Lessons about Protein Folding and Binding from Archetypal Folds

The function of proteins as biological nanomachines relies on their ability to fold into complex 3D structures, bind selectively to partners, and undergo conformational changes on cue. The native functional structures, and the rates of interconversion between conformational states (folded-unfolded, bound-free), are all encoded in the physical chemistry of their amino acid sequence. However, despite extensive research over decades, this code has proven difficult to fully crack, in terms of both prediction and understanding the molecular mechanisms at play. Earlier work on single-domain proteins reported a commonality of slow rates (10–2–102 s–1) and simple behavior in both kinetic and thermodynamic unfolding experiments, which suggested the process was all-or-none and thereby analogous to a chemical reaction (e.g., A ⇄ B). In the absence of a first-principles pre-exponential factor for protein (un)folding dynamics, the rates could only be interpreted in relative terms, e.g., the changes induced by mutation, and hence, neither the height of nor the entropic contribution to the free energy barriers was known. The rates were also many orders of magnitude too slow for direct atomistic simulations, and the computational focus was on predicting rate changes induced by mutation via coarse grained simulations. However, even the effects of mutation proved to be strikingly homogeneous with all experimental data clustering at ∼1/3 of the free energy perturbation recovered on folding and ∼2/3 on unfolding. The implementation of ultrafast kinetic methods turned the field upside down because they allowed researchers to measure the time scales of elementary (un)folding motions, which set the pre-exponential factor for protein conformational transitions at ∼1 μs. In parallel, we and others set out to investigate the simplest possible protein structures capable of autonomous folding, which we defined as archetypal folds. The rationale was to recapitulate the hierarchical organization of protein structure, starting from the bottom up. The study of fold archetypes ended up opening new research avenues in protein (un)folding, but also making unexpected connections with the folding upon binding of intrinsically disordered proteins and suggesting their functioning as conformational rheostats. This Account describes our work on the kinetic, thermodynamic, mechanistic, and functional analysis of fold archetypes. We first discuss the kinetic studies, emphasizing their impact on our understanding of (un)folding rates, of barrierless (downhill) folding, and as benchmarks for atomistic simulations. We continue with the thermodynamic analysis, introducing the differential scanning calorimetry, multiprobe, and NMR approaches that we developed to dissect their gradual, minimally cooperative (un)folding transitions and to probe the underlying mechanisms with unprecedented detail. The last two sections cover single-molecule analyses and some recent, mostly computational, results on the exploration of possible biological and technological roles for the gradual conformational transitions of fold archetypes.
V.M. acknowledges support from the European Research Council (ERC-2012-ADG- 323059), the National Science foundation (NSF-MCB-1616759), the CREST Center for Cellular and Biomolecular Machines (NSF-CREST-1547848), and the W.M. Keck Foundation.