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NMR spectroscopy brings invisible protein states into focus

Nature Chemical Biology volume 5, pages 808–814 (2009)Cite this article

Abstract

Molecular dynamics are essential for protein function. In some cases these dynamics involve the interconversion between ground state, highly populated conformers and less populated higher energy structures ('excited states') that play critical roles in biochemical processes. Here we describe recent advances in NMR spectroscopy methods that enable studies of these otherwise invisible excited states at an atomic level and that help elucidate their important relation to function. We discuss a range of examples from molecular recognition, ligand binding, enzyme catalysis and protein folding that illustrate the role that motion plays in 'funneling' conformers along preferred pathways that facilitate their biological function.

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Figure 1: A simple schematic of a CPMG relaxation dispersion experiment.
Figure 2: Modulation of spin-relaxation from conformational changes affecting the distance between a paramagnet and NMR probes.
Figure 3: Probing sparsely populated states involved in molecular interactions.
Figure 4: New NMR methods for determining the structures of excited states.
Figure 5: Relaxation dispersion studies of catalysis.
Figure 6: Sparsely populated states along protein folding pathways.

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References

  1. Hammes, G.G. Mechanism of enzyme catalysis. Nature 204, 342–343 (1964).

    CAS  PubMed  Google Scholar 

  2. Fersht, A.R. Structure and Mechanism in Protein Science (W.H. Freeman, New York, 1998).

    Google Scholar 

  3. Karplus, M. & Kuriyan, J. Molecular dynamics and protein function. Proc. Natl. Acad. Sci. USA 102, 6679–6685 (2005).

    CAS  PubMed  Google Scholar 

  4. Perutz, M.F. & Mathews, F.S. An X-ray study of azide methaemoglobin. J. Mol. Biol. 21, 199–202 (1966).

    CAS  PubMed  Google Scholar 

  5. Hvidt, A. & Linderstrom-Lang, K. Exchange of hydrogen atoms in insulin with deuterium atoms in aqueous solutions. Biochim. Biophys. Acta 14, 574–575 (1954).

    CAS  PubMed  Google Scholar 

  6. Wüthrich, K. & Wagner, G. Internal motion in globular proteins. Trends Biochem. Sci. 3, 227–230 (1978).

    Google Scholar 

  7. Snyder, G.H., Rowan, R. III, Karplus, S. & Sykes, B.D. Complete tyrosine assignments in the high field 1H nuclear magnetic resonance spectrum of the bovine pancreatic trypsin inhibitor. Biochemistry 14, 3765–3777 (1975).

    CAS  PubMed  Google Scholar 

  8. Campbell, I.D., Dobson, C.M. & Williams, R.J. Proton magnetic resonance studies of the tyrosine residues of hen lysozyme-assignment and detection of conformational mobility. Proc. R. Soc. Lond. B 189, 503–509 (1975).

    CAS  PubMed  Google Scholar 

  9. Austin, R.H., Beeson, K.W., Eisenstein, L., Frauenfelder, H. & Gunsalus, I.C. Dynamics of ligand binding to myoglobin. Biochemistry 14, 5355–5373 (1975).

    CAS  PubMed  Google Scholar 

  10. McCammon, J.A., Gelin, B.R. & Karplus, M. Dynamics of folded proteins. Nature 267, 585–590 (1977).

    CAS  PubMed  Google Scholar 

  11. Tokuriki, N. & Tawfik, D.S. Protein dynamism and evolvability. Science 324, 203–207 (2009).

    CAS  PubMed  Google Scholar 

  12. Bryngelson, J.D. & Wolynes, P.G. Spin glasses and the statistical mechanics of protein folding. Proc. Natl. Acad. Sci. USA 84, 7524–7528 (1987).

    CAS  PubMed  Google Scholar 

  13. Chan, H.S. & Dill, K.A. Polymer principles in protein structure and stability. Annu. Rev. Biophys. Biophys. Chem. 20, 447–490 (1991).

    CAS  PubMed  Google Scholar 

  14. Wolynes, P.G. Recent successes of the energy landscape theory of protein folding and function. Q. Rev. Biophys. 38, 405–410 (2005).

