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）Dyson-equilibrium NMR

NMR supplement Hendrickson, W-A& Wuthrich, K Macromolecular 16. Leahy, D.J., Aukhil, I.& Erickson, H.P. 2.0 Cell 84 A K.D. thanks the Wellcome Trust and The queens 2D0i9 Riddiough. G. Nature Struct. Biol 18. sakaning. AK Kean. cel l8r.597-605i(eRw& by the Wellcome Trust. The authors acknowledge Biol. Chem. 266 P. Handford for critical reading of the The oxford centre for molecular science 4. Kuriyan, J. &Cowburn, D. Annu. Rev. Biophys. 19. Cardy. C M.& Handford. P.A. J. Mol. BioL. 276, by the Biology and Biotechnology Science 296-298(199 Trends Biochem. 20. Handford PA. et al. J. Biol. Chem. 270. 6751-6756 Council and medical Research Council 73-75 5:1997. uney. 3.K. curt. opin. struc, Blo. .2-71.671-678(1992). 5. Baron, Mr, Boy lain D. Campbell and A. Kristina Downing 8. 3135:120 R196) Mardon. H. J. Biol. Chem. 272. 6159-6 are at the Department of Biochemistry. University of Oxford, South Parks Road, Bork, P, Downing. A.K. Kieffer, B. Campbell, LD 25. Bax, A&Tjandra, N. J Biomol. NMR 10, 289-292 Oxford OXI3QU, England and the Oxford10m.be地m面Mcm2.01 Centre for Molecular Sciences, New 11. Sakai, L.Y. Keene, D.R.& Engvall, E.Cell Biol.103 26. Tjandra, N.& Bax, A. Science 278. 1111-111 hemistry Laboratory, University of Oxford, 2499-2509(19 roud. G. et al. Nucleic Acids Res. 26 South Parks Road, Oxford OX1 3QT, England. 、DGM2 lag. New 28. Merritt, E.A.& Murphey, M.E.P. Acta Crystallogr. Correspon hould be addressed to owning, A.K., Knott, V& Handford. P.A. 29. Kraulis, P.J. J. Appl. Crystallogr. 24, 946-950(1991). LD C email. idc@bioch ox ac uk EMBO16,6659-6666(1997) 15. Yuan, x. et al. Prot. Sci., in the press(1998) Campbell, L.D. J Mol. BioL. 265, 565-579(1997) Equilibrium NMR studies of unfolded and partially folded proteins Jane Dyson and Peter E. Wright Multidimensional NMR studies of proteins in unfolded and partially folded states give unique insights into their structures and dynamics and provide new understanding of protein folding and function In recent years NMR has developed into of the free energy landscape at the very tion of equilibrium folding intermediates, one of the two leading technologies, beginning of the folding process. Finally, partly folded states, peptide fragments, together with X-ray crystallography, for it is now recognized that many proteins and fully denatured states of proteins. For determining the three-dimensional struc- and protein domains are only partially most proteins, refolding is very rapid and tures of folded proteins at atomic resolu- structured or are unstructured under any intermediates formed are populated tion. However, NMR is unequaled in its physiological conditions and only become only transiently and are therefore difficult ability to characterize the structure and structured upon binding to their biologi- to study by direct real-time NMR experi dynamics of unfolded and partially folded cal targets. Knowledge of the structural ments. An especially powerful method for states of proteins. Such non-native pro- propensities of these domains is essential obtaining site-specific information on the tein states do not adopt unique three- to a proper understanding at the molecu- structure of folding intermediates is dimensional structures in solution but lar level of their biological functions and hydrogen exchange pulse labeling com fluctuate rapidly over an ensemble of con- interactions. bined with 2D NMR detection. 4. A typi formations. Structural characterization of Understanding the fundamental molec- cal experiment involves rapid dilution of non-native states is of great interest ular mechanisms by which proteins fold denatured protein in H2o buffer to initi because of their importance in protein into the complex structures required for ate refolding: the protein is allowed to folding, in the tran ular processes such as tral challenges in structural biology. NMR liseconds-seconds)before mixing with a sport of p roteins across biological activity remains one of the cen- refold for a short period(typically mil membranes. in signal transduction, and in the develop- has emerged especially important high pH labeling buffer in D2O solution ment of amyloid diseases(Fig. 1). tool for studies of protein folding because Amide protons that become involved in Knowledge of the structure of protein of the unique structural insights it can hydrogen bonded secondary structures folding intermediates is of central impor- provide into many aspects of the folding during the refolding period are protected tance for a detailed understanding of pro- process. Applications range from direct from exchange, whereas amide protons in tein folding mechanisms. Likewise, or indirect characterization of kinetic regions of the polypeptide that remain characterization of the ensemble of con- folding events (reviewed in the accompa- unfolded are exchanged with deuterium formations sampled by denatured pro- nying article by Dobson and colleagues) by the labeling pulse. After quenching of teins can provide insights into the nature to structural and dynamic characteriza- exchange and completion of folding, 2D nature structural biology. NMR supplement. july 1998

