Revisiting Protein Folding at the Single Molecule Level

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Determining the mechanism by which a protein folds remains a primary goal in biology. Statistical theories of protein folding have long predicted plausible mechanisms for reducing the vast conformational space through distinct ensembles of structures. However, these predictions have remained untested experimentally, since the multiplicity of trajectories and folding structures is averaged out using bulk techniques. Moreover, most intermediate conformations are only transiently present, rendering their isolation and characterization difficult by commonly used spectroscopic methods. Owing to recent advances in single molecule force-clamp spectroscopy, we are now able to probe the structure and dynamics of the small protein ubiquitin by measuring its length, mechanical stability and effect of solvent environment during each stage of folding. Here we discover that upon hydrophobic collapse, the protein rapidly selects a subset of non-native like, minimum energy structures that are mechanically weak and insensitive to the solvent environment. From this much reduced ensemble, the native state is acquired through a barrier-limited transition. The existence of such heterogeneous ensemble of minimum energy collapsed states was theoretically proposed by lattice simulations to be a milestone in the process of narrowing the available conformational space of a protein during its journey to the native fold, and a general feature of proteins that are naturally designed through evolution to fold on biological timescales. Here we demonstrate that such ensemble of collapsed states is also apparent in our experiments in the well-characterized I27 and Protein L proteins, albeit on different timescales, thus suggesting that their presence is ubiquitous to other mechanically stable proteins with a well-defined fold. Our results present the first experimental evidence for the validity of statistical mechanics models in describing the folding of small proteins on biological timescales.