Overview of Protein Folding Mechanisms: Experimental and Theoretical Approaches to Probing Energy Landscapes

Elizabeth R. Morris1, Mark S. Searle1

1 Centre for Biomolecular Sciences, School of Chemistry, University of Nottingham, Nottingham, United Kingdom
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 28.2
DOI:  10.1002/0471140864.ps2802s68
Online Posting Date:  April, 2012
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Abstract

We present an overview of the current experimental and theoretical approaches to studying protein folding mechanisms, set against current models of the folding energy landscape. We describe how stability and folding kinetics can be determined experimentally and how this data can be interpreted in terms of the characteristic features of various models from the simplest two‐state pathway to a multi‐state mechanism. We summarize the pros and cons of a range of spectroscopic methods for measuring folding rates and present a theoretical framework, coupled with protein engineering approaches, for elucidating folding mechanisms and structural features of folding transition states. A series of case studies are used to show how experimental kinetic data can be interpreted in the context of non‐native interactions, populated intermediates, parallel folding pathways, and sequential transition states. We also show how computational methods now allow transient species of high energy, such as folding transition states, to be modeled on the basis of experimental ϕ‐value analysis derived from the effects of point mutations on folding kinetics. Curr. Protoc. Protein Sci. 68:28.2.1‐28.2.22. © 2012 by John Wiley & Sons, Inc.

Keywords: protein folding; folding kinetics; energy landscape; protein engineering; folding transition states; molecular dynamics

     
 
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Table of Contents

  • The Protein Folding Problem
  • Protein Folding Pathways
  • Folding Energy Landscape
  • Measuring Protein Stability and Folding Kinetics
  • Choice of Denaturation Technique
  • Detection Methods
  • Equilibrium Methods to Determine Folding Stability
  • Kinetic Methods to Study Protein Folding
  • Kinetic Models for Probing Folding Pathways
  • Case Studies—Populated Intermediates and Sequential Folding Pathways
  • Unifying Folding Mechanisms
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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Literature Cited

Literature Cited
   Anfinsen, C.B., Haber, E., Sela, M., and White, F.H. 1961. The kinetics of formation of native ribonuclease during oxidation of the reduced polypeptide chain. Proc. Natl. Acad. Sci. U.S.A. 47:1309‐1304.
   Anfinsen, C.B. 1973. Principles that govern the folding of protein chains. Science 181:223‐230.
   Bachmann, A. and Kiefhaber, T. 2001. Apparent two‐state tendamistat folding is a sequential process along a defined route. J. Mol. Biol. 306:375‐386.
   Bachmann, A. and Kiefhaber, T. 2005. Kinetic mechanisms in protein folding. In Protein Folding Handbook. Part I. (J. Buchner and T. Kiefhaber, eds.) pp. 379‐410. Wiley, Weinheim, Germany.
   Balakrishnan, G., Weeks, C.L., Ibrahim, M., Soldatova, A.V., and Spiro, T.G. 2008. Protein dynamics from time resolved UV Raman spectroscopy. Curr. Opin. Struct. Biol. 18:1‐7.
   Baldwin, R.L. 1995. The nature of protein folding pathways: The classical versus the new view. J. Bimolec. NMR 5:103‐109.
   Baryshnikova, E.N., Melnik, B.S., Finkelstein, A.V., Semisotnov, G.V., and Bychkova, V.E. 2005. Three‐state protein folding: Experimental determination of free energy profile. Protein Sci. 14:2658‐2667.
   Bennion, B.J. and Daggett, V. 2003. The molecular basis for the chemical denaturation of proteins by urea. Proc. Natl. Acad. Sci. U.S.A. 100:5142‐5147.
   Bieri, O., Wildegger, G., Bachmann, A., Wagner, C., and Kiefhaber, T. 1999. A salt‐induced kinetic intermediate is on a new parallel pathway of lysozyme folding. Biochemistry 38:12460‐12470.