NMR supplement nature structural biology • NMR supplement • july 1998 499 Equilibrium NMR studies of unfolded and partially folded proteins H. Jane Dyson and Peter E. Wright Multidimensional NMR studies of proteins in unfolded and partially folded states give unique insights into their structures and dynamics and provide new understanding of protein folding and function. Acknowledgments A.K.D. thanks the Wellcome Trust and The Queen’s College, Oxford for support. I.D.C. is also supported by the Wellcome Trust. The authors acknowledge P. Handford for critical reading of the manuscript. The Oxford Centre for Molecular Sciences is funded by the Biology and Biotechnology Sciences Research Council, Engineering and Physical Sciences Research Council and Medical Research Council. Iain D. Campbell and A. Kristina Downing are at the Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, England and the Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QT, England. Correspondence should be addressed to I.D.C. email: idc@bioch.ox.ac.uk 1. Hendrickson, W.A. & Wüthrich, K. Macromolecular structures (Current Biology, London, 1996). 2. Cowburn, D. & Riddihough, G. Nature Struct. Biol. 4, 761–762 (1997). 3. Doolittle, R.F. Annu. Rev. Biochem. 64, 287–314 (1995). 4. Kuriyan, J. & Cowburn, D. Annu. Rev. Biophys. Biomol. Struct. 26, 259–288 (1997). 5. Bork, P., Schultz, J. & Ponting, C.P. Trends Biochem. Sci. 22, 296–298 (1997). 6. Rhodes, D. & Burley, S.K. Curr. Opin. Struct. Biol. 7, 73–75 (1997). 7. Chothia, C. & Jones, E.Y. Annu. Rev. Biochem. 66, 823–862 (1997). 8. Potts, J.R. & Campbell, I.D. Matrix Biology 15, 313–320 (1996). 9. Bork, P., Downing, A.K., Kieffer, B. & Campbell, I.D. Quarterly Rev. Biophys. 29, 119–167 (1996). 10. Pereira, L. et al. Human Mol. Gen. 2, 961–968 (1993). 11. Sakai, L.Y., Keene, D.R. & Engvall, E. J. Cell Biol. 103, 2499–2509 (1986). 12. Collod-Béroud, G. et al. Nucleic Acids Res. 26, 229–233 (1998). 13. Hynes, R.O. Fibronectins. (Springer-Verlag, New York, 1990). 14. Yuan, X., Downing, A.K., Knott, V. & Handford, P.A. EMBO J. 16, 6659–6666 (1997). 15. Yuan, X. et al. Prot. Sci., in the press (1998). 16. Leahy, D.J., Aukhil, I. & Erickson, H.P. 2.0 Cell 84, 155–164 (1996). 17. Downing, A.K. et al. Cell 85, 597–605 (1996). 18. Sakai, L.Y., Keene, D.R., Glanville, R.W. & Bächinger, H.P. J. Biol. Chem. 266, 14763–14770 (1991). 19. Cardy, C.M. & Handford, P.A. J. Mol. Biol. 276, 855–860 (1998). 20. Handford, P.A. et al. J. Biol. Chem. 270, 6751–6756 (1995). 21. Main, A.L., Harvey, T.S., Baron, M., Boyd, J. & Cell 71, 671–678 (1992). 22. Grant, R.P., Spitzfaden, C., Altroff, H., Campbell, I.D. & Mardon, H.J. J. Biol. Chem. 272, 6159–6166 (1997). 23. Copié, V. et al. J. Mol. Biol. 277, 663–682 (1998). 24. Wiles, A.P. et al. J. Mol. Biol. 272, 253–265 (1997). 25. Bax, A. & Tjandra, N. J. Biomol. NMR 10, 289–292 (1997). 26. Tjandra, N. & Bax, A. Science 278, 1111–1114 (1997). 27. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. Nucleic Acids Res. 25, 4876–4882 (1997). 28. Merritt, E.A. & Murphey, M.E.P. Acta Crystallogr. 50, 869–873 (1994). 29. Kraulis, P.J. J. Appl. Crystallogr. 24, 946–950 (1991). 30. Spitzfaden, C., Grant, R.P., Mardon, H.J. & Campbell, I.D. J. Mol. Biol. 265, 565–579 (1997). In recent years NMR has developed into one of the two leading technologies, together with X-ray crystallography, for determining the three-dimensional struc￾tures of folded proteins at atomic resolu￾tion. However, NMR is unequaled in its ability to characterize the structure and dynamics of unfolded and partially folded states of proteins. Such non-native pro￾tein states do not adopt unique three￾dimensional structures in solution but fluctuate rapidly over an ensemble of con￾formations. Structural characterization of non-native states is of great interest because of their importance in protein folding, in the transport of proteins across membranes, in cellular processes such as signal transduction, and in the develop￾ment of amyloid diseases (Fig. 1). Knowledge of the structure of protein folding intermediates is of central impor￾tance for a detailed understanding of pro￾tein folding mechanisms. Likewise, characterization of the ensemble of con￾formations sampled by denatured pro￾teins can provide insights into the nature of the free energy landscape at the very beginning of the folding process. Finally, it is now recognized that many proteins and protein domains are only partially structured or are unstructured under physiological conditions and only become structured upon binding to their biologi￾cal targets. Knowledge of the structural propensities of these domains is essential to a proper understanding at the molecu￾lar level of their biological functions and interactions. Understanding the fundamental molec￾ular mechanisms by which proteins fold into the complex structures required for biological activity remains one of the cen￾tral challenges in structural biology. NMR has emerged as an especially important tool for studies of protein folding because of the unique structural insights it can provide into many aspects of the folding process1. Applications range from direct or indirect characterization of kinetic folding events (reviewed in the accompa￾nying article by Dobson and colleagues2) to structural and dynamic characteriza￾tion of equilibrium folding intermediates, partly folded states, peptide fragments, and fully denatured states of proteins. For most proteins, refolding is very rapid and any intermediates formed are populated only transiently and are therefore difficult to study by direct real-time NMR experi￾ments. An especially powerful method for obtaining site-specific information on the structure of folding intermediates is hydrogen exchange pulse labeling com￾bined with 2D NMR detection3,4. A typi￾cal experiment involves rapid dilution of denatured protein in H2O buffer to initi￾ate refolding; the protein is allowed to refold for a short period (typically mil￾liseconds–seconds) before mixing with a high pH labeling buffer in D2O solution. Amide protons that become involved in hydrogen bonded secondary structures during the refolding period are protected from exchange, whereas amide protons in regions of the polypeptide that remain unfolded are exchanged with deuterium by the labeling pulse. After quenching of exchange and completion of folding, 2D