    CAS  PubMed  Google Scholar 

  15. Hegler, J.A., Weinkam, P. & Wolynes, P.G. The spectrum of biomolecular states and motions. HFSP J. 2, 307–313 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Kay, L.E., Torchia, D.A. & Bax, A. Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. Biochemistry 28, 8972–8979 (1989).

    CAS  PubMed  Google Scholar 

  17. Baber, J.L., Szabo, A. & Tjandra, N. Analysis of slow interdomain motion of macromolecules using NMR relaxation data. J. Am. Chem. Soc. 123, 3953–3959 (2001).

    CAS  PubMed  Google Scholar 

  18. Rueda, M. et al. A consensus view of protein dynamics. Proc. Natl. Acad. Sci. USA 104, 796–801 (2007).

    CAS  PubMed  Google Scholar 

  19. Dobson, C.M. & Karplus, M. Internal motion of proteins: nuclear magnetic resonance measurements and dynamic simulations. Methods Enzymol. 131, 362–389 (1986).

    CAS  PubMed  Google Scholar 

  20. Peng, J.W. & Wagner, G. Investigation of protein motions via relaxation measurements. Methods Enzymol. 239, 563–596 (1994).

    CAS  PubMed  Google Scholar 

  21. Palmer, A.G., Williams, J. & McDermott, A. Nuclear magnetic resonance studies of biopolymer dynamics. J. Phys. Chem. 100, 13293–13310 (1996).

    CAS  Google Scholar 

  22. Ishima, R. & Torchia, D.A. Protein dynamics from NMR. Nat. Struct. Biol. 7, 740–743 (2000).

    CAS  PubMed  Google Scholar 

  23. Palmer, A.G., Grey, M.J. & Wang, C. Solution NMR spin relaxation methods for characterizing chemical exchange in high-molecular-weight systems. Methods Enzymol. 394, 430–465 (2005).

    CAS  PubMed  Google Scholar 

  24. Henzler-Wildman, K. & Kern, D. Dynamic personalities of proteins. Nature 450, 964–972 (2007).

    CAS  PubMed  Google Scholar 

  25. Mulder, F.A., Mittermaier, A., Hon, B., Dahlquist, F.W. & Kay, L.E. Studying excited states of proteins by NMR spectroscopy. Nat. Struct. Biol. 8, 932–935 (2001).

    CAS  PubMed  Google Scholar 

  26. Mittag, T., Schaffhausen, B. & Gunther, U.L. Direct observation of protein-ligand interaction kinetics. Biochemistry 42, 11128–11136 (2003).

    CAS  PubMed  Google Scholar 

  27. Tang, C., Iwahara, J. & Clore, G.M. Visualization of transient encounter complexes in protein-protein association. Nature 444, 383–386 (2006).

    CAS  PubMed  Google Scholar 

  28. Iwahara, J. & Clore, G.M. Detecting transient intermediates in macromolecular binding by paramagnetic NMR. Nature 440, 1227–1230 (2006).

    CAS  PubMed  Google Scholar 

  29. Sugase, K., Lansing, J.C., Dyson, H.J. & Wright, P.E. Tailoring relaxation dispersion experiments for fast-associating protein complexes. J. Am. Chem. Soc. 129, 13406–13407 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Sugase, K., Dyson, H.J. & Wright, P.E. Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447, 1021–1025 (2007).

    CAS  PubMed  Google Scholar 

  31. Tang, C., Schwieters, C.D. & Clore, G.M. Open-to-closed transition in apo maltose-binding protein observed by paramagnetic NMR. Nature 449, 1078–1082 (2007).

    CAS  PubMed  Google Scholar 

  32. Korzhnev, D.M., Bezsonova, I., Lee, S., Chalikian, T.V. & Kay, L.E. Alternate binding modes for a ubiquitin-SH3 domain interaction studied by NMR spectroscopy. J. Mol. Biol. 386, 391–405 (2009).

    CAS  PubMed  Google Scholar 

  33. Bruschweiler, S. et al. Direct observation of the dynamic process underlying allosteric signal transmission. J. Am. Chem. Soc. 131, 3063–3068 (2009)

    CAS  PubMed  Google Scholar 

  34. Ishima, R., Freedberg, D.I., Wang, Y.-X., Louis, J.M. & Torchia, D.A. Flap opening and dimer-interface flexibility in the free and inhibitor-bound HIV protease, and their implications for function. Structure 7, 1047–1055 (1999).

