcome the kinetics and chemical shifts suggest the formation of an intermediate that is unable to bind short periods of time. Unfolding reac- and 133, and little if any native side chain environment around tryptophans 22 and 74. The resonance tions frequently take place over minutes beled 47i is that of Trp 47 in the intermediate. (Taken from ref. 10 with permission) or hours, and some specific types of nature structural biology . NMR supplement. july 1998

NMR supplement 504 nature structural biology • NMR supplement • july 1998 In a cell, the starting point of protein folding is the nascent chain as it forms on the ribosome. The process of protein folding continues in a crowded molecu￾lar environment, in the presence of a variety of helper molecules, the most famous of which are the molecular chap￾erones whose major functions include the control of protein aggregation. Many small proteins, however, will refold effi￾ciently in dilute aqueous solutions fol￾lowing transfer from a denaturing environment (such as 6 M guanidinium chloride) into one where the native state is thermodynamically stable (such as that produced by dilution of the denatu￾rant). This allows the use of biophysical techniques to follow the folding process in real time. Two aspects of folding, how￾ever, make this task challenging. The first is that folding is usually fast; many small proteins fold in milliseconds or less, although others may take consider￾ably longer. The second, and perhaps most significant, is that the initial state from which the folding reaction is initi￾ated is extremely heterogeneous1. The ultimate starting point of folding is a random coil, and proteins in strong denaturants approach this rather close￾ly2. In the random coil state there are more accessible conformations for a polypeptide than there are molecules in the test tube. This means that the process of folding can involve extremely diverse structural ensembles until the very last stages of the reaction. This complicates substantially the analysis of the results of structural studies. In order to combat these problems, one approach has been to utilize a wide range of spectroscopic techniques, each able to monitor the formation of specific aspects of protein structure, in stopped and quenched flow mode3. NMR spec￾troscopy has played a significant role here through its ability to analyze the distributions of hydrogen and deuterium in labile sites in proteins, and through ‘pulse labeling’ to follow in a site-specific manner the formation of structure that protects against solvent exchange, for example as a result of the formation of hydrogen bonds involving amide hydro￾gens4. Much has been learned through these approaches, but recently, increas￾ing progress has been made on strategies to use NMR directly to follow folding. In principle such experiments could allow the detailed analysis of the structural ensembles populated at different stages of the folding reaction, and transform fundamentally the level of detail in which we are able to define the folding process. Kinetic NMR approaches The use of NMR to study reactions of proteins in ‘real time’ started nearly 30 years ago with the objective of studying enzymatic mechanisms5,6. Not long afterwards efforts were made to study protein folding and unfolding, and a variety of experimental strategies were developed for this purpose (reviewed in ref. 7). Particular emphasis was placed on slow reactions to overcome the intrinsic difficulties in accumulating NMR spectra of adequate quality in short periods of time. Unfolding reac￾tions frequently take place over minutes or hours, and some specific types of Kinetic studies of protein folding using NMR spectroscopy Christopher M. Dobson and Peter J. Hore Recent progress has advanced our abilities to use NMR spectroscopy to follow — in real time — the structural and dynamic changes taking place during protein folding. Fig. 1 Stopped-flow 19F NMR spectra of the refolding of 6-19F-tryptophan labeled Escherichia coli dihy￾drofolate reductase following dilution from 5.5 to 2.75 M urea at 5 °C in the presence of 3.8 mM NADP+. The disappearance of the five resonances of the unfolded state, clustered between -46.0 and -46.6 p.p.m., and the growth of the more widely dispersed native peaks are clearly seen in this well￾resolved set of spectra. Each spectrum represents the sum of 41 separate rapid dilution experiments. The kinetics and chemical shifts suggest the formation of an intermediate that is unable to bind NADP+ strongly, having a native-like side chain environment in the regions around tryptophans 30, 47 and 133, and little if any native side chain environment around tryptophans 22 and 74. The resonance labeled 47i is that of Trp 47 in the intermediate. (Taken from ref. 10 with permission)