NMR supplement NMR spectra are acquired to identify the protected amides and monitor the pro gressive stabilization of hydrogen bonded secondary structure during kinetic refold h its importance should not be underestimated, the primary limitation of the pulse labeling method is that it pro olten Globule Native Folded Protein vides information only on the location of amide protons that become protected from exchange during folding: the nature of the structures that give rise to protec- tion must be deduced indirectly and ele- ments of structure that are insufficiently stable to protect amides from exchange ill go undetected. Fortunately, for some proteins, partially folded states that corre Aggrega0s一 pond closely to kinetic folding interme- diates can be stabilized at equilibrium, thereby opening the way to direct NMR analysis. In addition, direct NMR studies Fig. 1 Schematic diagram summarizing the roles of unfolded, partially folded proteins, and mis. of fully denatured states provide valuable folded proteins in biology insights into the nature of the conforma- tional ensemble at the starting point of One significant advantage in NMR Main chain coupling constants also give protein folding, while studies of peptide studies of proteins in highly unfolded insights into the conformational ensemble ragments reveal the intrinsic conforma- states is that resonances are generally nar- populated by an unfolded or partly folded tional propensities of the polypeptide row due to the rapid fluctuations of the protein Careful analysis of NMR data chain and identify potential folding initia- polypeptide chain. As a result, high quali- for unfolded proteins and peptide frag tion sites ty 2D and 3D spectra can be obtained at ments of proteins has led to a description surprisingly low protein concentrations; of the random coil state as a statistical dis The challenge of assignments indeed, our own experience is that excel- tribution of backbone dihedral angles in Characterization of unfolded and partial- lent data can be obtained at concentra- o, w space5. It is becoming increasingly ly folded states of proteins by Nmr pre- tions of 0.1 mM or lower. In addition, clear that many unfolded proteins do not sents special challenges because the sequential assignments can be made using simply form statistical random coils but polypeptide chain in such states is inher- triple resonance experiments that other- exhibit measurable propensities to popu ly flexible and rapidly interconverts wise may be unsuitable for a folded late native-like backbone conformations between multiple conformations. protein of comparable molecular weight Consequently, the chemical shift disper- NMR spectroscopy of partially folded Secondary structure sion of most resonances is poor and proteins can be even more challenging in NMR is particularly useful for determining nances is difficult( Fig. 2). Exceptions are those of native folded proteins but residue-by-residue basis in unfolded and the backbone 15N and 3C(that is, car- with the limited dispersion found in com- partly folded proteins; this is necessary for bonyl carbon) resonances, which are pletely unfolded states in many cases, an understanding of the local interactions influenced both by residue type and by NMR studies are impeded by severe that are likely to participate in the initiation the local amino acid sequence and there- resonance broadening that results from of protein foldingl. 12. Information obtained fore remain well-dispersed, even in fully conformational fluctuations on a milli- under non-denaturing or very weakly unfolded states,6. Multi-dimensional second-microsecond time scale&, 10 denaturing conditions is most relevant triple resonance NMR experiments since it more closely relates to the condi- which establish sequential connectivities Structural characterization ions prevailing at the start of a protein hrough the well-resolved IN and 3C Once resonance assignments have been folding reaction. For many proteins, the resonances provide a robust method for completed, detailed information on the unfolded state can only be obtained in obtaining unambiguous resonance conformational propensities of the solutions of strong denaturants which will gnments7-9. The lack of 'H and polypeptide chain can be readily derived have a pronounced effect on the popula atic C chemical shift dispersion for from chemical shifts, NOEs or coupling tion of residual structured conformers. The unfolded or partially folded proteins constants. The patterns and relative inten- ensemble of conformations sampled by a neans that it is extremely difficult to sities of the sequential and medium range polypeptide can differ significantly assign unambiguously the NOEs that NOEs provide information on the between denaturing and non-denaturing could provide key information on sec- propensity of the polypeptide to populate conditions 6.17 and subtle differences in the ondary structure and tertiary contacts. the a and p regions of o, y space or to location of residual structure have been Fortunately, recently developed NMR form ordered helical structures! 12. The observed for different denaturants experiments help to overcome this prob- deviations of chemical shifts from random Because their tendency towards structure lem by transferring the NOE information coil values, especially for 3Ca and ' Ho, formation is governed by local rather than to the relatively well-resolved NH or provide a convenient and sensitive probe long-range interactions, short linear pep 13C resonances of the secondary structural propensities. tide fragments of proteins are an ideal vehi- nature structural biology. NMR supplementjuly 1998

NMR supplement NMR spectra are acquired to identify the protected amides and monitor the pro￾gressive stabilization of hydrogen bonded secondary structure during kinetic refold￾ing. Although its importance should not be underestimated, the primary limitation of the pulse labeling method is that it pro￾vides information only on the location of amide protons that become protected from exchange during folding; the nature of the structures that give rise to protec￾tion must be deduced indirectly and ele￾ments of structure that are insufficiently stable to protect amides from exchange will go undetected. Fortunately, for some proteins, partially folded states that corre￾spond closely to kinetic folding interme￾diates can be stabilized at equilibrium, thereby opening the way to direct NMR analysis. In addition, direct NMR studies of fully denatured states provide valuable insights into the nature of the