    CAS  PubMed  Google Scholar 

  35. Cole, R. & Loria, J.P. Evidence for flexibility in the function of ribonuclease A. Biochemistry 41, 6072–6081 (2002).

    CAS  PubMed  Google Scholar 

  36. Eisenmesser, E.Z., Bosco, D.A., Akke, M. & Kern, D. Enzyme dynamics during catalysis. Science 295, 1520–1523 (2002).

    CAS  PubMed  Google Scholar 

  37. Venkitakrishnan, R.P. et al. Conformational changes in the active site loops of dihydrofolate reductase during the catalytic cycle. Biochemistry 43, 16046–16055 (2004).

    CAS  PubMed  Google Scholar 

  38. Wolf-Watz, M. et al. Linkage between dynamics and catalysis in a thermophilic-mesophilic enzyme pair. Nat. Struct. Mol. Biol. 11, 945–949 (2004).

    CAS  PubMed  Google Scholar 

  39. Eisenmesser, E.Z. et al. Intrinsic dynamics of an enzyme underlies catalysis. Nature 438, 117–121 (2005).

    CAS  PubMed  Google Scholar 

  40. Vallurupalli, P. & Kay, L.E. Complementarity of ensemble and single-molecule measures of protein motion: a relaxation dispersion NMR study of an enzyme complex. Proc. Natl. Acad. Sci. USA 103, 11910–11915 (2006).

    CAS  PubMed  Google Scholar 

  41. Watt, E.D., Shimada, H., Kovrigin, E.L. & Loria, J.P. The mechanism of rate-limiting motions in enzyme function. Proc. Natl. Acad. Sci. USA 104, 11981–11986 (2007).

    CAS  PubMed  Google Scholar 

  42. Kempf, J.G., Jung, J.Y., Ragain, C., Sampson, N.S. & Loria, J.P. Dynamic requirements for a functional protein hinge. J. Mol. Biol. 368, 131–149 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Tang, C., Louis, J.M., Aniana, A., Suh, J.Y. & Clore, G.M. Visualizing transient events in amino-terminal autoprocessing of HIV-1 protease. Nature 455, 693–696 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Hill, R.B., Bracken, C., DeGrado, W.F. & Palmer, A.G. Molecular motions and protein folding: characterization of the backbone dynamics and folding equilibrium of α2D using 13C NMR spin relaxation. J. Am. Chem. Soc. 122, 11610–11619 (2000).

    CAS  Google Scholar 

  45. Korzhnev, D.M. et al. Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR. Nature 430, 586–590 (2004).

    CAS  PubMed  Google Scholar 

  46. Choy, W.Y., Zhou, Z., Bai, Y. & Kay, L.E. An 15N NMR spin relaxation dispersion study of the folding of a pair of engineered mutants of apocytochrome b562. J. Am. Chem. Soc. 127, 5066–5072 (2005).

    CAS  PubMed  Google Scholar 

  47. Zeeb, M. & Balbach, J. Millisecond protein folding studied by NMR spectroscopy. Protein Pept. Lett. 12, 139–146 (2005).

    CAS  PubMed  Google Scholar 

  48. Korzhnev, D.M., Neudecker, P., Zarrine-Afsar, A., Davidson, A.R. & Kay, L.E. Abp1p and Fyn SH3 domains fold through similar low-populated intermediate states. Biochemistry 45, 10175–10183 (2006).

    CAS  PubMed  Google Scholar 

  49. Tang, Y., Grey, M.J., McKnight, J., Palmer, A.G. III & Raleigh, D.P. Multistate folding of the villin headpiece domain. J. Mol. Biol. 355, 1066–1077 (2006).

    CAS  PubMed  Google Scholar 

  50. Neudecker, P. et al. Identification of a collapsed intermediate with non-native long-range interactions on the folding pathway of a pair of Fyn SH3 domain mutants by NMR relaxation dispersion spectroscopy. J. Mol. Biol. 363, 958–976 (2006).

    CAS  PubMed  Google Scholar 

  51. Neudecker, P., Zarrine-Afsar, A., Davidson, A.R. & Kay, L.E. Phi-value analysis of a three-state protein folding pathway by NMR relaxation dispersion spectroscopy. Proc. Natl. Acad. Sci. USA 104, 15717–15722 (2007).

