Observing and Characterizing Early Folding Intermediates of E.Coli Rnase H using Kinetic and Equilibrium Approaches

Abstract

E. coli ribonuclease H has been an important model system for protein folding for many years. It has long been known that it folds through an intermediate (Icore) en route to the native state. However, because folding to Icore occurs on the fast millisecond timescale, and there is very little Icore populated under equilibrium conditions, key questions have remained unanswered: What is the nature of the folding pathway en route to Icore, and how well packed is Icore?In this work, a new ultrarapid mixing technique coupled with time-correlated single photon counting detection of intrinsic fluorescence allows observation of the millisecond folding to Icore, including new early intermediates formed along the way. Mutagenesis and hydrogen exchange methods provide structural information about these new early intermediates. Additionally, two equilibrium models of Icore are made and characterized, providing new insight into the nature of this intermediate. This work increases our knowledge about the folding pathway and energy landscape of an important protein folding model system, at a time- and size-scale that is amenable to comparison with protein folding simulations.