NMR supplement monomers to structured trimers( Fig. 2) POGPOGPOGITGARGLAGPOGPdGPOGPOG As well as enabling the mechanism of this process to be defined, these experi ments are providing key information bout the molecular basis of diseases associated with mutations in the 0.8 encoding the collagen sequence (J Baum, pers. comm. An attractive alternative presents itself for proteins whose folding can be initiat- ed photochemically. A nice example is N0.4 the very recent study by Kaptein and coworkers of photoactive yellow protein (PYP), the motile bacterium Ectothiorhodospil 60 Light excitation induces the trans-cis iso merization of the p-coumaric acid cofac Time(min tor, which triggers a cycle of structural hanges in PYP yielding an intermediate, pB, that reverts to the native state pG in I s. Characterization of pB by revealed that it exhibits extensive struc. al and d ontrast to pG. The conversion of pb to pG can therefore be considered to be a folding reaction. It was monitored in detail by observing the recovery of pG cross peaks in a series of(H, 5N)HsQC spectra recorded at different times after a laser pulse. Considerable variation in the build-up rates was found, with more 2 NMR folding profiles of a peptide(top) labeled with N at Gly 24(circles)and Ala 13(squares) id recovery for the more disorganized hows the time dependence regions of the protein. The major exce he tik. Thesisapsiar ance of the monomer peak sohie ties and the ap peafanme of the trimer peaks tion to this was in the neighborhood d onfor matia tf rv z4 titeat w ith mechanism (bottom) ivolving intermediates in which the correlates with high degrees of disorder. the unfolded state. (Taken with permission from ref. the chromophore controls the refolding of that part of the molecule. Although less generally applicable than stopped actions(such as those limited has provided novel insights into both flow methods, rapid photochemical trig ed to isomerize peptide bonds unfolding and folding reactions-lo. The gering of refolding (for example, usi very slow. In order to monitor rapid NMR enables data collection to begin tial to allow monitoring of very rapid reactions, stopped flow procedures within 100 ms of mixing, and has processes. 14 involving rapid mixing within the NMR allowed, for example, distinct steps in In our laboratories we have focused on ample tube are being developed?. the folding of dihydrofolate reductase to the development of a variety of comple xperiments of this type have recently be resolved and characterized through mentary NMR methods aimed at begun to transform NMR into a general repetitive collection of spectra during describing at the atomic level the struc and powerful technique for studying a the folding process(Fig. 1)0. A similar tural and dynamic changes taking place ide range of fundamental events in strategy is of course possible using two- during the folding of a protein from its folding dimensional(2D) approaches if the denatured state. The ultimate objective is One of the obvious requirements in reactions under investigation are suffi- to map out by experiment the energy these studies is obtaining sufficient reso- ciently slow. Baum and colleagues have surface of the folding reaction!. This lution to be able to monitor events at the exploited this in an extremely elegant requires the ability to monitor the envi- werful approach has been to use F bly of peptide fragments of collagen. 2. folding(for example, whether they are IR to study proteins in which specific By labeling the peptides with isN it has buried or exp o the inter-residue interac residues (particularly aromatic ones) been possible to record 2D HSQC spec- ularly to define have been replaced by fluorinated tra at intervals of as little as four min- tions or contacts that develop at analogs. This strategy has been pio- utes, and to observe the transition different stages of folding. The latter can peered by Frieden and coworkers, and of these peptides from disordered in principle be studied directly if nuclear nature structural biology. NMR supplement. july 1998