    CAS  PubMed  Google Scholar 

  52. Korzhnev, D.M., Religa, T.L., Lundstrom, P., Fersht, A.R. & Kay, L.E. The folding pathway of an FF domain: characterization of an on-pathway intermediate state under folding conditions by (15)N, (13)C(alpha) and (13)C-methyl relaxation dispersion and (1)H/(2)H-exchange NMR spectroscopy. J. Mol. Biol. 372, 497–512 (2007).

    CAS  PubMed  Google Scholar 

  53. Clore, G.M. Visualizing lowly-populated regions of the free energy landscape of macromolecular complexes by paramagnetic relaxation enhancement. Mol. Biosyst. 4, 1058–1069 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Carr, H.Y. & Purcell, E.M. Effects of diffusion on free precession in nuclear magnetic resonance experiments. Phys. Rev. 94, 630–638 (1954).

    CAS  Google Scholar 

  55. Meiboom, S. & Gill, D. Modified spin-echo method for measuring nuclear relaxation times. Rev. Sci. Instrum. 29, 688–691 (1958).

    CAS  Google Scholar 

  56. Solomon, I. Relaxation processes in a system of two spins. Phys. Rev. 99, 559–565 (1955).

    CAS  Google Scholar 

  57. Korzhnev, D.M. & Kay, L.E. Probing invisible, low-populated states of protein molecules by relaxation dispersion NMR spectroscopy: an application to protein folding. Acc. Chem. Res. 41, 442–451 (2008).

    CAS  PubMed  Google Scholar 

  58. Lundstrom, P., Vallurupalli, P., Hansen, D.F. & Kay, L.E. Isotope labeling methods for studies of excited protein states by relaxation dispersion NMR spectroscopy. Nat. Protoc. (in the press).

  59. Vallurupalli, P., Hansen, D.F. & Kay, L.E. Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy. Proc. Natl. Acad. Sci. USA 105, 11766–11771 (2008).

    CAS  PubMed  Google Scholar 

  60. Clore, G.M. & Iwahara, J. Theory, practice, and applications of paramagnetic relaxation enhancement for the characterization of transient low-population states of biological macromolecules and their complexes. Chem. Rev. 109, 4108–4139 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Bai, Y., Sosnick, T., Mayne, L. & Englander, S. Protein folding intermediates: native-state hydrogen exchange. Science 269, 192–197 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Englander, S.W., Mayne, L. & Krishna, M.M. Protein folding and misfolding: mechanism and principles. Q. Rev. Biophys. 40, 287–326 (2007).

    CAS  PubMed  Google Scholar 

  63. Smock, R.G. & Gierasch, L.M. Sending signals dynamically. Science 324, 198–203 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Verkhivker, G.M. et al. Complexity and simplicity of ligand-macromolecule interactions: the energy landscape perspective. Curr. Opin. Struct. Biol. 12, 197–203 (2002).

    CAS  PubMed  Google Scholar 

  65. Lange, O.F. et al. Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution. Science 320, 1471–1475 (2008).

    CAS  PubMed  Google Scholar 

  66. Wolfenden, R. & Snider, M.J. The depth of chemical time and the power of enzymes as catalysts. Acc. Chem. Res. 34, 938–945 (2001).

    CAS  PubMed  Google Scholar 

  67. Olsson, M.H., Parson, W.W. & Warshel, A. Dynamical contributions to enzyme catalysis: critical tests of a popular hypothesis. Chem. Rev. 106, 1737–1756 (2006).

    CAS  PubMed  Google Scholar 

  68. Nagel, Z.D. & Klinman, J.P. A 21st century revisionist's view at a turning point in enzymology. Nat. Chem. Biol. 5, 543–550 (2009).

    CAS  PubMed  Google Scholar 

  69. Pauling, L. Nature of forces between large molecules of biological interest. Nature 161, 707–709 (1948).

    CAS  PubMed  Google Scholar 

  70. Hammes, G.G. Multiple conformational changes in enzyme catalysis. Biochemistry 41, 8221–8228 (2002).

    CAS  PubMed  Google Scholar 

  71. Vamvaca, K., Vogeli, B., Kast, P., Pervushin, K. & Hilvert, D. An enzymatic molten globule: efficient coupling of folding and catalysis. Proc. Natl. Acad. Sci. USA 101, 12860–12864 (2004).

