Lipid Bilayer Induces Contraction of the Denatured State Ensemble of a Helical-Bundle Membrane Protein

Abstract

Defining the denatured state ensemble (DSE) and intrinsically disordered proteins is essential to understanding protein folding, chaperone action, degradation, translocation and cell signaling. While a majority of studies have focused on water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we reconstituted the DSE of a helical bundle membrane protein GlpG of Escherichia coli in native lipid bilayers and measured its conformation and compactness. The DSE was obtained using steric trapping, which couples spontaneous denaturation of a doubly biotinylated GlpG to binding of two bulky monovalent streptavidin molecules. Using limited proteolysis and mass spectrometry, we mapped the flexible regions in the DSE. Using our paramagnetic biotin derivative and double electron-electron resonance spectroscopy, we determined the dimensions of the DSE. Finally, we employed our Upside model for molecular dynamics simulations to generate the DSE including the collapsed and fully expanded states in a bilayer. We find that the DSE is highly dynamic involving the topology changes of transmembrane segments and their unfolding. The DSE is expanded relative to the native state, but only to 55-90% of the fully expanded condition. The degree of expansion depends on the chemical potential with regards to local packing and the lipid composition. Our result suggests that the native lipid bilayer promotes the association of helices in the DSE of membrane proteins and, probably in general, facilitating interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.

Methods to study folding of alpha-helical membrane proteins in lipids

“…For GlpG a similar trapping method was tested in detergent micelles and the fluorescence change of a pyrene/DABCYL FRET pair during steric trapping were extrapolated and calculated to be 4.7–5.8 kcal mol −1 , which was significantly lower than the SDS denaturation ΔGU in DDM micelles, which was calculated to be 8.4–8.7 kcal mol −1 [57]. In further work, double mSA bound and denatured GlpG was reconstituted from micelles into both bicelles composed of a 3 : 1 mix of DMPC and the negative charged DMPG (1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol)) lipids plus the detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), and liposomes of E. coli phospholipids [58]. The sterically trapped GlpG remained denatured in both bilayer environments, though could refold to an active conformation on the removal of the mSA.…”