Propagation of Load Through Proteins via Steered Molecular Dynamics: Effect on Fluctuation Dynamics and Architecture

Abstract

Mechanical load in the form of externally applied forces and/or moments are known to regulate biochemical activity in proteins through induced conformational changes exposing cryptic binding sites, altered kinetics, and, in extreme cases, unfolding. It is unknown, however, how load propagates through protein structure from remotely applied forces to local regions: do loads manifest as changes in fluctuations, architecture or both? Single molecule force spectroscopy (SMFS) studies have demonstrated protein unfolding with nanometer- and piconewton-level resolution, yet provide little information about the intra-protein pathways of load. SMFS studies of Green Flourescent Protein (GFP) have demonstrated the anisotropy of deformation, providing an ideal test system for studying the structural response of proteins to load. To explore the manifestation of load in proteins via simulation of a controlled experiment, constant-force steered molecular dynamics (SMD) are used to generate probability density functions (PDFs) at atomic-level resolution describing the structure of GFP when subjected to simple, sub-unfolding, externally applied loads along the vectors used in SMFS studies. Here the focus is on describing protein structure (both vibrational and architectural) with increasing forces; the experimental unfolding values provide a top-force validation of the simulation results. Total data include PDFs for both equilibrium and non-equilibrium loaded scenarios permitting comparison for the effect of loads. Data are analyzed for principal components (via PCA) to determine fluctuation dynamics and changes in structure architecture along the spectrum of applied loads. PCA data allow comparison of both fluctuation amplitude and direction through the power spectra of mode covariance overlap. Results indicate areas of increased strain under load, predicting locations of unfolding via increased strain and demonstrating the anisotropic pathways of load at sub-unfolding loads. Intra-molecular constitutive properties are calculated as derivatives of free energy.