NMR supplement nature structural biology • NMR supplement • july 1998 505 folding reactions (such as those limited by the need to isomerize peptide bonds involving proline residues) can also be very slow. In order to monitor rapid reactions, stopped flow procedures involving rapid mixing within the NMR sample tube are being developed7,8. Experiments of this type have recently begun to transform NMR into a general and powerful technique for studying a wide range of fundamental events in folding. One of the obvious requirements in these studies is obtaining sufficient reso￾lution to be able to monitor events at the level of single residues. One extremely powerful approach has been to use 19F NMR to study proteins in which specific residues (particularly aromatic ones) have been replaced by fluorinated analogs. This strategy has been pio￾neered by Frieden and coworkers, and has provided novel insights into both unfolding and folding reactions8–10. The ability to use one-dimemnsional (1D) NMR enables data collection to begin within 100 ms of mixing, and has allowed, for example, distinct steps in the folding of dihydrofolate reductase to be resolved and characterized through repetitive collection of spectra during the folding process (Fig. 1)10. A similar strategy is of course possible using two￾dimemnsional (2D) approaches if the reactions under investigation are suffi￾ciently slow. Baum and colleagues have exploited this in an extremely elegant manner to study the folding and assem￾bly of peptide fragments of collagen11,12. By labeling the peptides with 15N it has been possible to record 2D HSQC spec￾tra at intervals of as little as four min￾utes, and to observe the transition of these peptides from disordered monomers to structured trimers (Fig. 2). As well as enabling the mechanism of this process to be defined, these experi￾ments are providing key information about the molecular basis of diseases associated with mutations in the gene encoding the collagen sequence (J. Baum, pers. comm.). An attractive alternative presents itself for proteins whose folding can be initiat￾ed photochemically. A nice example is the very recent study by Kaptein and coworkers of photoactive yellow protein (PYP), the proposed photosensor of the motile bacterium Ectothiorhodospira halophilia (R. Kaptein, pers. comm.). Light excitation induces the trans-cis iso￾merization of the p-coumaric acid cofac￾tor, which triggers a cycle of structural changes in PYP yielding an intermediate, pB, that reverts to the native state pG in ~1 s. Characterization of pB by NMR revealed that it exhibits extensive struc￾tural and dynamic disorder, in strong contrast to pG. The conversion of pB to pG can therefore be considered to be a folding reaction. It was monitored in detail by observing the recovery of pG cross peaks in a series of (1H,15N) HSQC spectra recorded at different times after a laser pulse. Considerable variation in the build-up rates was found, with more rapid recovery for the more disorganized regions of the protein. The major excep￾tion to this was in the neighborhood of the chromophore, where slow refolding correlates with high degrees of disorder, suggesting that retro-isomerization of the chromophore controls the refolding of that part of the molecule. Although less generally applicable than stopped flow methods, rapid photochemical trig￾gering of refolding (for example, using nanosecond laser pulses) has the poten￾tial to allow monitoring of very rapid processes13,14. In our laboratories we have focused on the development of a variety of comple￾mentary NMR methods aimed at describing at the atomic level the struc￾tural and dynamic changes taking place during the folding of a protein from its denatured state. The ultimate objective is to map out by experiment the ‘energy surface’ of the folding reaction1. This requires the ability to monitor the envi￾ronments of individual residues during folding (for example, whether they are buried or exposed to solvent) and partic￾ularly to define the inter-residue interac￾tions or ‘contacts’ that develop at different stages of folding. The latter can in principle be studied directly if nuclear Fig. 2 NMR folding profiles of a peptide (top) labeled with 15N at Gly 24 (circles) and Ala 13 (squares). O is the one-letter amino acid code for hydroxyproline. The central panel shows the time dependence of the cross peaks in an (1H-15N) HSQC spectrum of the peptide as it folds to form a collagen-like triple helix. The disappearance of the monomer peaks (solid lines) and the appearance of the trimer peaks (dashed lines) are faster for Gly 24 than for Ala 13. Gly 24 and Ala 13 follow 2nd and 1st order kinetics respectively. The data are consistent with a mechanism (bottom) involving intermediates in which the local conformation of Gly 24 towards the chain end is largely helical while the more central Ala 13 is still in the unfolded state. (Taken with permission from ref. 12)