   Blake, C.C., Koenig, D.F., Mair, G.A., North, A.C., Phillips, D.C., and Sarma, V.R. 1965. Structure of hen egg‐white lysozyme. A three‐dimensional Fourier synthesis at 2 Angstrom resolution. Nature 206:757‐761.
   Bollen, Y.J.M., Sanchez, I.E., and van Mierlo, C.P.M. 2004. Formation of on‐ and off‐pathway intermediates in the folding kinetics of Azobacter Vinelandii apoflavodoxin. Biochemistry 43:10475‐10489.
   Brandts, J.F., Halvorson, H.R., and Brennan, M. 1975. Consideration of the possibility that the slow step in protein denaturation reactions is due to cis‐trans isomerism of praline residues. Biochemistry 14:4953‐4963.
   Bryngelson, J.D., Onuchic, J.N., Socci, N.D., and Wolynes, P.G. 1995. Funnels, pathways, and the energy landscape of protein folding: A synthesis. Proteins 21:167‐195.
   Calosci, N., Chi, C.N., Richter, B., Camilloni, C., Engstrom, A., Eklund, L., Travaglini‐Allocatelli, C., Gianni, S., Vendruscolo, M., and Jemth, P. 2008. Comparison of successive transition states for folding reveals alternative early folding pathways of two homologous proteins. Proc. Natl. Acad. Sci. U.S.A. 105:19240‐19245.
   Capaldi, A.P., Ferguson, S.J., and Radford, S.E. 1999. The Greek key protein apo‐pseudoazurin folds through an obligate on‐pathway intermediate. J. Mol. Biol. 286:1621‐1632.
   Capaldi, A.P., Kleanthous, C., and Radford, S.E. 2002. Im7 folding mechanism: Misfolding on a path to the native state. Nat. Struct. Biol. 9:209‐216.
   Chedad, A., Van Dael, H., Vanhooren, A., and Hanssens, I. 2005. Influence of Trp mutation on native, intermediate and transition states of goat alpha‐lactalbumin: An equilibrium and kinetic study. Biochemistry 44:15129‐15138.
   Chen, P., Long, J., and Searle, M.S. 2008. Sequential barriers and an obligatory metastable intermediate define the apparent two‐state folding pathway of the ubiquitin‐like PB1 domain of NBR1. J. Mol. Biol. 376:1463‐1477.
   Chen, P., Evans, C.‐L., Hirst, J.D., and Searle, M.S. 2011. Structural insights into the two sequential folding transition states of the PB1 domain of NBR1 from φ value analysis and biased molecular dynamics simulations. Biochemistry 50:125‐135.
   Conrad, J.C. and Flory, P.J. 1976. Moments and distribution functions for polypeptide chains. Poly‐L‐alanine. Macromolecules 9:41‐47.
   Crespo, M.D., Platt, G.W., Bofill, R., and Searle, M.S. 2004. Context‐dependent effects of proline residues on the stability and folding pathway of ubiquitin. Eur. J. Biochem. 271:4474‐4484.
   Crespo, M.D., Simpson, E.R., and Searle, M.S. 2006. Population of on‐pathway intermediates in the folding of ubiquitin. J. Mol. Biol. 360:1053‐1066.
   Daggett, V. and Fersht, A.R. 2003. Is there a unifying mechanism for protein folding? Trends Biochem. Sci. 28:18‐25.
   De los Rios, M.A., Daneshi, M., and Plaxco, K.W. 2005. Experimental investigation of the frequency and substitution dependence of negative phi‐values in SH3 folding. Biochemistry 44:12160‐12167.
   Dill, K.A. and Chan, H.S. 1997. From Levinthal to pathways to funnels. Nat. Struct. Biol. 4:10‐19.
   Dobson, C.M. 1992. Unfolded proteins, compact states and molten globules. Curr. Opin. Struct. Biol. 2:6‐12.
   Dobson, C.M. 1999. Protein misfolding, evolution and disease. Trends Biochem. Sci. 24:329‐332.
