Energetics-Based Methods for Protein Folding and Stability Measurements

Abstract

Over the past 15 years, a series of energetics-based techniques have been developed for the thermodynamic analysis of protein folding and stability. These techniques include Stability of Unpurified Proteins from Rates of amide H/D Exchange (SUPREX), pulse proteolysis, Stability of Proteins from Rates of Oxidation (SPROX), slow histidine H/D exchange, lysine amidination, and quantitative cysteine reactivity (QCR). The above techniques, which are the subject of this review, all utilize chemical or enzymatic modification reactions to probe the chemical denaturant– or temperature-induced equilibrium unfolding properties of proteins and protein-ligand complexes. They employ various mass spectrometry-, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)-, and optical spectroscopy–based readouts that are particularly advantageous for high-throughput and in some cases multiplexed analyses. This has created the opportunity to use protein folding and stability measurements in new applications such as in high-throughput screening projects to identify novel protein ligands and in mode-of-action studies to identify protein targets of a particular ligand.