Unfolding of the Villin Headpiece Domain: Revealing Structural Heterogeneity with Time-Resolved X-ray Solution Scattering and Markov State Modeling.

Abstract

Understanding protein folding pathways is crucial to deciphering the principles of protein structure and function. Here, we investigate the unfolding dynamics of the 35-residue villin headpiece (HP35) and a norleucine-substituted variant (2F4K) using a combination of experimental and computational techniques. Time-resolved X-ray solution scattering (TRXSS) coupled with equilibrium Molecular Dynamics (MD) simulations and Markov State modeling reveals distinct unfolding mechanisms between the two variants: HP35 and 2F4K. Specifically, HP35 exhibits a two-state unfolding process, whereas an intermediate state was identified for the 2F4K mutant. A Markov state model constructed from simulations was used to map atomic-level transitions to experimental observations, providing insights into the role of sequence variations in modulating folding pathways. Our findings underscore the importance of integrating experimental and computational approaches to unravel protein unfolding mechanisms between heterogenous structural ensembles.