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

Alvin C. K. Teo,Naomi L. Pollock,Andrew R. Pitt,Sarah C. Lee,Zoe Stroud,Stephen Hall,Timothy R. Dafforn,Corinne M. Spickett,David I. Roper,Alpesh Thakker

doi:10.1038/s41598-018-37962-0

Alvin C. K. Teo, Naomi L. Pollock + Show 8 more

Open Access

https://doi.org/10.1038/s41598-018-37962-0

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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.

Highlights

During division[7], and for FtsA and ZipA as well as PgpB, the local membrane lipid environment is likely to be critical for their correct structure and function
We showed that E. coli membrane proteins ZipA, FtsA and PgpB are amenable to the solubilisation and purification using the 2:1 styrene maleic acid co-polymer (SMA) copolymer (Supplementary Fig. 2); to the best of our knowledge, the work on PgpB represents the first report to-date of an integral membrane lipid phosphatase directly extracted and examined in a detergent-free membrane mimetic system
The solubilisation efficiency of the 2:1 SMA copolymer was previously reported to be comparable to DDM for several other bacterial membrane proteins overexpressed in E. coli, namely ZipA, BmrA, and LeuT38

Summary

Introduction

During division[7], and for FtsA and ZipA as well as PgpB, the local membrane lipid environment is likely to be critical for their correct structure and function. Until recently it has been difficult to study them in a native condition or determine the associated lipids, because of the extraction and reconstitution methods commonly used for membrane proteins. The SMA reagent extracts the protein within the portion of membrane that surrounds it, thereby in theory preserving the native lipid environment[18]. This assertion has been strengthened by the first high resolution cryo-EM structure of a SMALP solubilised protein, bacterial alternative complex III, which showed the electron densities of several phospholipids tightly bound to the protein, the precise phospholipid class was not confirmed[19]. A key goal was to determine whether the integral membrane protein (PgpB) associates with different membrane phospholipids to the bitopic protein ZipA and peripheral membrane protein FtsA

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