Detection of protein unfolding and fluctuations by native state hydrogen exchange

Abstract

Publisher Summary Hydrogen exchange can monitor protein fluctuations and opening events in many protons throughout the molecule simultaneously. Protein amide hydrogen exchange is an extremely powerful technique for studies on protein folding, stability, and structure. It allows probing different regions of a protein simultaneously and has a high degree of sensitivity. Because of this, amide hydrogen exchange presents a unique merging of energetic and structural information. Recently, several studies have examined protection as a function of dilute amounts of denaturant. This technique is termed “native state hydrogen exchange” as the small levels of denaturant have no influence on the protein when monitored by standard probes of stability, such as circular dichroism or fluorescence. These new studies reveal that hydrogen exchange from an amide site in proteins can be modeled by two different processes. This chapter shows how this new method greatly increases the understanding of protein energetics and dynamics using the example of E. coli ribonuclease H1* (RNase H*). The data on RNase H* were collected at guanidine concentrations in which RNase H* appears fully native by circular dichroism. The hydrogen exchange samples contained up to 1.3 M guanidine, but the unfolding transition starts around 1.5 M guanidine. These data demonstrate the problem with trying to interpret hydrogen exchange results without following exchange as a function of denaturant.