To understand how proteins fold, assemble and function, it is necessary to characterize the structure and dynamics of each state they adopt during their lifetime. Experimental characterization of the transient states of proteins remains a major challenge because high-resolution structural techniques, including NMR and X-ray crystallography, cannot be directly applied to study short-lived protein states. To circumvent this limitation, we show that transient states during protein folding can be characterized by measuring the fluorescence of tryptophan residues, introduced at many solvent-exposed positions to determine whether each position is native-like, denatured-like or non-native-like in the intermediate state. We use this approach to characterize a late-folding-intermediate state of the small globular mammalian protein ubiquitin, and we show the presence of productive non-native interactions that suggest a 'flycatcher' mechanism of concerted binding and folding.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Protein Data Bank
Fersht, A.R. Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding (W.H. Freeman, New York, 1999).
Vendruscolo, M. & Dobson, C.M. Towards complete descriptions of the free-energy landscapes of proteins. Philos. Transact. A Math. Phys. Eng. Sci. 363, 433–452 (2005).
Karplus, M., Gao, Y.Q., Ma, J., van der Vaart, A. & Yang, W. Protein structural transitions and their functional role. Philos. Transact. A Math. Phys. Eng. Sci. 363, 331–356 (2005).
Jahn, T.R. & Radford, S.E. Folding versus aggregation: polypeptide conformations on competing pathways. Arch. Biochem. Biophys. 469, 100–117 (2008).
Schaeffer, R.D., Fersht, A. & Daggett, V. Combining experiment and simulation in protein folding: closing the gap for small model systems. Curr. Opin. Struct. Biol. 18, 4–9 (2008).
Bowman, G.R., Voelz, V.A. & Pande, V.S. Taming the complexity of protein folding. Curr. Opin. Struct. Biol. 21, 4–11 (2011).
Fleishman, S.J. & Baker, D. Role of the biomolecular energy gap in protein design, structure, and evolution. Cell 149, 262–273 (2012).
Korzhnev, D.M. et al. Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR. Nature 430, 586–590 (2004).
Schotte, F. et al. Watching a protein as it functions with 150-ps time-resolved x-ray crystallography. Science 300, 1944–1947 (2003).
Brockwell, D.J. & Radford, S.E. Intermediates: ubiquitous species on folding energy landscapes? Curr. Opin. Struct. Biol. 17, 30–37 (2007).
Plaxco, K.W., Simons, K.T. & Baker, D. Contact order, transition state placement and the refolding rates of single domain proteins. J. Mol. Biol. 277, 985–994 (1998).
Sanchez, I.E. & Kiefhaber, T. Evidence for sequential barriers and obligatory intermediates in apparent two-state protein folding. J. Mol. Biol. 325, 367–376 (2003).
Zarrine-Afsar, A. et al. Theoretical and experimental demonstration of the importance of specific non-native interactions in protein folding. Proc. Natl. Acad. Sci. USA 105, 9999–10004 (2008).
Capaldi, A.P., Kleanthous, C. & Radford, S.E. Im7 folding mechanism: misfolding on a path to the native state. Nat. Struct. Biol. 9, 209–216 (2002).
Krishna, M.M. & Englander, S.W. A unified mechanism for protein folding: predetermined pathways with optional errors. Protein Sci. 16, 449–464 (2007).
Friel, C.T., Smith, D.A., Vendruscolo, M., Gsponer, J. & Radford, S.E. The mechanism of folding of Im7 reveals competition between functional and kinetic evolutionary constraints. Nat. Struct. Mol. Biol. 16, 318–324 (2009).
Weber, G. Fluorescence-polarization spectrum and electronic-energy transfer in proteins. Biochem. J. 75, 345–352 (1960).
Royer, C.A. Probing protein folding and conformational transitions with fluorescence. Chem. Rev. 106, 1769–1784 (2006).
Smith, C.J. et al. Detection and characterization of intermediates in the folding of large proteins by the use of genetically inserted tryptophan probes. Biochemistry 30, 1028–1036 (1991).
Vallée-Bélisle, A. & Michnick, S.W. Multiple tryptophan probes reveal that ubiquitin folds via a late misfolded intermediate. J. Mol. Biol. 374, 791–805 (2007).
Beechem, J.M. Picosecond fluorescence decay curves collected on millisecond time scale: direct measurement of hydrodynamic radii, local/global mobility, and intramolecular distances during protein-folding reactions. Methods Enzymol. 278, 24–49 (1997).
Gu, H. et al. Robustness of protein folding kinetics to surface hydrophobic substitutions. Protein Sci. 8, 2734–2741 (1999).
Jackson, S.E. Ubiquitin: a small protein folding paradigm. Org. Biomol. Chem. 4, 1845–1853 (2006).
Hicke, L., Schubert, H.L. & Hill, C.P. Ubiquitin-binding domains. Nat. Rev. Mol. Cell Biol. 6, 610–621 (2005).
Went, H.M., Benitez-Cardoza, C.G. & Jackson, S.E. Is an intermediate state populated on the folding pathway of ubiquitin? FEBS Lett. 567, 333–338 (2004).
Khorasanizadeh, S., Peters, I.D. & Roder, H. 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 (1996).
Krantz, B.A. & Sosnick, T.R. Distinguishing between two-state and three-state models for ubiquitin folding. Biochemistry 39, 11696–11701 (2000).
Roder, H., Maki, K., Cheng, H. & Shastry, M.C. Rapid mixing methods for exploring the kinetics of protein folding. Methods 34, 15–27 (2004).
Sosnick, T.R., Dothager, R.S. & Krantz, B.A. Differences in the folding transition state of ubiquitin indicated by phi and psi analyses. Proc. Natl. Acad. Sci. USA 101, 17377–17382 (2004).