NMR supplement Overhauser effects (NOEs) can be detected between specific nuclei. It will be necessary of course to interpret these in terms of structural ensembles, as we have discussed above. and to information about the dynamic ssociated with the polypeptide c出 B We have used the family of c-type mm1500 lysozymes and their structural homologs, the a-lactalbumins, as test systems for many of these experiments because the folding of these proteins has been studied in detail using a wide variety of other biophysical methods. 15, 16. In addi it is possible to alter the folding kinet ics of some members of this family by factors of -100 simply by changing the Ca2+ concentration in the refolding buffer. This turns out to be an extremely valuable factor in devising NMR experiments to different aspects of the folding rocess. One approach we have adopted to extract structural information from ID experiments is to exploit photo-CIDNPI7 photochemically induced dynamic nuclear polarization). This technique, in which photo-excitation of a dye molecule rTTT can result in enhanced nuclear polariza- tion of tryptophan, tyrosine and histidine residues to which it has accessis has been δ/ppm used to probe the accessibility of these Fig 3"H photo CIDNP spectra(B)of the refolding of hen lysozyme after a simultaneous pH jump states. We have found that it can be par- end of the 30 ms injection of protein solution into the refolding buffer and the beginnning of t ticularly powerful in time-resolved exper- 50ms light flash that generates photo-CIDNP. Each spectrum is the result of a single injection. Th iments(Fig. 3). Because polarization is suggests a rapidly formed disordered collapsed state which reorganizes within a second to gener. induced in only a small number of ate the native state whose spectrum is shown as(c) residues, the resulting spectra are relative ly well resolved. The approach also has a shorter experimantal dead time than con- kinetic events. It turns out that this infor- remains a major challenge. In many cases ventional NMR, firstly because the polar- mation can be extracted from a single 2d the intrinsic problems of dealing with het ization is produced during a-50 ms light spectrum recorded while the time-depen- erogeneous systems are compounded by flash, a somewhat faster process than the dent process takes place. If a reaction the existence of extensive exchange broad spin-lattice relaxation required to polar- occurs during the accumulation of data in ening of resonances resulting from slow ize spins transferred into the NMR probe the experiment, it perturbs the line shapes interconversion of conformers within com- from a lower field region of the magnet. and intensities of the cross-peaks in the pact but disordered systems. Nevertheless, Secondly, efficient mixing is only needed resulting 2D spectrum. Computer simula- we have been able to show that extensive in the small portion of the sample tion and kinetic model-fitting of these NOEs do exist at the earliest detectable exposed to the laser flash, from which the spectral features gives residue-specific rate stages of the folding of a-lactalbumin, and nal is detected i? onstants for the folding reaction. This their characteristics indicate that the mole- This rapid mixing approach, coupled approach has been used already to probe cules have native-like compactness 22. In with more conventional ID experiments the cooperativity of the formation of order to begin to identify the specific NOEs has enabled probing of the disordered col- native-like structure in bovine a-lactalbu- within such species we have made use of psed state, formed rapidly after the initi- min during folding using a('H-I5N) the fact mentioned above that Ca2+can ation of refolding of these proteins, and HSQC experiment(Fig 4), and to probe profoundly change the folding kinetics of monitoring of the rearrangement process- the structure of a folding intermediate the protein. The idea is to generate NOEs in es that occur subsequently. We are present- with a non-native proline isomer formed the partially folded state, and then to refold ly engaged in attempts to increase in the refolding of ribonuclease Tl (. the protein rapidly to its native state2z significantly the sensitivity of this experi- Ballach, pers. comm) Provided this refolding can be done rapidly ment, and to develop 2D variants. This Although such experiments ar d with the nuclear relaxation rates task has been substantially aided by the forming the possibilities for NMR it is possible to transfer the NoEs to the recognition that it is not necessary to ing folding, the detection of ell resolved spectrum of the native state record sequential spectra to monitor collapsed and partially folded for detection. Initial attempts to implement nature structural biology . NMR supplement. july 1998

