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Visualizing transient protein-folding intermediates by tryptophan-scanning mutagenesis


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.

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Figure 1: A tryptophan-scanning strategy to characterize the transient intermediate states of proteins.
Figure 2: Tryptophan substitution at solvent-exposed sites on a protein surface minimally perturbs protein stability and serves as a sensitive probe to monitor folding and unfolding of a protein.
Figure 3: Folding kinetics of ubiquitin probed using 27 solvent-exposed tryptophan mutants.
Figure 4: Fluorescence-based representation of the IL using ω values.
Figure 5: Ubiquitin folds via a late intermediate (IL) through the formation of non-native interactions.

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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.

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A.V.-B. performed experiments and mathematical modeling. A.V.-B. and S.W.M. designed experiments, analyzed results, and wrote the manuscript.

Corresponding author

Correspondence to Stephen W Michnick.

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The authors declare no competing financial interests.

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Supplementary Figures 1–5, Supplementary Tables 1–3 and Supplementary Notes (PDF 8825 kb)

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Vallée-Bélisle, A., Michnick, S. Visualizing transient protein-folding intermediates by tryptophan-scanning mutagenesis. Nat Struct Mol Biol 19, 731–736 (2012).

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