Site-Specific Determination of Conformational Flexibility from a Side Chain Perspective: Native State Thiol Exchange of E. Coli Ribonuclease H

Abstract

Tools such as crystallography and hydrogen exchange (HX) have revealed a wealth of knowledge about protein function, folding, and dynamics. Here we explore the use of thiol exchange (SX) to gain further insight into the energy landscape of E. coli ribonuclease H (RNase H). Similar to HX, SX investigates the solvent accessibility and conformational fluctuations of specific positions in a protein, but while HX measures exchange of the backbone amide proton, SX takes advantage of cysteine's unique reactivity to measure solvent accessibility of the side chain. Native state SX results for a hyperstabilized mutant of E. coli RNase H reveal a partially unfolded form (PUF) at equilibrium with the native state, as is seen by HX. The structured regions of the PUFs measured by the two techniques agree overall, with some slight differences due to probing the side chain rather than the backbone. Moreover, while for some positions the SX experiments revealed this equilibrium information, the same experiments yielded direct kinetic information about protein opening events. Thus, in one set of experiments we have measured both kinetic and equilibrium parameters describing the folding of E. coli RNase H.