In Cell Footprinting Coupled with Mass Spectrometry for the Structural Characterization of a Membrane Protein

Abstract

Membrane proteins are assembled through balanced interactions among protein, lipids and water. Studying their folding while maintaining the native lipid environment has been a necessary but challenging. Here we present a set of methods for analyzing key elements in membrane protein folding, including thermodynamic stability, compactness of the unfolded state and unfolding cooperativity under native conditions. The methods are based on steric trapping which couples unfolding of a doublybiotinylated protein to binding of monovalent streptavidin (mSA). We further advanced this technology for general application by developing versatile biotin probes possessing spectroscopic reporters that are sensitized by mSA binding or protein unfolding. By applying those methods to an intramembrane protease GlpG, we elucidated a widely unraveled unfolded state, subglobal unfolding of the region encompassing the active site, and a network of cooperative and localized interactions for maintaining the stability. These findings provide crucial insights into the folding energy landscape of membrane proteins.