Vallée-Bélisle, A., Turcotte, J.F. & Michnick, S.W. raf RBD and ubiquitin proteins share similar folds, folding rates and mechanisms despite having unrelated amino acid sequences. Biochemistry 43, 8447–8458 (2004).
Rea, A.M., Simpson, E.R., Crespo, M.D. & Searle, M.S. Helix mutations stabilize a late productive intermediate on the folding pathway of ubiquitin. Biochemistry 47, 8225–8236 (2008).
Khorasanizadeh, S., Peters, I.D., Butt, T.R. & Roder, H. Folding and stability of a tryptophan-containing mutant of ubiquitin. Biochemistry 32, 7054–7063 (1993).
Went, H.M. & Jackson, S.E. Ubiquitin folds through a highly polarized transition state. Protein Eng. Des. Sel. 18, 229–237 (2005).
Semisotnov, G.V. et al. Study of the “molten globule” intermediate state in protein folding by a hydrophobic fluorescent probe. Biopolymers 31, 119–128 (1991).
Jennings, P.A. Speeding along the protein folding highway, are we reading the signs correctly? Nat. Struct. Biol. 5, 846–848 (1998).
Callis, P.R. & Liu, T. Quantitative prediction of fluorescence quantum yields for tryptophan in proteins. J. Phys. Chem. B 108, 4248–4259 (2004).
Briggs, M.S. & Roder, H. Early hydrogen-bonding events in the folding reaction of ubiquitin. Proc. Natl. Acad. Sci. USA 89, 2017–2021 (1992).
Clementi, C. & Plotkin, S.S. The effects of non-native interactions on protein folding rates: theory and simulation. Protein Sci. 13, 1750–1766 (2004).
Wolynes, P.G., Onuchic, J.N. & Thirumalai, D. Navigating the folding routes. Science 267, 1619–1620 (1995).
Onuchic, J.N. & Wolynes, P.G. Theory of protein folding. Curr. Opin. Struct. Biol. 14, 70–75 (2004).
Oliveberg, M. & Wolynes, P.G. The experimental survey of protein-folding energy landscapes. Q. Rev. Biophys. 38, 245–288 (2005).
Sorenson, J.M. & Head-Gordon, T. Toward minimalist models of larger proteins: a ubiquitin-like protein. Proteins 46, 368–379 (2002).
Sugase, K., Dyson, H.J. & Wright, P.E. Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447, 1021–1025 (2007).
Shoemaker, B.A., Portman, J.J. & Wolynes, P.G. Speeding molecular recognition by using the folding funnel: the fly-casting mechanism. Proc. Natl. Acad. Sci. USA 97, 8868–8873 (2000).
Baker, D. A surprising simplicity to protein folding. Nature 405, 39–42 (2000).
Watters, A.L. et al. The highly cooperative folding of small naturally occurring proteins is likely the result of natural selection. Cell 128, 613–624 (2007).
Korzhnev, D.M., Religa, T.L., Banachewicz, W., Fersht, A.R. & Kay, L.E. A transient and low-populated protein-folding intermediate at atomic resolution. Science 329, 1312–1316 (2010).
Wensley, B.G. et al. Experimental evidence for a frustrated energy landscape in a three-helix-bundle protein family. Nature 463, 685–688 (2010).
Krantz, B.A., Dothager, R.S. & Sosnick, T.R. Discerning the structure and energy of multiple transition states in protein folding using psi-analysis. J. Mol. Biol. 337, 463–475 (2004).
Sosnick, T.R., Krantz, B.A., Dothager, R.S. & Baxa, M. Characterizing the protein folding transition state using psi analysis. Chem. Rev. 106, 1862–1876 (2006).
Vijay-Kumar, S., Bugg, C.E. & Cook, W.J. Structure of ubiquitin refined at 1.8 A resolution. J. Mol. Biol. 194, 531–544 (1987).
Maxwell, K.L. et al. Protein folding: defining a “standard” set of experimental conditions and a preliminary kinetic data set of two-state proteins. Protein Sci. 14, 602–616 (2005).
Bofill, R., Simpson, E.R., Platt, G.W., Crespo, M.D. & Searle, M.S. Extending the folding nucleus of ubiquitin with an independently folding beta-hairpin finger: hurdles to rapid folding arising from the stabilisation of local interactions. J. Mol. Biol. 349, 205–221 (2005).
The authors acknowledge A. Bonham for help with Mathematica; T. Sosnick, H. Roder, K. Plaxco, F.-X. Campbell-Valois, S. Chteinberg, J.W. Keillor, H. Bhaskarah, C. Lawrence and H. Watkins for helpful discussions; M. Fyfe for sequencing; and J.W. Keillor for providing access to the stopped-flow apparatus. This work was supported by the National Science and Engineering Research Council of Canada (Grant 194582-SWM). A.V.-B. acknowledges the financial support of the Fonds Québécois de Recherche Nature et Technologies.
The authors declare no competing financial interests.
About this article
Cite this article
Vallée-Bélisle, A., Michnick, S. Visualizing transient protein-folding intermediates by tryptophan-scanning mutagenesis. Nat Struct Mol Biol 19, 731–736 (2012). https://doi.org/10.1038/nsmb.2322
Evolution of Conformation and Dynamics of Solvents in Hydration Shell along the Urea-induced Unfolding of Ubiquitin
Chinese Journal of Polymer Science (2019)
Scientific Reports (2018)
The effects of organic solvents on the folding pathway and associated thermodynamics of proteins: a microscopic view
Scientific Reports (2016)
Nature Communications (2015)