conforma￾tional ensemble at the starting point of protein folding, while studies of peptide fragments reveal the intrinsic conforma￾tional propensities of the polypeptide chain and identify potential folding initia￾tion sites. The challenge of assignments Characterization of unfolded and partial￾ly folded states of proteins by NMR pre￾sents special challenges because the polypeptide chain in such states is inher￾ently flexible and rapidly interconverts between multiple conformations. Consequently, the chemical shift disper￾sion of most resonances is poor and sequence-specific assignment of reso￾nances is difficult (Fig. 2). Exceptions are the backbone 15N and 13C' (that is, car￾bonyl carbon) resonances, which are influenced both by residue type and by the local amino acid sequence and there￾fore remain well-dispersed, even in fully unfolded states5,6. Multi-dimensional triple resonance NMR experiments which establish sequential connectivities through the well-resolved 15N and 13C' resonances provide a robust method for obtaining unambiguous resonance assignments7–9. The lack of 1H and aliphatic 13C chemical shift dispersion for unfolded or partially folded proteins means that it is extremely difficult to assign unambiguously the NOEs that could provide key information on sec￾ondary structure and tertiary contacts. Fortunately, recently developed NMR experiments help to overcome this prob￾lem by transferring the NOE information to the relatively well-resolved 15NH or 13C' resonances6. One significant advantage in NMR studies of proteins in highly unfolded states is that resonances are generally nar￾row due to the rapid fluctuations of the polypeptide chain. As a result, high quali￾ty 2D and 3D spectra can be obtained at surprisingly low protein concentrations; indeed, our own experience is that excel￾lent data can be obtained at concentra￾tions of 0.1 mM or lower. In addition, sequential assignments can be made using triple resonance experiments that other￾wise may be unsuitable for a folded protein of comparable molecular weight. NMR spectroscopy of partially folded proteins can be even more challenging in that resonances are at least as broad as those of native folded proteins but with the limited dispersion found in com￾pletely unfolded states; in many cases, NMR studies are impeded by severe resonance broadening that results from conformational fluctuations on a milli￾second–microsecond time scale8,10. Structural characterization Once resonance assignments have been completed, detailed information on the conformational propensities of the polypeptide chain can be readily derived from chemical shifts, NOEs or coupling constants. The patterns and relative inten￾sities of the sequential and medium range NOEs provide information on the propensity of the polypeptide to populate the α and β regions of φ,ψ space or to form ordered helical structures11,12. The deviations of chemical shifts from random coil values, especially for 13Cα and 1Hα, provide a convenient and sensitive probe of the secondary structural propensities13. Main chain coupling constants also give insights into the conformational ensemble populated by an unfolded or partly folded protein14. Careful analysis of NMR data for unfolded proteins and peptide frag￾ments of proteins has led to a description of the random coil state as a statistical dis￾tribution of backbone dihedral angles in φ,ψ space15. It is becoming increasingly clear that many unfolded proteins do not simply form statistical random coils but exhibit measurable propensities to popu￾late native-like backbone conformations. Secondary structure NMR is particularly useful for determining secondary structural propensities on a residue-by-residue basis in unfolded and partly folded proteins; this is necessary for an understanding of the local interactions that are likely to participate in the initiation of protein folding1,12. Information obtained under non-denaturing or very weakly denaturing conditions is most relevant since it more closely relates to the condi￾tions prevailing at the start of a protein folding reaction. For many proteins, the unfolded state can only be obtained in solutions of strong denaturants which will have a pronounced effect on the popula￾tion of residual structured conformers. The ensemble of conformations sampled by a polypeptide can differ significantly between denaturing and non-denaturing conditions16,17 and subtle differences in the location of residual structure have been observed for different denaturants7. Because their tendency towards structure formation is governed by local rather than long-range interactions, short linear pep￾tide fragments of proteins are an ideal vehi￾Fig. 1 Schematic diagram summarizing the roles of unfolded, partially folded proteins, and mis￾folded proteins in biology. 500 nature structural biology • NMR supplement • july 1998

NMR supplement b C 110 11515 F 12o(ppm) 10.09.08.07.010.0908.07.010.0908.07.0 H(ppm) cle for elucidation of the intrinsic propen- contain native-like secondary structure bin molten globule is of particular interest sities of sequences to fold under non-dena- but which lack the unique side chain and importance because it corresponds turing conditions 2. Studies of peptide interactions that characterize the tertiary closely to an intermediate formed during fragments of proteins and of proteins that structure of the native protein. kinetic refolding of the protein2. High are unfolded under non-denaturing or Equilibrium molten globules are formed quality NMR spectra can often be the intrinsic conformational propensities ing conditions. Unfortunately, the confor- species formed by denaturation of pro of the polypeptide backbone frequently mational heterogeneity and complex teins with alcohols however, the rele reflect the secondary structure found in dynamics of these species frequently result vance of such states to protein folding the native folded protein&, 18-20. In other in extremely broad and featureless Nmr remains to be established. words, the conformations populated