Probing Determinants of Protein Folding Cooperativit

The problem of how a protein's three dimensional structure is encoded in its primary sequence of amino acids has long been studied yet remains to be elucideated. It has been known for over a century that pressure leads to the unfolding of proteins. High pressure is used to probe protein folding cooperativity. Pressure leads to unfolding of proteins because of solvent excluded voids that are present and inhomogeneously distributed in the folded state and that are eliminated in the unfolded state. The inhomogeneous distribtioin of voids leads to a local effect of pressure, in contrast to chemical denaturants whose effects are proportional to the accessible surface area of the unfolded state, leading to a global unfolding effect. Here we probe the effects of internal cavities on folding pathways and cooperativity of a model linear repeat protein, pp32. For WT pp32, high pressure NMR experiments revealed that cooperativity was highest at 293K, and deviation from two-state behavior unfolding at higher and lower temperatures. Coarse-grained simulations constrained by NMR data allowed full characterization of the free energy profile confirming folding via intermediate states that had only been previously inferred from kinetic data. High pressure NMR studies of cavity variants showed that the effect of the introduction of internal voids on folding cooperativity depended on the context in which the cavity was placed. Creating a cavity in the less stable N-terminal region of the protein caused a large deviation from two-state behavior and the population of an intermediate. Placing a cavity in the center repeat of the protein led to general destabilization in all regions of the protein, and the population of a distinct intermediate. Finally, creation of a cavity in the stable C-terminal region of the protein destabilized the more stable C-terminal region, such that folding cooperativity was enhanced with respect to the WT protein. Preliminary high pressure studies have been initiated on two model globular proteins, ACBP and protein G, which were shown to have very low change in volume. By performing high pressure experiments at multiple urea concentrations, we compared the pressure "m-value" to that of force, urea or temperature unfolding. For protein G, high pressure NMR was used to determine folding cooperativity on the amino acid level. These results show that high pressure studies can greatly aid studies of protein folding cooperativity.