Analysis of protein stability by hydrogen exchange

Stability measurements are frequently performed to establish a polypeptide as a folded protein and to study the forces that lead to its folding. Conventional denaturation methods used for the analysis of protein stability have the following experimental limitations: (1) stability can only be measured under conditions where the protein is partially unfolded, (2) the energetics of localized fluctuations can not be easily measured, (3) many proteins can not be analyzed because they aggregate during the course of denaturation, (4) stability measurements can not be obtained in complex mixtures, extracts or living cells and (5) denaturation experiments are time-consuming and not amenable to high-throughput analysis. These constraints limit the number of biological problems that can be addressed through stability measurements. Here, we develop a number of hydrogen exchange based approaches to overcome these limitations. We demonstrate the accuracy and practicality of the methods by analyzing the thermodynamics of a number of model proteins. The developed techniques are then used in a number of biologically relevant applications.; Through fluorescence stopped-flow analysis we show that monomeric lambda repressor (λ_6–85) folds very rapidly in a two state fashion. These properties allow us to obtain thermodynamic hydrogen exchange information in the presence of a range of denaturant concentrations by NMR. This information is used to elucidate the mechanisms of hydrogen exchange. We show that NMR-detected hydrogen exchange (NMR-HX) can be used to accurately measure the global stability as well as the folding thermodynamics of localized regions within the protein. We then develop a matrix assisted laser desorption/ionization mass spectrometry protocol for measuring hydrogen exchange (MALDI-HX). The residue-specific information obtained by NMR-HX is used to develop the equations necessary for interpretation of MALDI-HX data in terms of protein stability. We use MALDI-HX to rapidly measure stabilities in crude extracts, examine protein-protein interactions and analyze the stability of proteins within the cytoplasm of E. coli. Finally, we correlate thermodynamic stabilities to in vivo degradation rates. Together, the results make a number of novel observations about the nature of hydrogen exchange and protein folding thermodynamics.