Conformational Fluctuations in GTP-Bound K-Ras: A Metadynamics Perspective with Harmonic Linear Discriminant Analysis.

Biomacromolecules often undergo significant conformational rearrangements during function. In proteins, these motions typically consist in nontrivial, concerted rearrangement of multiple flexible regions. Mechanistic, thermodynamics, and kinetic predictions can be obtained via molecular dynamics simulations, provided that the simulation time is at least comparable to the relevant time scale of the process of interest. Because of the substantial computational cost, however, plain MD simulations often have difficulty in obtaining sufficient statistics for converged estimates, requiring the use of more-advanced techniques. Central in many enhanced sampling methods is the definition of a small set of relevant degrees of freedom (collective variables) that are able to describe the transitions between different metastable states of the system. The harmonic linear discriminant analysis (HLDA) has been shown to be useful for constructing low-dimensional collective variables in various complex systems. Here, we apply HLDA to study the free-energy landscape of a monomeric protein around its native state. More precisely, we study the K-Ras protein bound to GTP, focusing on two flexible loops and on the region associated with oncogenic mutations. We perform microsecond-long biased simulations on the wild type and on G12C, G12D, G12 V mutants, describe the resulting free-energy landscapes, and compare our predictions with previous experimental and computational studies. The fast interconversion between open and closed macroscopic states and their similar thermodynamic stabilities are observed. The mutation-induced effects include the alternations of the relative stabilities of different conformational states and the introduction of many microscopic metastable states. Together, our results demonstrate the applicability of the HLDA-based protocol for the conformational sampling of multiple flexible regions in folded proteins.