    CAS  PubMed  Google Scholar 

  72. Roca, M., Messer, B., Hilvert, D. & Warshel, A. On the relationship between folding and chemical landscapes in enzyme catalysis. Proc. Natl. Acad. Sci. USA 105, 13877–13882 (2008).

    CAS  PubMed  Google Scholar 

  73. Fierke, C.A., Johnson, K.A. & Benkovic, S.J. Construction and evaluation of the kinetic scheme associated with dihydrofolate reductase from Escherichia coli. Biochemistry 26, 4085–4092 (1987).

    CAS  PubMed  Google Scholar 

  74. Boehr, D.D., McElheny, D., Dyson, H.J. & Wright, P.E. The dynamic energy landscape of dihydrofolate reductase catalysis. Science 313, 1638–1642 (2006).

    CAS  PubMed  Google Scholar 

  75. Boehr, D.D., Dyson, H.J. & Wright, P.E. Conformational relaxation following hydride transfer plays a limiting role in dihydrofolate reductase catalysis. Biochemistry 47, 9227–9233 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. Kubelka, J., Chiu, T.K., Davies, D.R., Eaton, W.A. & Hofrichter, J. Sub-microsecond protein folding. J. Mol. Biol. 359, 546–553 (2006).

    CAS  PubMed  Google Scholar 

  77. Farrow, N.A., Zhang, O., Forman-Kay, J.D. & Kay, L.E. Comparison of the backbone dynamics of a folded and an unfolded SH3 domain existing in equilibrium in aqueous buffer. Biochemistry 34, 868–878 (1995).

    CAS  PubMed  Google Scholar 

  78. Aronsson, G., Brorsson, A.-C., Sahlman, L. & Jonsson, B.-H. Remarkably slow folding of a small protein. FEBS Lett. 411, 359–364 (1997).

    CAS  PubMed  Google Scholar 

  79. Zarrine-Afsar, A. et al. Theoretical and experimental demonstration of the importance of specific nonnative interactions in protein folding. Proc. Natl. Acad. Sci. USA 105, 9999–10004 (2008).

    CAS  PubMed  Google Scholar 

  80. Plaxco, K.W. et al. The folding kinetics and thermodynamics of the Fyn-SH3 domain. Biochemistry 37, 2529–2537 (1998).

    CAS  PubMed  Google Scholar 

  81. Di Nardo, A.A. et al. Dramatic acceleration of protein folding by stabilization of a nonnative backbone conformation. Proc. Natl. Acad. Sci. USA 101, 7954–7959 (2004).

    CAS  PubMed  Google Scholar 

  82. Maity, H., Maity, M., Krishna, M.M., Mayne, L. & Englander, S.W. Protein folding: the stepwise assembly of foldon units. Proc. Natl. Acad. Sci. USA 102, 4741–4746 (2005).

    CAS  PubMed  Google Scholar 

  83. Fersht, A.R. From the first protein structures to our current knowledge of protein folding: delights and scepticisms. Nat. Rev. Mol. Cell Biol. 9, 650–654 (2008).

    CAS  PubMed  Google Scholar 

  84. Whittaker, S.B., Spence, G.R., Gunter Grossmann, J., Radford, S.E. & Moore, G.R. NMR analysis of the conformational properties of the trapped on-pathway folding intermediate of the bacterial immunity protein Im7. J. Mol. Biol. 366, 1001–1015 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  85. Sprangers, R., Gribun, A., Hwang, P.M., Houry, W.A. & Kay, L.E. Quantitative NMR spectroscopy of supramolecular complexes: dynamic side pores in ClpP are important for product release. Proc. Natl. Acad. Sci. USA 102, 16678–16683 (2005).

    CAS  PubMed  Google Scholar 

  86. Sprangers, R. & Kay, L.E. Quantitative dynamics and binding studies of the 20S proteasome by NMR. Nature 445, 618–622 (2007).

    CAS  PubMed  Google Scholar 

  87. Loria, J.P., Rance, M. & Palmer, A.G.A. Relaxation-compensated (Carr Purcell Meiboom Gill) sequence for characterizing chemical exchange by NMR spectroscopy. J. Am. Chem. Soc. 121, 2331–2332 (1999).