   Dyson, H.J. and Wright, P.E. 2004. Unfolded proteins and protein folding studies by NMR. Chem. Rev. 104:3607‐3622.
   Epstein, C.J., Goldberger, R.F., and Anfinsen, C.B. 1963. The genetic control of tertiary protein structure: studies with model systems. Cold Spring Harb. Symp. Quant. Biol. 28:439‐449.
   Fabian, H. and Naumann, D. 2004. Methods to study protein folding by stopped‐flow FT‐IR. Methods 34:28‐40.
   Ferguson, N., Capaldi, A.P., James, R., Kleanthous, C., and Radford, S.E. 1999. Rapid folding with and without populated intermediates in the homologous four‐helix proteins Im7 and Im9. J. Mol. Biol. 286:1597‐1608.
   Fersht, A.R. 1995. Optimization of rates of protein folding: The nucleation‐condensation mechanism and its implications. Proc. Natl. Acad. Sci. U.S.A. 92:10869‐10873.
   Fersht, A.R. 1997. Nucleation mechanisms in protein folding. Curr. Opin. Struct. Biol. 7:3‐9.
   Fersht, A. R. and Sato, S. 2004. ϕ‐Value analysis and the nature of protein‐folding transition states. Proc. Natl. Acad. Sci. U.S.A. 101:7976‐7981.
   Fersht, A. R., Matouschek, A., and Serrano, L. 1992. The folding of an enzyme. 1. Theory of protein engineering analysis of stability and pathway of protein folding. J. Mol. Biol. 224:771‐782.
   Fersht, A.R., Itzhaki, L.S., El Masry, N.F., Matthews, J.M., and Otzen, D.E. 1994. Single versus parallel pathways of protein folding and fractional formation of structure in the transition state. Proc. Natl. Acad. Sci. U.S.A. 91:10426‐10429.
   Flory, P.J. 1969. Statistical mechanics of chain molecules.Wiley Interscience, New York.
   Friel, C.T., Capaldi, A.P., and Radford, S.E. 2003. Structural analysis of the rate‐limiting transition state in the folding of Im7 and Im9: Similarities and differences in the folding of homologous proteins. J. Mol. Biol. 326:293‐305.
   Friel, C.T., Beddard, G.S., and Radford, S.E. 2004. Switching two‐state to three‐state kinetics in the helical protein Im9 via the optimisation of stabilising non‐native interactions by design. J. Mol. Biol. 342:261‐273.
   Fulton, K.F., Main, E.R.G., Daggett, V., and Jackson, S.E. 1999. Mapping the interactions present in the transition state for folding/unfolding of FKB12. J. Mol. Biol. 291:445‐461.
   Garcia‐Maria, M.M., Boehringer, D., and Schmid, F.X. 2004. The folding transition state of the cold shock protein is stongly polarised. J. Mol. Biol. 339:555‐569.
   Garel, J.‐R. and Baldwin, R.L. 1973. Both the fast and slow refolding reactions of ribonuclease A yield native enzyme. Proc. Natl. Acad. Sci. U.S.A. 70:3347‐3351.
   Gianni, S., Guydosh, N.R., Khan, F., Caldas, T.D., Mayor, U., White, G.W.N., DeMarco, M.L., Daggett, V., and Fersht, A.R. 2003. Unifying features in protein‐folding mechanisms. Proc. Natl. Acad. Sci. U.S.A. 100:13286‐13291.
   Gianni, S., Geierhaas, C.D., Calosci, N., Jemth, P., Vuister, G.W., Travaglini‐Allocatelli, C., Vendruscolo, M., and Brunori, M. 2007. A PDZ domain recapitulates a unifying mechanism for protein folding. Proc. Natl. Acad. Sci. U.S.A. 103:128‐133.
   Geierhaas, C.D., Best, R.B., Paci, E., Vendruscolo, M., and Clarke, J. 2006. Structural comparison of the two alternative transition states for folding of TI I27. Biophys. J. 91:263‐275.