NMR supplement 506 nature structural biology • NMR supplement • july 1998 Overhauser effects (NOEs) can be detected between specific nuclei. It will be necessary of course to interpret these in terms of structural ensembles, as we have discussed above, and to obtain information about the dynamic events associated with the polypeptide chain as folding takes place. We have used the family of c-type lysozymes and their structural homologs, the α-lactalbumins, as test systems for many of these experiments because the folding of these proteins has been studied in detail using a wide variety of other biophysical methods1,15,16. In addi￾tion, it is possible to alter the folding kinet￾ics of some members of this family by factors of ~100 simply by changing the Ca2+ concentration in the refolding buffer. This turns out to be an extremely valuable factor in devising NMR experiments to probe different aspects of the folding process. One approach we have adopted to extract structural information from 1D experiments is to exploit photo-CIDNP17 (photochemically induced dynamic nuclear polarization). This technique, in which photo-excitation of a dye molecule can result in enhanced nuclear polariza￾tion of tryptophan, tyrosine and histidine residues to which it has access18 has been used to probe the accessibility of these residues in both native and denatured states19. We have found that it can be par￾ticularly powerful in time-resolved exper￾iments (Fig. 3). Because polarization is induced in only a small number of residues, the resulting spectra are relative￾ly well resolved. The approach also has a shorter experimantal dead time than con￾ventional NMR, firstly because the polar￾ization is produced during a ~50 ms light flash, a somewhat faster process than the spin-lattice relaxation required to polar￾ize spins transferred into the NMR probe from a lower field region of the magnet. Secondly, efficient mixing is only needed in the small portion of the sample exposed to the laser flash, from which the signal is detected17. This rapid mixing approach, coupled with more conventional 1D experiments20 has enabled probing of the disordered col￾lapsed state, formed rapidly after the initi￾ation of refolding of these proteins, and monitoring of the rearrangement process￾es that occur subsequently. We are present￾ly engaged in attempts to increase significantly the sensitivity of this experi￾ment, and to develop 2D variants. This task has been substantially aided by the recognition that it is not necessary to record sequential spectra to monitor kinetic events21. It turns out that this infor￾mation can be extracted from a single 2D spectrum recorded while the time-depen￾dent process takes place. If a reaction occurs during the accumulation of data in the experiment, it perturbs the line shapes and intensities of the cross-peaks in the resulting 2D spectrum. Computer simula￾tion and kinetic model-fitting of these spectral features gives residue-specific rate constants for the folding reaction. This approach has been used already to probe the cooperativity of the formation of native-like structure in bovine α-lactalbu￾min during folding using a (1H-15N) HSQC experiment21 (Fig. 4), and to probe the structure of a folding intermediate with a non-native proline isomer formed in the refolding of ribonuclease T1 (J. Ballach, pers. comm.). Although such experiments are trans￾forming the possibilities for NMR in study￾ing folding, the detection of NOEs in collapsed and partially folded states remains a major challenge. In many cases the intrinsic problems of dealing with het￾erogeneous systems are compounded by the existence of extensive exchange broad￾ening of resonances resulting from slow interconversion of conformers within com￾pact but disordered systems. Nevertheless, we have been able to show that extensive NOEs do exist at the earliest detectable stages of the folding of α-lactalbumin, and their characteristics indicate that the mole￾cules have native-like compactness22. In order to begin to identify the specific NOEs within such species we have made use of the fact mentioned above that