Using HDX-MS to probe the dynamics and denatured state of GlpG, a helical membrane protein.

Abstract

The denatured state ensemble (DSE) of membrane proteins remains poorly characterized despite its importance in understanding protein folding, thermodynamics, degradation, chaperone action, and other cellular processes. While recent strides have been made in understanding the relationship between the lipid bilayer and the transmembrane domains of helical proteins in the DSE, open questions remain, including whether helices within the membrane can unfold and whether they can leave the membrane entirely or in part. Here, we apply Hydrogen-Deuterium Exchange (HDX) mass spectrometry to probe the dynamics of E. coli helical membrane protein GlpG to complement our previous study that used a combination of proteolysis, DEER and simulations to probe the DSE. Our HDX-MS data provide a much higher resolution picture of the DSE and permit the quantification of the free energies of the structural openings that lead to HDX of helices. Notably, we find that some helices are extremely stable while others are seen to exit the bilayer, if only temporarily, via pistoning motions. Moreover, in order to exchange, these helices are also undergoing at least temporary unfolding as they move in and out of the bilayer. These results expand on the canonical model of membrane protein folding in which helices fold before entering the membrane by characterizing the dynamics of transmembrane helices moving between the native and denatured states.