Mapping protein energy landscapes with X-ray footprinting mass spectrometry.

Abstract

Understanding how the linear sequence of a protein can encode the diversity of protein structures and functions has been a central goal of biology for the last half-century. Recent advances now allow one to predict the three-dimensional structure for a given protein sequence with incredible accuracy; the sequence of a protein, however, encodes much more than just this native structure - it encodes the entire energy landscape - an ensemble of conformations whose populations (energetics) and dynamics are finely tuned. Therefore, proteins should not be thought of as single static structures but as statistical ensembles. To truly predict protein function from sequence, we must also understand protein energy landscapes. Unfortunately, current methods for measuring protein stability suffer from several limitations, requiring milligram quantities of purified protein in isolation at micromolar concentrations. We have developed an approach based on x-ray radical footprinting mass spectrometry (XF-MS), in combination with chemical denaturation, that overcomes these challenges and can accurately measure protein stabilities using ∼103 fold less protein, including following the stabilities of individual proteins in complex mixtures. Importantly, we can follow the energetics of individual regions in a protein allowing us to study complex multidomain proteins refractory to standard tools for measuring protein stability. Together, this approach enables us to dramatically increase the number of proteins for which we can quantitatively measure energy landscapes.