by spectra which make direct NMR structur the unfolded polypeptide are not distrib- al analysis difficult. Nevertheless, numer- Tertiary structure uted randomly over the low energy regions ous NMR experiments have been devised Characterization of residual tertiary struc- of o, y space but are biased in a way that to provide structural information on ture in unfolded and partially folded pro reflects the secondary structural propensi- molten globule states, including hydrogen teins is extremely challenging given their ies of the local amino acid sequence. In exchange measurements, magnetization intrinsic flexibility While observation of a ly populated in unfolded states of proteins titrations to allow residue-specific charac- definitively indicates that they must be in nd in peptide fragments. The observation terization of the hydrophobic core 22. The close proximity in at least some structures of conformational preferences for forma- molten globule state formed by apomyo- in the conformational ensemble, determi- tion of secondary structure or hydropho- globin at pH 4 is exceptional in the quality nation of the nature of the folded struc- bic clusters in short peptides shows that of the NMR spectra that it yields; this ture is difficult unless an extensive local interactions determined by the species is stable at relatively high tempera- network of NOEs can be observed. Newly amino acid sequence bias the conforma- ture where there is sufficient internal developed methods for assigning NOE tional search toward specific structured motion to give rise to narrow resonances peaks in partly folded states may eventual forms, even in the absence of stabilization and permit use of multidimensional nmr ly provide sufficient data in favorable cases by long-range interactions with the experiments. As a Fois yete backbone populated structures zo, For the 434 repres. detailed description of highl remainder of the protein been possible to make com NMR assignments and obtain highly sor, for example, enough NOEs were The molten globule detailed insights into secondary structure observed to permit distance geometry cal- Molten globules are compact states that and backbone dynamics. The apomyoglo- culations of the three-dimensional struc nature structural biology. NMR supplement. july 1998

NMR supplement nature structural biology • NMR supplement • july 1998 501 cle for elucidation of the intrinsic propen￾sities of sequences to fold under non-dena￾turing conditions12. Studies of peptide fragments of proteins and of proteins that are unfolded under non-denaturing or weakly denaturing conditions show that the intrinsic conformational propensities of the polypeptide backbone frequently reflect the secondary structure found in the native folded protein8,9,18–20. In other words, the conformations populated by the unfolded polypeptide are not distrib￾uted randomly over the low energy regions of φ,ψ space but are biased in a way that reflects the secondary structural propensi￾ties of the local amino acid sequence. In addition, turn-like structures are frequent￾ly populated in unfolded states of proteins and in peptide fragments. The observation of conformational preferences for forma￾tion of secondary structure or hydropho￾bic clusters in short peptides shows that local interactions determined by the amino acid sequence bias the conforma￾tional search toward specific structured forms, even in the absence of stabilization by long-range interactions with the remainder of the protein. The molten globule Molten globules are compact states that contain native-like secondary structure but which lack the unique side chain interactions that characterize the tertiary structure of the native protein. Equilibrium molten globules are formed by many proteins under partially denatur￾ing conditions. Unfortunately, the confor￾mational heterogeneity and complex dynamics of these species frequently result in extremely broad and featureless NMR spectra which make direct NMR structur￾al analysis difficult. Nevertheless, numer￾ous NMR experiments have been devised to provide structural information on molten globule states, including hydrogen exchange measurements21, magnetization transfer experiments10, and denaturant titrations to allow residue-specific charac￾terization of the hydrophobic core22. The molten globule state formed by apomyo￾globin at pH 4 is exceptional in the quality of the NMR spectra that it yields; this species is stable at relatively high tempera￾ture where there is sufficient internal motion to give rise to narrow resonances and permit use of multidimensional NMR experiments9. As a consequence, it has been possible to make complete backbone NMR assignments and obtain highly detailed insights into secondary structure and backbone dynamics. The apomyoglo￾bin molten globule is of particular interest and importance because it corresponds closely to an intermediate formed during kinetic refolding of the protein23. High quality NMR spectra can often be obtained from partially folded compact species formed by denaturation of pro￾teins with alcohols24; however, the rele￾vance of such states to protein folding remains to be established. Tertiary structure Characterization of residual tertiary struc￾ture in unfolded and partially folded pro￾teins is extremely challenging given their intrinsic flexibility. While observation of a long-range NOE between two protons definitively indicates that they must be in close proximity in at least some structures in the conformational ensemble, determi￾nation of the nature of the folded struc￾ture is difficult unless an extensive network of NOEs can be observed. Newly developed methods for assigning NOE peaks in partly folded states may eventual￾ly provide sufficient data in favorable cases to allow a detailed description of highly populated structures20. For the 434 repres￾sor, for example, enough NOEs were observed to permit distance geometry cal￾culations of the three-dimensional struc￾a b c Fig. 2 1H-15N HSQC spectra of apomyoglobin at three pHs, illustrating the decrease in resonance dispersion in the 1H dimension as the protein unfolds. Note that the 15N dimension remains relatively well-dispersed, an important factor in successful assignment of resonances of unfolded pro￾teins. a, pH 2.0 (acid-unfolded state); b, pH 4.0 (equilibrium molten globule intermediate state); c, pH 6.0 ( folded native apoprotein). (Reproduced from ref. 9 with permission)