   Greenfield, N.J. 2007a. Using circular dichroism spectra to estimate protein secondary structure. Nat. Prot. 1:2876‐2890.
   Greenfield, N.J. 2007b. Determination of the folding of proteins as a function of denaturants, osmolytes or ligands using circular dichroism. Nat. Prot. 1:2733‐2741.
   Greenfield, N.J. 2007c. Analysis of the kinetics of folding of proteins and peptides using circular dichroism. Nat. Prot. 1:2891‐2899.
   Griffiths‐Jones, S.R., Sharman, G.J., Maynard, A.J., and Searle, M.S. 1998. Modulation of intrinsic phi, psipropensities of amino acids by neighbouring residues in the coil regions of protein structures: NMR analysis and dissection of a beta‐hairpin peptide. J. Mol. Biol. 284:1597‐1609.
   Hubner, I.A., Edmonds, K.A., and Shakhnovich, E.I. 2005. Nucleation and the transition state of the SH3 of the SH3 domain. J. Mol. Biol. 349:424‐434.
   Hughson, F.M., Wright, P.E., and Baldwin, R.L. 1990. Structural characterisation of a partly folded apomyoglobin intermediate. Science 249:1544‐1548.
   Hummer, G., Garde, S., García, A.E., Paulaitis, M.E., and Pratt, L.R. 1998. The pressure dependence of hydrophobic interactions is consistent with the observed pressure denaturation of proteins. Proc. Natl. Acad. Sci. U.S.A. 95:1552‐1555.
   Ibarra‐Molero, B., Loladze, V.V., Makhatadze, G.I., and Sanchez‐Ruiz, J.M. 1999. Thermal versus guanidine‐induced unfolding of ubiquitin. An analysis in terms of the contributions from charge‐charge interactions to protein stability. Biochemistry 38:8138‐8149.
   Itzhaki, L.S., Otzen, D.E., and Fersht, A.R. 1995. The structure of the transition state for folding of chymotrypsin inhibitor 2 analysed by protein engineering methods: evidence for a nucleation‐condensation mechanism for protein folding. J. Mol. Biol. 254:260‐288.
   Jackson, S.E. 1998. How do small single‐domain proteins fold? Folding Design 3:R81‐R91.
   Jacob, J., Krantz, B., Dothager, R.S., Thiyagarajan, P., and Sosnick, T.R. 2004. Early collapse is not an obligate step in protein folding. J. Mol. Biol. 338:369‐382.
   Jemth, P., Gianni, S., Day, R., Li, B., Johnson, C.M., Daggett, V., and Fersht, A.R. 2004. Demonstration of a low‐energy on‐pathway intermediate in a fast‐folding protein by kinetics, protein‐engineering and simulation. Proc. Natl. Acad. Sci. U.S.A. 101:6450‐6455.
   Kamagata, K., Sawano, Y., Tanokura, M., and Kuwajima, K. 2003. Multiple parallel‐pathway folding of proline‐free staphyloccal nuclease. J. Mol. Biol. 332:1143‐1153.
   Kamagata, K., Arai, M., and Kuwajima, K. 2004. Unification of the folding mechanisms of non‐two‐state and two‐state proteins. J. Mol. Biol. 339:951‐965.
   Karplus, M. and Weaver, D.L. 1979. Diffusion‐collision model for protein folding. Biopolymers 18:1421‐1437.
   Kelly, S.M. and Price, N.C. 1997. The application of circular dichroism to studies of protein folding and unfolding. Biochim. Biophys. Acta 1338:161‐185.
   Khorasanizadeh, S., Peters, I.D., and Roder, H. 1996. Evidence for a three‐state model of protein folding from kinetic analysis of ubiquitin variants with altered core residues. Nat. Struct. Biol. 3:193‐205.
   Kiefhaber, T. 1995. Kinetic traps in lysozyme folding. Proc. Natl. Acad. Sci. U.S.A. 92:9029‐9033.
   Kim, P.S. and Baldwin, R.L. 1982. Specific intermediates in the folding reactions of small proteins and the mechanism of protein folding. Annu. Rev. Biochem. 51:459‐489.
