Analysis of SMALP co-extracted phospholipids shows distinct membrane environments for three classes of bacterial membrane protein

Abstract

Biological characterisation of membrane proteins lags behind that of soluble proteins. This reflects issues with the traditional use of detergents for extraction, as the surrounding lipids are generally lost, with adverse structural and functional consequences. In contrast, styrene maleic acid (SMA) copolymers offer a detergent-free method for biological membrane solubilisation to produce SMA-lipid particles (SMALPs) containing membrane proteins together with their surrounding lipid environment. We report the development of a reverse-phase LC-MS/MS method for bacterial phospholipids and the first comparison of the profiles of SMALP co-extracted phospholipids from three exemplar bacterial membrane proteins with different topographies: FtsA (associated membrane protein), ZipA (single transmembrane helix), and PgpB (integral membrane protein). The data showed that while SMA treatment per se did not preferentially extract specific phospholipids from the membrane, SMALP-extracted ZipA showed an enrichment in phosphatidylethanolamines and depletion in cardiolipins compared to the bulk membrane lipid. Comparison of the phospholipid profiles of the 3 SMALP-extracted proteins revealed distinct lipid compositions for each protein: ZipA and PgpB were similar, but in FtsA samples longer chain phosphatidylglycerols and phosphatidylethanolamines were more abundant. This method offers novel information on the phospholipid interactions of these membrane proteins.

Structure and Dynamics of GPCRs in Lipid Membranes: Physical Principles and Experimental Approaches

“…When studying endogenously expressed protein, the physiological accuracy of SMALPs is improved over other systems, as the solubilised protein is isolated with a sample of mixed lipid species, representative of the membrane into which the protein was expressed. Coupled with lipidomics, this has helped to delineate the different lipid environments within a cell and their relevance for protein function [ 289 , 290 ].…”

Conserved His-Gly motif of acid-sensing ion channels resides in a reentrant ‘loop’ implicated in gating and ion selectivity

“…While a reconstitution approach provides for a controlled and defined lipid environment, the necessity of an initial detergent-based extraction step may disrupt protein-lipid interactions integral to the structural integrity of transmembrane segments. In contrast with nanodisc reconstitution, SMA copolymers extract membrane proteins directly from the lipid bilayer, eschewing detergent entirely and permitting the study of membrane proteins in the presence of endogenous lipids (51, 52) and, in principle, maintaining the protein-lipid interactions that occur at the cell membrane (53).…”

CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups

“…It is important to note that the lipid fingerprint of yeast membranes depends on growth conditions such as temperature, carbon source and oxygen supply, and these could contribute to the quantitative difference observed between studies [ [51] , [52] , [53] ]. Furthermore, it was previously shown that SMA solubilisation is not preferential for any particular lipid subset [ [13] , [14] , [15] , 54 ]. Nevertheless, in SMA-solubilised membranes we have observed loss of the signals for sphingomyelin (d36:3) and triacylglycerol, higher diversity of phosphatidylethanolamine and phosphatidylserine lipids, and the change in signal intensities of phosphatidylcholine lipids while their heterogeneity remained the same.…”

“…It is well established that specific lipid environments are important not just for stability of many proteins but also for their function [ [10] , [11] , [12] ], including CD81. In recent years an alternative approach to detergents has been developed using styrene maleic acid co -polymer (SMA), which extracts membrane proteins with their surrounding lipids [ [13] , [14] , [15] ]. The SMA polymer inserts into a membrane and extracts a disc of lipid bilayer with the polymer wrapping around the outer edge [ 16 ].…”