Protein folding at single-molecule resolution

Abstract

The protein folding reaction carries great significance for cellular function and hence continues to be the research focus of a large interdisciplinary protein science community. Single-molecule methods are providing new and powerful tools for dissecting the mechanisms of this complex process by virtue of their ability to provide views of protein structure and dynamics without associated ensemble averaging. This review briefly introduces common FRET and force methods, and then explores several areas of protein folding where single-molecule experiments have yielded insights. These include exciting new information about folding landscapes, dynamics, intermediates, unfolded ensembles, intrinsically disordered proteins, assisted folding and biomechanical unfolding. Emerging and future work is expected to include advances in single-molecule techniques aimed at such investigations, and increasing work on more complex systems from both the physics and biology standpoints, including folding and dynamics of systems of interacting proteins and of proteins in cells and organisms. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.

Fig. 1

Several areas of interest in the protein folding field linked to the concept of the protein folding funnel.

Fig. 2

T4 lysozyme folding cooperativity investigated using optical tweezers. A. The experimental setup, with the protein attached to beads via long DNA handles, and using optical trap for manipulation. B. Unfolding (red) and refolding (blue) force-extension curves, with the inset showing data at higher resolution, showing variability in folding-unfolding behavior. Adapted by permission from Macmillan Publishers Ltd: Nature, Shank et al. [36] 465:637 (2010), copyright 2010.

Fig. 3

Equilibrium, dynamic and binding-folding behavior of an intrinsically disordered protein studied using single-molecule fluorescence. α-Synuclein denaturation demonstrates non-cooperative expansion (top, adapted from [26], Ferreon et al. Methods Enzymol. (2010) 472:179). SDS binding reveals multistate folding and varied dynamics (middle, adapted from [55], Ferreon et al. Proc. Natl. Acad. Sci. USA, (2009) 106:5645). The Parkinson’s disease-linked mutation A30P substantially alters α-synuclein binding-folding landscape (bottom, adapted from [56], Ferreon et al. Angew. Chem. Int. Ed. (2010) 49:3469).