Computational modelling of protein structure and stability using molecular dynamics simulations with enhanced sampling*

Protein structure and stability are frequently studied in vitro, while proteins naturally occur in crowded cellular environments that include other proteins, DNA, and numerous organelles. As a result, we lack a quantitative understanding of protein structure and stability in cells. In this work, we carry out extensive molecular dynamics simulations with enhanced sampling methods based on potential energy rescaling and Hamiltonian replica exchange to determine the accuracy of measurements of the melting temperature T_m. The ability to accurately measure the melting temperature will allow us to determine changes in protein structure and stability caused by different cellular environments. We show that we can accurately determine the change in melting temperature ΔT_m in vitro between wildtype peptides and those with point mutations, e.g. chignolin and cln025 and wwdomain and several single-point mutants. We also illustrate the usage of a small number of FRET experimental restraints in MD simulations to pin down the complete structure of the peptides. These techniques will be applied to proteins in the presence of large crowding molecules in future studies.