   Kim, P.S. and Baldwin, R.L. 1990. Intermediates in the folding reactions of small proteins. Annu. Rev. Biochem. 59:631‐660.
   Klein‐Seetharaman, J., Oikawa, M., Grimshaw, S.B., Wirmer, J., Duchardt, E., Ueda, T., Imoto, T., Smith, L.J., Dobson, C.M., and Schwalbe, H. 2002. Long‐range interactions within a nonnative protein. Science 295:1719‐1722.
   Kong, J. and Yu, S. 2007. Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochim. Biophys. Sin. 39:549‐559.
   Koradi, R., Billeter, M., and Wüthrich, K. 1996. MOLMOL: A program for display and analysis of macromolecular structure. J. Mol. Graph. 14:51‐55.
   Krantz, B.A., Mayne, L., Rumbley, J., Englander, S.W., and Sosnick, T.R. 2002. Fast and slow intermediate accumulation and the initial barrier mechanism in protein folding. J. Mol. Biol. 324:359‐371.
   Krantz, B.A., Dothager, R.S., and Sosnick, T.R. 2004. Discerning the energy and structure of multiple transition states innproteinn folding using psi‐analysis. J. Mol. Biol. 337:463‐475.
   Krishna, M.M.G. and Englander, S.W. 2007. A unified mechanism for protein folding: Predetermined pathways with optional errors. Protein Sci. 16:449‐464.
   Lange, R. and Balny, C. 2002. UV‐visible derivative spectroscopy under high pressure. Biochim. Biophys. Acta 1595:80‐93.
   Levinthal, C. 1968. Are there pathways for protein folding? J. Chim. Phys. Phys.‐Chim. Biol. 65:44‐45.
   Main, E.R.G., Fulton, K.F., and Jackson, S.E. 1999. Folding pathway of the FKBP12 and characterisation of the transition state. J. Mol. Biol. 291:429‐444.
   Matouschek, A., Otzen, D.E., Itzhaki, L.S., Jackson, S.E., and Fersht, A.R. 1995. Movement of the position of the transition state in protein folding. Biochemistry 34:13656‐13662.
   Mayor, U., Guydosh, N.R., Johnson, C.M., Grossmann, J.G., Sato, S., Jas, G.S., Freund, S.M.V., Alonso, D.O.V., Daggett, V., and Fersht, A.R. 2003. The complete folding pathway of a protein from nanoseconds to microseconds. Nature 421:863‐867.
   Mogensen, J.E., Ipsen, H., Holm, J., and Otzen, D.E. 2004. Elimination of a misfolded folding intermediate by a single point mutation. Biochemistry 43:3357‐3367.
   Munoz, V. and Serrano, L. 1997. Development of the multiple sequence approximation within the AGADIR model of alpha‐helix formation: Comparison with Zimm‐Bragg and Lifson‐Roig formalisms. Biopolymers 41:495‐509.
   Nelson, E.D. and Grishin, N.V. 2006. Alternate pathways for folding in the flavodoxin fold family revealed by a nucleation‐growth model. J. Mol. Biol. 358:646‐653.
   Nölting, B. 2006. Protein Folding Kinetics (2nd edition). Biophysical Methods. Springer, New York.
   O'Brien, E.P., Dima, R.I., Brooks, B., and Thirumalai, D. 2007. Interactions between hydrophobic and ionic solutes in aqueous guanidinium chloride and urea solutions: Lessons for protein denaturation mechanism. J. Am. Chem. Soc. 129:7346‐7353.
   Odefey, C., Mayr, L.M., and Schmid, F.X. 1995. Non‐prolyl cis‐trans peptide bond isomerization as a rate‐determining step in protein unfolding and refolding. J. Mol. Biol. 245:69‐78.
   Oliveberg, M., Tan, Y.‐J., Silow, M., and Fersht, A.R. 1998. The changing nature of the protein folding transition state: Implications for the shape of the free energy profile for folding. J. Mol. Biol. 277:933‐943.
