Interrogating Membrane Protein Conformational Dynamics within Native Lipid Compositions

Abstract

The interplay between membrane proteins and the lipids of the membrane is important for cellular function, however, tools enabling the interrogation of protein dynamics within native lipid environments are scarce and often invasive. We show that the styrene–maleic acid lipid particle (SMALP) technology can be coupled with hydrogen–deuterium exchange mass spectrometry (HDX‐MS) to investigate membrane protein conformational dynamics within native lipid bilayers. We demonstrate changes in accessibility and dynamics of the rhomboid protease GlpG, captured within three different native lipid compositions, and identify protein regions sensitive to changes in the native lipid environment. Our results illuminate the value of this approach for distinguishing the putative role(s) of the native lipid composition in modulating membrane protein conformational dynamics.

Mass spectrometry-enabled structural biology of membrane proteins

“…For example, the hydrophobic transmembrane segments of MPs can be inherently difficult to digest and observe by LC-MS, although this is often protein and digestion condition dependent [189], and does not always preclude structural analyses. Recent studies on detergent and lipid solubilized proteins have demonstrated that both are compatible with HDX-MS [181,[190][191][192][193][194][195].…”

“…The conformational dynamics of the F 0 F 1 ATP synthase during catalysis have also been studied by HDX-MS using protein solubilized in inside-out membrane vesicles, revealing that the rotor shaft is destabilized when pumping protons against a transmembrane gradient [194]. More recently, the SMALP platform was implemented to study the rhomboid protease GlpG in different lipid conditions, to identify regions of the protein sensitive to the lipid environment [195].…”

“…nanodiscs, bicelles or liposomes), the lipids are often removed prior to MS analyses to prevent them interfering with ionization and LC separation [43,198], though more recent work suggests that this is not always necessary [181,[192][193][194][195]199]. HDX-MS in nanodiscs has been used to study propeptide binding to c glutamyl carboxylase [192], and to study the conformational dynamics underlying the alternating access mechanism of LeuT [193].…”

Structural Mass Spectrometry of Membrane Proteins within Their Native Lipid Environments

“…Recently,H DX-MSh as been employed to study membrane proteins within native nanodiscs. [13] Readinga nd colleagues successfully analyzedt he conformational dynamics of the Escherichiacoli rhomboid protease, GlpG, within native nanodiscs ( Figure 2). Sufficient sequence coverageo fupt o8 0% was achieved through optimizationo fq uenching and digestion conditions, asw ell as the overall HDX-MS workflow.M oreover,t he utility of native nanodiscs enabledt he HDX structural interrogation of GlpG within different native lipid environments (generated using cell lines with differing lipid compositions or by altering cell growth temperatures during protein expression).…”

“…There were significant differences in fatty acid chainunsaturation and small differences in chain length between the two systems-C 16 possessed higher abundances of unsaturated chainsa nd as malli ncrease in longer chain length quantityc omparedt oC 37 .M onitoring the temporal differences in HDX( DHDX) of the GlpG membraneprotein within the two differentn ativelipid systems revealed regions that were conformationallysensitive to alterationstot heir native lipid environment.P arts of this Figurewas previously publishedb yW iley-VCH by Readingetal. [13] Membrane proteins have been studied within isolated biological membranes using structural MS, albeit not extensively. Ap ioneering study by Konermann and colleagues was the first to demonstrate that FPOP-MS could inform on membrane protein structure within an atural lipide nvironment (Figure 3).…”