Abstract
A fundamental question in protein folding is whether proteins fold through one or multiple trajectories. While most experiments indicate a single pathway, simulations suggest proteins can fold through many parallel pathways. Here, we use a combination of chemical denaturant, mechanical force and site-directed mutations to demonstrate the presence of multiple unfolding pathways in a simple, two-state folding protein. We show that these multiple pathways have structurally different transition states, and that seemingly small changes in protein sequence and environment can strongly modulate the flux between the pathways. These results suggest that in vivo, the crowded cellular environment could strongly influence the mechanisms of protein folding and unfolding. Our study resolves the apparent dichotomy between experimental and theoretical studies, and highlights the advantage of using a multipronged approach to reveal the complexities of a protein's free-energy landscape.
Highlights
A fundamental question in protein folding is whether proteins fold through one or multiple trajectories
A fundamental question in protein folding is whether proteins fold through a single pathway or many parallel pathways
Our study resolves the apparent conflict between simulations and experiments—even though most experiments suggest a single robust pathway, we demonstrate that proteins have a choice of multiple pathways
Summary
A fundamental question in protein folding is whether proteins fold through one or multiple trajectories. We use a combination of chemical denaturant, mechanical force and site-directed mutations to demonstrate the presence of multiple unfolding pathways in a simple, two-state folding protein We show that these multiple pathways have structurally different transition states, and that seemingly small changes in protein sequence and environment can strongly modulate the flux between the pathways. Theoretical studies and molecular dynamics simulations suggest that proteins access multiple folding and unfolding trajectories, describing the native state as a kinetic hub or the bottom of a funnel[1,2,3,4,5,6] This heterogeneity of the energy landscape is rarely detected experimentally; most protein-folding experiments can be described by a simple one-dimensional reaction coordinate indicative of a single pathway[7,8,9,10]. The relationship between these potentially different trajectories is not known, and it is not clear how they relate to the pathway accessed in traditional, bulk experiments in the absence of force (the zeroforce pathway, which we will refer to as pathway B)
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