   Onuchic, J.N., Luthey‐Schulten, Z., and Wolynes, P.G. 2005. Theory of protein folding: The energy landscape perspective. Annu. Rev. Phys. Chem. 48:545‐600.
   Otzen, D.E. and Oliveberg, M. 1999b. Salt‐induced detour through compact regions of the protein folding landscape. Proc. Natl. Acad. Sci. U.S.A. 96:11746‐11751.
   Otzen, D.E., Itzhaki, L.S., Elmasry, N.F., Jackson, S.E., and Fersht, A.R. 1994. Structure of the transition state for the folding/unfolding of the barley chymotrypsin inhibitor 2 and its implications for mechanisms of protein folding. Proc. Natl. Acad. Sci. U.S.A. 91:10422‐10425.
   Otzen, D.E. Kristensen, O., Proctor, M., and Oliveberg, M. 1999. Structural changes in the transition state of protein folding: Alternative interpretations of curved chevron plots. Biochemistry 38:6499‐6511.
   Paci, E. and Karplus, M. 1999. Forced unfolding of fibronectin type 3 modules: An analysis by biased molecular dynamics simulations. J. Mol. Biol. 288:441‐459.
   Paci, E., Vendruscolo, M., Dobson, C.M., and Karplus, M. 2002. Determination of a transition state at atomic resolution from protein engineering data. J. Mol. Biol. 324:151‐163.
   Pappenberger, G., Aygun, H., Engels, J.W., Reimer, U., Fischer, G., and Kiefhaber, T. 2001. Nonprolyl cis peptide bonds in unfolded proteins cause complex folding kinetics. Nat. Struct. Biol. 8:452‐458.
   Ptitsyn, O.B. 1973. Stages in the mechanism of self‐organization in protein molecules. Dokl. Akad. Nauk SSSR 210:1213‐1215.
   Radford, S.E., Dobson, C.M., and Evans, P.A. 1992. The folding of hen lysozyme involves partially structured intermediates and multiple pathways. Nature 358:302‐307.
   Rea, A.M., Simpson, E.R., Crespo, M.D., and Searle, M.S. 2008a. Helix mutations stabilise a late productive intermediate on the folding pathway of ubiquitin. Biochemistry 47:8225‐8236.
   Rea, A.M., Simpson, E.R., Meldrum, J.K., Williams, H.E.L., and Searle, M.S. 2008b. Aromatic residues engineered into the β‐turn nucleation site of ubiquitin lead to a complex folding landscape, non‐native side chain interactions and kinetic traps. Biochemistry 47:12910‐12922.
   Riddle, D.S., Gantcharova, V.P., Santiago, J.V., Alm, E., Ruczinski, I., and Baker, D. 1999. Experimental and theoretical highlight role of native state topology on SH3 folding. Nature Struct. Biol. 6:1016‐1024.
   Roder, H. and Colon, W. 1997. Kinetic role of early intermediates in protein folding. Curr. Opin. Struct. Biol. 7:15‐28.
   Roder, H., Maki, K., Latypov, R.F., Cheng, H., and Ramachandra Shastry, M.C. 2005. Early events in protein folding explored by rapid mixing methods. In Protein Folding Handbook. Part I (T. Kiefhaber and J. Buchner, eds.) pp. 491‐535. Wiley‐VCH, Weinheim, Germany.
   Royer, C.A. 2006. Probing protein folding and conformational transitions with fluorescence. Chem. Rev. 106:1769‐1784.
   Sanchez, I.E. and Kiefhaber, T. 2003. Evidence for sequential barriers and obligatory intermediates in apparent two‐state protein folding. J. Mol. Biol. 325:367‐376.
   Scalley‐Kim, M. and Baker, D. 2004. Characterisation of the folding energy landscape of computer generated proteins suggests high folding free energy barriers and co‐operativity may be consequences of natural selection. J. Mol. Biol. 338:573‐583.