Ca2+ can profoundly change the folding kinetics of the protein. The idea is to generate NOEs in the partially folded state, and then to refold the protein rapidly to its native state22. Provided this refolding can be done rapidly compared with the nuclear relaxation rates, it is possible to transfer the NOEs to the well resolved spectrum of the native state for detection. Initial attempts to implement Fig. 3 1H photo-CIDNP spectra (B) of the refolding of hen lysozyme after a simultaneous pH jump from 1.1 to 5.2, and dilution from 10 to 1.4 M urea17. The delay times are the intervals between the end of the 30 ms injection of protein solution into the refolding buffer and the beginnning of the 50 ms light flash that generates photo-CIDNP. Each spectrum is the result of a single injection. The first spectrum, at 30 ms, differs markedly from that of the denatured state in 10 M urea (A) and suggests a rapidly formed disordered collapsed state which reorganizes within a second to gener￾ate the native state whose spectrum is shown as (C)

NMR supplement 110 115 120 EE95z @ 135 908.07.0 9.08.07.0 08.07.0 H Chemical Shift(ppm)H Chemical Shift(ppm)H Chemical Shift(ppm) Fig 4(H 2C spectra of bovine a-lactalbumin at 3 C during different stages of the folding process. a, Poorly resolved spectrum of the dena tured state at pH 2.0 recorded before the initiation of refolding. b, Kinetic spectrum accumulated during folding(30 min). c, Well resolved (N)state at pH 7. 0 recorded after the refolding reaction. The insets show enlargements of the region containing the Val 92 tral peak ci b)compared to(c), and the negative features above and below the cen- formation on the local rate of formation of native structure this radio frequency pulse-labeling method NMR approaches based on magnetiza- 1. Dobson C Mi ali a Kaoplus, M Angew. Chem indicate the viability of this approach, sug- tion transfer and line shape analysis that 2. Foith, L 4 iein, .s- ch asbe H& Dobson, C.M. rategy could result in the information kinetics, including events taking place on 630-636(1996) required to characterize the protein folding microsecond time scales26. In combina- 4. Baoxin, R. L curr Opin. Struct. Bio. rocess in detail 2. We are also developing a tion with other experimental approaches 5.Grimald ldi, J.J., Baldo, J, McMurray, C& Sykes, B D closely related experiment in which accessi- and theoretical advances, NMR is likely ldi, J.J.& Sykes, B. D. Re ble aromatic side chains in the partially to play an increasingly important role in 1201-1205(1975) mmp一如2 Concluding remarks Hoetzli, S.D.& Frieden, C Biochemistry 37, 387-398 We have outlined briefly in this article van Nuland and S L. Winder, for many of the ideas 11. Liu, x. Siegel, D L Fan, P. Brodsky. B.& Baum, J some of the approaches that are being that have gone into the work from our own developed to exploit the potential of laboratories and that is discussed here. We are 12. Baum, J& Brodsky, B Folding Design 2, R53-R6O NMR in kinetic studies of protein fold- grateful to. Baum, H. B Gray, R Kaptein and J 3. Pascher, T, Chesick, J.P., Winkler, J.R.&Gray, H.B. ing. These experiments are in the early unpublished work, and to C. Freiden andJBaum Balbach for kindly providing reprints of stages of development and there are for copies of Figs i and 2. The oxford Centre for 15. Dobson, C: M. Evans, P A Radford, s. E. Trends in the future, including novel methods of and MRC. The research of C. M.D. is also supported 12. Horeapima winder s. 1B, bets cH&bobson.c ple by using electrochemical or photo 18. Hore, P J& Broadhurst, R. W. Prog. NMR Spec 25, chemical techniques or temperature 5-402(1993) jumps, as well as innovations in NMR Christopher M. Dobson is at the Oxford (1910d, S.E. Rees. obus biochemistry 3 methodology. The information emerging Centre for Molecular Sciences, New 20 Balbach, I et al from these experiments is highly comple- Chemistry Laboratory and Peter J. Hore is 21.Blach.. et al. Scier studies of equilibrium folding intermedi- Laboratory University of Oxford, South 23. Nolting. B, Golbik, R.& Fersht, A.R.Proc.Natl ates, including those that probe the sta- Parks Road, Oxford OX1 3QR, UK. bilities of interactions by subjecting these addition to the real-time kinetic experi- C.M.D. email: chris. dobson@chem. ox. ac uk 26. Huang, G s, a r 630-631(90)&Redfield.c. species to progressive denaturation. In Correspondence should be addressed to 25. Schulman, B, Kim, P S, Dobson, CM nents described here, there are exciting or PJ. H. email: peter hore@chem. ox ac uk,6878-6882(1995). Proc. NatlAcad. Sci.USA nature structural biology. NMR supplement. july 1998