    CAS  Google Scholar 

  88. Tollinger, M., Skrynnikov, N.R., Mulder, F.A., Forman-Kay, J.D. & Kay, L.E. Slow dynamics in folded and unfolded states of an SH3 domain. J. Am. Chem. Soc. 123, 11341–11352 (2001).

    CAS  PubMed  Google Scholar 

  89. Ishima, R. & Torchia, D.A. Extending the range of amide proton relaxation dispersion experiments in proteins using a constant-time relaxation-compensated CPMG approach. J. Biomol. NMR 25, 243–248 (2003).

    CAS  PubMed  Google Scholar 

  90. Lundstrom, P. et al. Fractional 13C enrichment of isolated carbons using [1–13C]- or [2- 13C]-glucose facilitates the accurate measurement of dynamics at backbone Calpha and side-chain methyl positions in proteins. J. Biomol. NMR 38, 199–212 (2007).

    PubMed  Google Scholar 

  91. Hansen, D.F., Vallurupalli, P., Lundstrom, P., Neudecker, P. & Kay, L.E. Probing chemical shifts of invisible states of proteins with relaxation dispersion NMR spectroscopy: how well can we do? J. Am. Chem. Soc. 130, 2667–2675 (2008).

    CAS  PubMed  Google Scholar 

  92. Ishima, R., Baber, J., Louis, J.M. & Torchia, D.A. Carbonyl carbon transverse relaxation dispersion measurements and ms-micros timescale motion in a protein hydrogen bond network. J. Biomol. NMR 29, 187–198 (2004).

    CAS  PubMed  Google Scholar 

  93. Vallurupalli, P., Hansen, D.F., Stollar, E., Meirovitch, E. & Kay, L.E. Measurement of bond vector orientations in invisible excited states of proteins. Proc. Natl. Acad. Sci. USA 104, 18473–18477 (2007).

    CAS  PubMed  Google Scholar 

  94. Hansen, D.F., Vallurupalli, P. & Kay, L.E. Quantifying two-bond 1HN-13CO and one-bond 1H(alpha)-13C(alpha) dipolar couplings of invisible protein states by spin-state selective relaxation dispersion NMR spectroscopy. J. Am. Chem. Soc. 130, 8397–8405 (2008).

    CAS  PubMed  Google Scholar 

  95. Baldwin, A., Hansen, D.F., Vallurupalli, P. & Kay, L.E. Measurement of methyl axis orientations in invisible, excited states of proteins by relaxation dispersion NMR spectroscopy. J. Am. Chem. Soc. 131, 11939–11948 (2009).

    CAS  PubMed  Google Scholar 

  96. Vallurupalli, P., Hansen, D.F. & Kay, L.E. Probing structure in invisible protein states with anisotropic NMR chemical shifts. J. Am. Chem. Soc. 130, 2734–2735 (2008).

    CAS  PubMed  Google Scholar 

  97. Hansen, D.F., Vallurupalli, P. & Kay, L.E. Using relaxation dispersion NMR spectroscopy to determine the structures of excited, invisible protein states. J. Biomol. NMR 41, 113–120 (2008).

    CAS  Google Scholar 

  98. Lippens, G., Caron, L. & Smet, C. A microscopic view of chemical exchange: Monte Carlo simulation of molecular association. Concepts Magn. Reson. A 21A, 1–9 (2004).

    Google Scholar 

  99. Skrynnikov, N.R., Dahlquist, F.W. & Kay, L.E. Reconstructing NMR spectra of “invisible” excited protein states using HSQC and HMQC experiments. J. Am. Chem. Soc. 124, 12352–12360 (2002).

    CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada and the Canadian Institutes of Health Research (CIHR). The authors are grateful to Y. Bai (US National Cancer Institute) for providing Figure 6b. A.J.B. acknowledges support in the form of European Molecular Biology Organization and CIHR postdoctoral fellowships.

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  1. Department of Molecular Genetics, Department of Biochemistry and Department of Chemistry, The University of Toronto, Toronto, Ontario, Canada

    Andrew J Baldwin & Lewis E Kay

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Baldwin, A., Kay, L. NMR spectroscopy brings invisible protein states into focus. Nat Chem Biol 5, 808–814 (2009). https://doi.org/10.1038/nchembio.238

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