NMR supplement ture of a local hydrophobic However, it may often prove to be that backbone or side chain cor tional averaging is so extensive in partially range NOEs is difficult, precluding deter- onventional noe-based met The paucity of long-range NOEs in a denatured fragment of staphylococcal nuclease(termed△l3l△ led Gillespie and Shortle to develop an innovative method ngara ange distance constraints by measuring the enhancement of amide pro- nitroxide spin labels,z7. Spin labels were coupled to unique cysteine residues intro- duced at 14 different sites on the polypep- tide chain and -700 long-range distance constraints were derived from measure- ments of T2 relaxation enhancement(that is. bl ng of the close in space to the spin label). The calcu- Fig. 3 Ca backbone superposition of residues 56-140 lated ensemble of structures of this dena. tube)and five structures calculated for the fragment A131A(thin line). The three helices are col tured state has a global topology that is very from ref. 27 with permission) ilar to that of the native folded pro- tein26.27(Fig 3). These results suggest that the correct folding topology can be estab- surements can be used to probe the ping22. On the basis of the relaxation lished in denatured states even in the dynamics of the polypeptide backbone in measurements reported to date, it is clear absence of cooperative interactions and a these species. Interpretation of 15N relax- that unfolded states of proteins vary con tightly packed hydrophobic core. This spin ation rates and ('H)-I5N heteronuclear siderably in their dynamical properties. At labeling approach is highly promising and NOEs is not straightforward because the one extreme, the backbone fluctuations should be generally applicable to the eluci- motions are complex and the common show little variation as a function of lation of the folding topologies of other assumption of isotropic tumbling with a sequence 0 while for other proteins there tially folded proteins single correlation time is unlikely to be are clear indications of local interactions alid. Nevertheless, valuable insights into that lead to motional restriction29, 31. Fo the backbone motions can be obtained, molten globule states and other partially Unfolded and partially folded proteins are using either an extended model-free folded species, the molecular motions are highly flexible. 15N spin relaxation mea- analysis or reduced spectral density map- highly heterogeneous and relaxation mea- Fig. 4 Schematic diagram illustrating the increas. easingly structured an E D id), to the pH 4.1 molten globul acid MG153 Nolo+Napo he various partly folde icated by nm he smoothed(H-N heteronuclear NOE at each esidue is shown, on a color scale from dark bl of apomyoglobin is colored gray, since no 153 53 formation is available for it.(Adapted from ref 9 with permission nature structural biology . NMR supplement. july 1998

NMR supplement 502 nature structural biology • NMR supplement • july 1998 ture of a local hydrophobic cluster25. However, it may often prove to be the case that backbone or side chain conforma￾tional averaging is so extensive in partially folded states that observation of long￾range NOEs is difficult, precluding deter￾mination of the folding topology by conventional NOE-based methods. The paucity of long-range NOEs in a denatured fragment of staphylococcal nuclease (termed ∆131∆) led Gillespie and Shortle to develop an innovative method to obtain long-range distance constraints by measuring the enhancement of amide pro￾ton relaxation induced by paramagnetic nitroxide spin labels26,27. Spin labels were coupled to unique cysteine residues intro￾duced at 14 different sites on the polypep￾tide chain and ~700 long-range distance constraints were derived from measure￾ments of T2 relaxation enhancement (that is, broadening of the resonances of protons close in space to the spin label). The calcu￾lated ensemble of structures of this dena￾tured state has a global topology that is very similar to that of the native folded pro￾tein26,27 (Fig. 3). These results suggest that the correct folding topology can be estab￾lished in denatured states even in the absence of cooperative interactions and a tightly packed hydrophobic core. This spin labeling approach is highly promising and should be generally applicable to the eluci￾dation of the folding topologies of other partially folded proteins. Dynamics Unfolded and partially folded proteins are highly flexible. 15N spin relaxation mea￾surements can be used to probe the dynamics of the polypeptide backbone in these species. Interpretation of 15N relax￾ation rates and {1H}-15N heteronuclear NOEs is not straightforward because the motions are complex and the common assumption of isotropic tumbling with a single correlation time is unlikely to be valid. Nevertheless, valuable insights into the backbone motions can be obtained, using either an extended model-free analysis or reduced spectral density map￾ping28,29. On the basis of the relaxation measurements reported to date, it is clear that unfolded states of proteins vary con￾siderably in their dynamical properties. At one extreme, the backbone fluctuations show little variation as a function of sequence30 while for other proteins there are clear indications of local interactions that lead to motional restriction29,31. For molten globule states and other partially folded species, the molecular motions are highly heterogeneous and relaxation mea￾Fig. 3 Cα backbone superposition of residues 56–140 of folded staphylococcal nuclease (thick tube) and five structures calculated for the fragment ∆131∆ (thin line). The three helices are col￾ored