Volume changes in protein folding: A Modular Approach

Abstract

Pressure effects of protein conformational transitions, which are manifest via the associated changes in molar volume, remain rather poorly understood, and regrettably so given that they are clearly linked to changes in the magnitude and type of hydration that accompany such transitions. Given that the role of solvent in protein energetics and structural dynamics remains one of the key questions in the field of protein folding, a better understanding of the physical basis for the volume changes that accompany folding reactions could provide direct means to quantify this differential solvation. We have recently begun high pressure studies on the folding of the ankyrin repeat domain of the protein Notch bearing 7 ankyrin repeats (Nank7) and a series of smaller constructs differing is the number of repeats, and/or their sequences. We reasoned that such a system would provide a means of incrementally testing the role of the size of the hydrophobic core, and the importance of the specific amino acid sequence in determining the volume change upon unfolding, ΔVu. We present here a complete P-T equilibrium and p-jump kinetic study of the full-length Nank7 construct, as well as on a number of the smaller constructs. We find that the volume change of the full-length construct depends strongly upon temperature, and that the expansivity of the transition state ensemble is similar to that of the unfolded state, while its molar volume is closer to that of the folded state. Preliminary results on two very small constructs indicate that the total volume change is a linear combination of the volume change associated with the unfolding of each individual repeat.