NMR supplement nature structural biology • NMR supplement • july 1998 507 this radio frequency pulse-labeling method indicate the viability of this approach, sug￾gesting that further development of this strategy could result in the information required to characterize the protein folding process in detail22. We are also developing a closely related experiment in which accessi￾ble aromatic side chains in the partially folded state are labeled by photo-CIDNP and then identified in the native state after rapid refolding. Concluding remarks We have outlined briefly in this article some of the approaches that are being developed to exploit the potential of NMR in kinetic studies of protein fold￾ing. These experiments are in the early stages of development and there are many opportunities for major advances in the future, including novel methods of initiating the folding process, for exam￾ple by using electrochemical or photo￾chemical techniques13,14, or temperature jumps23, as well as innovations in NMR methodology. The information emerging from these experiments is highly comple￾mentary to that emerging from NMR studies of equilibrium folding intermedi￾ates24, including those that probe the sta￾bilities of interactions by subjecting these species to progressive denaturation25. In addition to the real-time kinetic experi￾ments described here, there are exciting NMR approaches based on magnetiza￾tion transfer and line shape analysis that are providing information about folding kinetics, including events taking place on microsecond time scales26. In combina￾tion with other experimental approaches and theoretical advances, NMR is likely to play an increasingly important role in the quest to understand the mechanisms by which proteins fold. Acknowledgments We thank J. Balbach, V. Forge, J. A. Jones, N. A. J. van Nuland and S. L. Winder, for many of the ideas that have gone into the work from our own laboratories and that is discussed here. We are grateful to J. Baum, H. B. Gray, R. Kaptein and J. Balbach for kindly providing reprints of unpublished work, and to C. Freiden and J. Baum for copies of Figs 1 and 2. The Oxford Centre for Molecular Sciences is supported by BBSRC, EPSRC and MRC. The research of C.M.D. is also supported by the Howard Hughes Medical Institute and the Wellcome Trust. Christopher M. Dobson is at the Oxford Centre for Molecular Sciences, New Chemistry Laboratory and Peter J. Hore is at the Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QR, UK. Correspondence should be addressed to C.M.D. email: chris.dobson@chem.ox.ac.uk or P.J.H. email: peter.hore@chem.ox.ac.uk 1. Dobson, C.M., Sali, A. & Karplus, M. Angew. Chem. Int. Ed. Eng. 37, 868–893 (1998). 2. Smith, L.J, Fiebig, K.M., Schwalbe, H. & Dobson, C.M. Folding & Design 1, 95–106 (1996). 3. Plaxco, K. & Dobson, C.M. Curr. Opin. Struct. Biol. 6, 630–636 (1996). 4. Baldwin, R. L. Curr. Opin. Struct. Biol. 3, 84–91 (1993). 5. Grimaldi, J. J., Baldo, J., McMurray, C. & Sykes, B. D. J. Amer. Chem. Soc. 94, 7641–7645 (1972). 6. Grimaldi, J. J. & Sykes, B. D. Rev. Sci. Instr. 46, 1201–1205 (1975). 7. van Nuland, N.A.J., Forge, V., Balbach, J. & Dobson, C.M. Accts. Chem. Res,, in the press. 8. Frieden, C., Hoetzli, S. D. & Ropson, I. J. Prot. Sci. 2, 2007–2014 (1993). 9. Hoetzli, S.D. & Frieden, C. Biochemistry 35, 16843–16851 (1996). 10. Hoetzli, S.D. & Frieden, C. Biochemistry 37, 387–398 (1998). 11. Liu, X., Siegel, D. L., Fan, P., Brodsky, B. & Baum, J. 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USA 92, 10668–10672 (1995). 24. Dyson, H.J. & Wright, P.E. Nature Struct. Biol. 5, 499–503 (1998). 25. Schulman, B., Kim, P. S., Dobson, C. M. & Redfield, C. Nature Struct. Biol. 4, 630–634 (1997). 26. Huang, G. S. & Oas, T. G. Proc. Natl. Acad. Sci. USA 92, 6878–6882 (1995) Fig. 4 (1H-15N) HSQC spectra of bovine α-lactalbumin at 3 oC during different stages of the folding process. a, Poorly resolved spectrum of the dena￾tured state (A-state) at pH 2.0 recorded before the initiation of refolding. b, Kinetic spectrum accumulated during folding (30 min). c, Well resolved spectrum of the native (N) state at pH 7.0 recorded after the refolding reaction. The insets show enlargements of the region containing the Val 92 resonance of the N-state. The lower intensity of this resonance in spectrum (b) compared to (c), and the negative features above and below the cen￾tral peak contain information on the local rate of formation of native structure21. a b c