Experimental and theoretical high pressure strategies for investigating protein-nucleic acid assemblies

A method was developed to investigate the stability of protein-nucleic acid complexes using hydrostatic pressure during electrophoretic gel mobility shift analysis. The initial system probed by this technique was the well-characterized cognate BamHI-DNA complex. Band shift analysis at several elevated pressures found the equilibrium dissociation (K_d) constant to be dependent on pressure, which allowed the volume change of dissociation (ΔV) to be calculated. In order to describe the effects of pressure on the specific BamHI-DNA complex at the molecular level, molecular dynamics simulations at both ambient and elevated pressure was performed. Comparison of the simulation trajectories identified several individual BamHI-DNA contacts that are disrupted due to pressure. The disruption of these contacts can be attributed to an observed pressure-induced increase in hydration at the protein-DNA interface during the elevated pressure simulation.