   Shortle, D.R. 1996. Structural analysis of non‐native states of proteins by NMR methods. Curr. Opin. Struct. Biol. 1:24‐30.
   Silow, M. and Oliveberg, M. 1997. Transient aggregates in protein folding are easily mistaken for folding intermediates. Proc. Natl. Acad. Sci. U.S.A. 94:6084‐6086.
   Simpson, E.R., Meldrum, J.K., and Searle, M.S. 2006. Engineering diverse changes in β‐turn propensities in the N‐terminal β‐hairpin of ubiquitin reveals significant effects on stability and kinetics but a robust folding transition state. Biochemistry 45:4220‐4230.
   Sosnick, T.R., Mayne, L., Hiller, R., and Englander, S.W. 1994. The barriers in protein folding. Nat. Struct. Biol. 1:149‐156.
   Sugase, K., Dyson, H.J., and Wright, P.E. 2007. Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447:1021‐1025.
   Swindells, M.B., MacArthur, M.W., and Thornton, J.M. 1995. Intrinsic phi, psi propensities of amino acids derived from the coil regions of known structures. Nat. Struct. Biol. 2:596‐603.
   Tanford, C. 1962. Contribution of hydrophobic interactions to the stability of the globular conformation of proteins. J. Am. Chem. Soc. 84:4240‐4247.
   Tang, Y.F., Grey, M.J., McKnight, J., Palmer, A.G., and Raleigh, D.P. 2006. Multi‐state folding of the Villin headpiece domain. J. Mol. Biol. 355:1066‐1077.
   Taverna, D.M. and Goldstein, R.A. 2002. Why are proteins marginally stable? Proteins 46:105‐119.
   Watters, A.L., Deka, P., Corrent, C., Callender, D., Varani, G., Sosnick, T., and Baker, D. 2007. The highly co‐operative folding of small naturally occurring proteins is likely the result of natural selection. Cell 128:613‐624.
   Wetlaufer, D.B. 1973. Nucleation, rapid folding and globular intrachain regions in proteins. Proc. Natl. Acad. Sci. U.S.A. 70:697‐701.
   Went, H.M. and Jackson, S.E. 2004. Ubiquitin folds through a highly polarised transition state. Protein Eng. 18:239‐246.
   White, G.W.N., Gianni, S., Grossman, J.G., Jemth, P., Fersht, A.R., and Daggett, V. 2005. Simulation and experiment conspire to reveal cryptic intermediates and a slide from the nucleation‐condensation to framework mechanism of folding. J. Mol. Biol. 350:757‐775.
   Wilson, C.J., Das, P., Clementi, C., Matthews, K.S., and Wittung‐Stashed, P. 2005. The experimental folding landscape of monomeric lactose repressor, a large two‐domain proytein, involves two kinetic intermediates. Proc. Natl. Acad. Sci. U.S.A. 102:14563‐14568.
   Wlodarski, T. and Zagrovic, B. 2009. Conformational selection and induced fit mechanism underlie underlie specificity in non‐covalent interactions with ubiquitin. Proc. Natl. Acad. Sci. U.S.A. 106:19346‐19351.
   Wong, K.B., Clarke, J., Bond, C.J., Neira, J.L., Freund, S.M., Fersht, A.R., and Daggett, V. 2000. Towards a complete description of the structural and dynamic properties of the denatured state of barnase and the role of residual structure in folding. J. Mol. Biol. 296:1257‐1282.
   Wright, C.F., Lindorff‐Larsen, K., Randles, L.G., and Clarke, J. 2003. Parallel protein‐unfolding pathways revealed and mapped. Nat. Struct. Biol. 8:658‐662.
   Wright, C.F., Teichmann, S.A., Clarke, J., and Dobson, C.M. 2005. The importance of sequence evolution in the aggregation and evolution of proteins. Nature 438:878‐881.
   Wüthrich, K. 1994. NMR assignments as a basis for structural characterization of denatured states of globular proteins. Curr. Opin. Struct. Biol. 4:93‐99.
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