red and the three β-strands are shown in yellow, yellow-green, and orange. (Reproduced from ref. 27 with permission). Fig. 4 Schematic diagram illustrating the increas￾ing restriction of backbone flexibility as myoglo￾bin folds to increasingly structured and increasingly compact states, from the acid-unfold￾ed state (Uacid), to the pH 4.1 molten globule state (IMG), to native apomyoglobin (Napo), and finally to fully folded holomyoglobin (Nholo). Except for holomyoglobin, the structures are purely schematic, shown only to indicate the location of secondary structure in the various partly folded states of apomyoglobin, as indicated by NMR data9. The polypeptide fluctuates over an ensem￾ble of conformations in all of these states, and no single structure suffices to describe its behavior. The smoothed {1H-15N} heteronuclear NOE at each residue is shown, on a color scale from dark blue (least flexible) to red (most flexible). The F helix region of apomyoglobin is colored gray, since no NMR information is available for it9. (Adapted from ref. 9 with permission)

NMR supplement surements can provide key insights into the understanding of the molecular assistance with preparation of the figures. Th the structural organization of such states. mechanisms of protein folding and mis- was supported by grants from the National For example, IsN relaxation studies of the folding, provide insights into the subtle ot Healin. 4 molten globule state of apomyoglo- relationship between amino acid H. Jane Dyson and Peter E. Wright are at bin show that backbone motions are sequence and protein structure, and lead the Department of Molecular Biology and ghly restricted within a compact to new understanding of the behavior Skaggs Institute for Chemical Biology, The hydrophobic core formed by packing of and biological function of intrinsically Scripps Research Institute, 10550 North three helices whilst other parts of the chain unstructured protein domains. Clearly it Torrey Pines Road, La Jolla, California remain highly fuctional Similarly, is of vital importance to understand the 92037, USA. nuclear spin relaxation studies of a partial- kinetics of collapse and structure forma ly folded state of ubiquitin formed in 60% tion that accompany the folding process, Correspondence should be addressed to methanol reveal the presence of three and NMR has important contributions PE W email: wright@scripps. edu or H.J.D loosely coupled secondary structural ele- to make in this field2. However, the email: dyson@scripps. edu lents with enhanced mobility relative to intrinsically long time scale of the NMR the native proteins experiment makes real-time kinetic 1 H.J.& Wright, P.E. Annu. Rev. Phys. Chem observations problematic. For many pro- 2 M. Nature Struct. Biol. 5. 504-50 Intrinsically unstructured proteins equilibrium NMR stu It is now recognized that many proteins vide valuable and extensive information 3. RI998 are intrinsically unstructured under phys- on the conformational propensities of 4.Udgaonkar,JB.&Baldwin,RL.Nature33 been known for certain polypeptide hor- tion that is directly applicable to an Som.o- Foro Wgy rich, K,IAm.Chem iological conditions 2. While this has long unfolded or partly folded states, informa mones such as glucagon, there is an understanding of the folding process hartle, D& Kay L.E. creasing awareness that many eukaryot. Recent studies of apomyoglobin pro- 7.Logan, T.M. Theriault,Y&Fesik,sWJMo.Biol ic proteins or protein domains involved in vide an illustrative example of the funda- 8. 236, 863d-648(1941) nal transduction, transcriptional acti- mental insights into protein folding shortle. D Biochemistry 33. 1063-1072(1994) cycle regulation adopt stable folded struc- obtained from equilibrium NMR experi- 10. Biome 3: D, 148-1 5. Evans, PA 8 Hamby 3 tures only upon binding to their molecu- ments. By careful manipulation of the 11. Dyson, H.J.& Wright, PE lar targets. Indeed, many genes in solution conditions, several states of Biophys. Chem. 20. 519-538( Ann. Rev. Biophys. eukaryotic genomes contain regions of apomyoglobin that differ in structural 12 27916 -717501088) Lerner, RA Biochemistry low sequence complexity that encode bio- content and degree of compaction can be 13. Wishart, D.S. Sykes, B.D. Meth. Enz. 239 ogically functional domains which would stabilized for NMR analysis In this way, 14. Smith, LI et ai. 1 Mol Biol. 255, 494-506(1996) not be expected to fold spontaneously into detailed insights can be obtained, at the 15.Smith, LJ,Fiebig KMSchwalbe, H.&Dobson tional stabilizing interactions. NMR is the gressive accumulation of secondary struc such domains, many of which probably do backbone dynamics as the chain collapses 18. Dyson, Hu, Merutka, G Waltho. I, Ler not exist as statistical random coils but during folding to form more compact 19. ./right, PE. J MOl. Biol. 226, 795-817(1992:RA. ang, O, Kay, L.E., Shortle, D.& Forman-Kay propensities that may presage the confor- a beginning, and the prospects for obtain 9-20(1997) mation stabilized upon binding. Recent ing even deeper insights into the folding 249 . M., Wright, P.E. Baldwin, R L. Science tein domains include the anti-sigma factor compact, partially folded states formed by 23. Jennings, .A. wright E science 2 FIgMS3, the SH3 domain of the Drosophila apomyoglobin and other proteins are 4 ha9 arding, M.M., williams, D.H. Woolfson, D.N. signal transduction protein Drk16, a excellent. Future work in the area will be fibronectin-binding protein from aided in no small part by the continued 25. Neri D 5 7 5s. M 3 ider, G& wuthrich, K Staphylococcus aureus, and the kinase development of novel NMR methodolo- 26. Gillence 257 inducible transactivation domain (KiD) gies and improvements in NMR instru- a, Gillespie,1997). Shortle. D. Mo. Bio. 268 from the transcription factor CREB. In mentation, especially the anticipated latter example nmr analysis shows development of spectrometers braun Bruschweiler, R.& Ernst. R.R. that the phosphorylated Kid domain is at or above 900 MHz which will provide 29. Farrow. N.A. Zhan 0390-1G5 」.D.&Kay folding transition to form a pair of a- consequence, more detailed insights into helices upon binding its target domain the nature of unfolded and partially fold- 31. aexa460190: A norte. D. J. Mol. from the CREB binding protein(CBP)35. ed states Plaxco, K.w.& Gross, M. Nature 386 Future perspectives 33. Daughdrill, G.W., Hanely, L.& Dahlquist, F.W. There can be little doubt that nmr will ield. C. Dodd, I. et al. J. Mol. Bio.274.152-159(199 continue to make major contributions to nk D Eliezer, P. Jennings and S. Cavagnero for 35. Radhakrishnan, L et al. Cel191, 741-752(1997) nature structural biology. NMR supplement. july 1998

