The Effect of Detergent, Temperature, and Lipid on the Oligomeric State of MscL Constructs: Insights from Mass Spectrometry

Abstract

The mechanosensitive channel of large conductance (MscL) acts as an emergency release valve for osmotic shock of bacteria preventing cell lysis. The large pore size, essential for function, requires the formation of oligomers with tetramers, pentamers, or hexamers observed depending on the species and experimental approach. We applied non-denaturing (native) mass spectrometry to five different homologs of MscL to determine the oligomeric state under more than 50 different experimental conditions elucidating lipid binding and subunit stoichiometry. We found equilibrium between pentameric and tetrameric species, which can be altered by detergent, disrupted by binding specific lipids, and perturbed by increasing temperature (37°C). We also established the presence of lipopolysaccharide bound to MscL and other membrane proteins expressed in Escherichia coli, revealing a potential source of heterogeneity. More generally, we highlight the use of mass spectrometry in probing membrane proteins under a variety of detergent-lipid environments relevant to structural biology.

Structural and Functional Basis for Lipid Synergy on the Activity of the Antibacterial Peptide ABC Transporter McjD

“…In addition to phospholipids, a second unexpected series of peaks with adduct masses of ϳ3.3 kDa was observed. We attribute these adducts to lipopolysaccharides (LPS), in agreement with a recently published study where it was shown that MscL, an inner membrane protein, has the tendency to bind to outer membrane lipids or lipopolysaccharides (21). Given that LPS are larger than phospholipids or CARD, consisting of several hydrocarbon chains, their size implies hydrophobic binding to the surface of the protein.…”

Spectator no more, the role of the membrane in regulating ion channel function

“…The closed crystal structure of pentameric M. tuberculosis MscL [4] has been known for several years. Native mass spectrometry of MscL suggested the in vitro oligomerisation state (pentamer vs tetramer) is influenced by temperature, detergent and amino acid variability as well as by the precise construct used [18]. Protein stability has been shown to be influenced by the presence of particular lipids [19].…”

Mass spectrometry-enabled structural biology of membrane proteins

“…It is becoming more and more apparent (from EM, X-ray crystallography and MS studies [22,96,[98][99][100]) that there is an intimate relationship between MPs and the surrounding lipid environment, whereby the binding of key lipids is important for maintaining MP structure and function [98]. Detergents are a poor mimic of the native bilayer, may not permit the retention of MP-lipid interactions [101], and in many cases disturb the correct, functional oligomeric state of MP assemblies [90,95,96].…”

“…Detergents are a poor mimic of the native bilayer, may not permit the retention of MP-lipid interactions [101], and in many cases disturb the correct, functional oligomeric state of MP assemblies [90,95,96]. Consequently, a variety of detergent-free methods have been developed to solubilize MPs for biophysical analyses, namely nanodiscs [102], bicelles [103], liposomes [104], styrene maleic acid lipid particles (SMALPs) (also called lipodisq) [93] and amphipols (Fig.…”

“…Recently there has been an upsurge in the use of other non-ionic detergents for native MS analysis of MPs, for example, Triton X-100, tetraethylene glycol monooctyl ether (C8E4), octaethylene glycol monododecyl ether (C12E8), lauryldimethylamine N-oxide (LDAO), and n-octyl-b-D-glucoside (b-OG) (Fig. 4) as it has been shown that these detergent micelles dissociate at much lower activation energies, often resulting in MP ions that are more native-like [95][96][97]. These detergent micelles may be dissociated more easily than those formed by DDM as their non-ionic nature means that the only stabilizing forces are hydrophobic interactions which are weakened in the gas-phase, whilst DDM and b-OG micelles are also stabilized by hydrogenbonding [95].…”