Structural biology relies on several powerful techniques, but these tend to be limited in their ability to characterize protein fluctuations and mobility. Over-reliance on structural approaches can lead to omission of critical information regarding biological function. Currently there is a need for complementary biophysical methods to visualize these mobile aspects of protein function. Here we review hydrostatic and osmotic pressure-based techniques to address this shortcoming for the paradigm of rhodopsin. Hydrostatic and osmotic pressure data contribute important examples which are interpreted in terms of an energy landscape for hydration-mediated protein dynamics. We find that perturbations of rhodopsin conformational equilibria by force-based methods are not unrelated phenomena; rather they probe various hydration states involving functional proton reactions. Hydrostatic pressure acts on small numbers of strongly interacting structural or solvent-shell water molecules with relatively high energies, while osmotic pressure acts on large numbers of weakly interacting bulk-like water molecules with low energies. Local solvent fluctuations due to the hydration shell and collective water interactions affect hydrogen-bonded networks and domain motions that are explained by a hierarchical energy landscape model for protein dynamics.
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