A Novel Trp Cage Conformer Revealed by Combining High Pressure NMR and MD Simulations

Abstract

We have used the tryptophan cage variant Tc5b, a widely used model protein for benchmarking of force fields and water models, to probe pressure effects on proteins using a combination of high pressure 2D proton NMR TOCSY and molecular dynamics simulations. Understanding the key factors of the pressure stability of protein is an essential step towards the development of better force fields and increased predictive power of simulation. We show that results of MD simulations on Tc5b as a function of pressure using the Amber 99sb force field show remarkable agreement with the pressure-dependent NMR data. Pressure perturbation in different solvent conditions yielded novel information on the structural changes in Tc5b induced by pressure. We identified a sub-ensemble in the folded basin that is strongly destabilized by pressure. Hence, most of the pressure effect on Tc5b between 1-2500 bar arises from modulation of the sub-populations of the folded state. The residues observed in the NMR experiments to be strongly affected by pressure coincided precisely with those implicated in the pressure-dependent change in Tc5b secondary structure revealed by the simulations. The relative populations of two folded state conformers, in which the central 3-10 helix and the following bend are inverted, changed upon pressurization, while the total secondary structural content remained the same. These results highlight the existence of significant plasticity in the secondary structure of folded proteins.