NMR supplement nature structural biology • NMR supplement • july 1998 503 surements can provide key insights into the structural organization of such states. For example, 15N relaxation studies of the pH 4 molten globule state of apomyoglo￾bin show that backbone motions are highly restricted within a compact hydrophobic core formed by packing of three helices whilst other parts of the chain remain highly fluctional9. Similarly, nuclear spin relaxation studies of a partial￾ly folded state of ubiquitin formed in 60% methanol reveal the presence of three loosely coupled secondary structural ele￾ments with enhanced mobility relative to the native protein28. Intrinsically unstructured proteins It is now recognized that many proteins are intrinsically unstructured under phys￾iological conditions32. While this has long been known for certain polypeptide hor￾mones such as glucagon, there is an increasing awareness that many eukaryot￾ic proteins or protein domains involved in signal transduction, transcriptional acti￾vation, nucleic acid recognition or cell cycle regulation adopt stable folded struc￾tures only upon binding to their molecu￾lar targets. Indeed, many genes in eukaryotic genomes contain regions of low sequence complexity that encode bio￾logically functional domains which would not be expected to fold spontaneously into ordered structures in the absence of addi￾tional stabilizing interactions. NMR is the method of choice for characterization of such domains, many of which probably do not exist as statistical random coils but will exhibit intrinsic conformational propensities that may presage the confor￾mation stabilized upon binding. Recent examples of functional yet unfolded pro￾tein domains include the anti-sigma factor FlgM33, the SH3 domain of the Drosophila signal transduction protein Drk16, a fibronectin-binding protein from Staphylococcus aureus34, and the kinase inducible transactivation domain (KID) from the transcription factor CREB35. In the latter example, NMR analysis shows that the phosphorylated KID domain is intrinsically unstructured but undergoes a folding transition to form a pair of α- helices upon binding its target domain from the CREB binding protein (CBP)35. Future perspectives There can be little doubt that NMR will continue to make major contributions to the understanding of the molecular mechanisms of protein folding and mis￾folding, provide insights into the subtle relationship between amino acid sequence and protein structure, and lead to new understanding of the behavior and biological function of intrinsically unstructured protein domains. Clearly it is of vital importance to understand the kinetics of collapse and structure forma￾tion that accompany the folding process, and NMR has important contributions to make in this field2. However, the intrinsically long time scale of the NMR experiment makes real-time kinetic observations problematic. For many pro￾teins, equilibrium NMR studies can pro￾vide valuable and extensive information on the conformational propensities of unfolded or partly folded states, informa￾tion that is directly applicable to an understanding of the folding process. Recent studies of apomyoglobin pro￾vide an illustrative example of the funda￾mental insights into protein folding mechanisms that can potentially be obtained from equilibrium NMR experi￾ments. By careful manipulation of the solution conditions, several states of apomyoglobin that differ in structural content and degree of compaction can be stabilized for NMR analysis9. In this way, detailed insights can be obtained, at the level of individual residues, into the pro￾gressive accumulation of secondary struc￾ture and increasing restriction of backbone dynamics as the chain collapses during folding to form more compact states (Fig. 4). Studies such as this are only a beginning, and the prospects for obtain￾ing even deeper insights into the folding topology, hydration, and dynamics of the compact, partially folded states formed by apomyoglobin and other proteins are excellent. Future work in the area will be aided in no small part by the continued development of novel NMR methodolo￾gies and improvements in NMR instru￾mentation, especially the anticipated development of spectrometers operating at or above 900 MHz which will provide greater sensitivity and dispersion and, as a consequence, more detailed insights into the nature of unfolded and partially fold￾ed states. Acknowledgments We thank D. Eliezer, P. Jennings and S. Cavagnero for assistance with preparation of the figures. This work was supported by grants from the National Institutes of Health. H. Jane Dyson and Peter E. Wright are at the Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA. Correspondence should be addressed to P.E.W. email: wright@scripps.edu or H.J.D. email: dyson@scripps.edu 1. Dyson, H.J. & Wright, P.E. Annu. Rev. Phys. Chem. 47, 369–395 (1996). 2. Dobson, C.M. Nature Struct. Biol. 5, 504–507 (1998). 3. Roder, H., Elöve, G.A. & Englander, S.W. Nature 335, 700–704 (1988). 4. Udgaonkar, J.B. & Baldwin, R.L. Nature 335, 694–699 (1988). 5. Braun, D., Wider, G. & Wüthrich, K. J. Am. Chem. Soc. 116, 8466–8469 (1994). 6. Zhang, O., Forman-Kay, J.D., Shortle, D. & Kay, L.E. J. Biomol. NMR 9, 181–200 (1997). 7. Logan, T.M., Thériault, Y. & Fesik, S.W. J. Mol. Biol. 236, 637–648 (1994). 8. 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