An engineered thermal-shift screen reveals specific lipid preferences of eukaryotic and prokaryotic membrane proteins

Emmanuel Nji,Michael Landreh,David Drew,Yurie Chatzikyriakidou

doi:10.1038/s41467-018-06702-3

Emmanuel Nji, Michael Landreh + Show 2 more

Open Access

https://doi.org/10.1038/s41467-018-06702-3

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Abstract

Membrane bilayers are made up of a myriad of different lipids that regulate the functional activity, stability, and oligomerization of many membrane proteins. Despite their importance, screening the structural and functional impact of lipid–protein interactions to identify specific lipid requirements remains a major challenge. Here, we use the FSEC-TS assay to show cardiolipin-dependent stabilization of the dimeric sodium/proton antiporter NhaA, demonstrating its ability to detect specific protein-lipid interactions. Based on the principle of FSEC-TS, we then engineer a simple thermal-shift assay (GFP-TS), which facilitates the high-throughput screening of lipid- and ligand- interactions with membrane proteins. By comparing the thermostability of medically relevant eukaryotic membrane proteins and a selection of bacterial counterparts, we reveal that eukaryotic proteins appear to have evolved to be more dependent to the presence of specific lipids.

Highlights

Membrane bilayers are made up of a myriad of different lipids that regulate the functional activity, stability, and oligomerization of many membrane proteins
We have recently investigated lipid binding to sodium/proton antiporters using a combination of nondenaturing mass spectrometry (MS) and molecular dynamics (MD) simulations
Dodecyl-β-D-maltopyranoside (DDM) purified NhaA– and NapA–green fluorescent protein (GFP) fusions were heated over a range of different temperatures, aggregates sedimented by centrifugation, and the supernatant subjected to size-exclusion chromatography on an high performance liquid chromatography (HPLC)-system coupled with an in-line fluorescence detector

Summary

Introduction

Membrane bilayers are made up of a myriad of different lipids that regulate the functional activity, stability, and oligomerization of many membrane proteins. Mass spectrometry (MS) has gained traction as a tool for unraveling the intricate connections between membrane proteins and lipids[4], shedding light on different lipid binding modes[5], lipid-mediated protein stabilization[6] and even thermodynamics and allosteric effects of protein–lipid interactions[7,8] It has proven powerful for studying individual protein–lipid contacts and is progressing toward the analysis of complex mixtures[9], MS, like other spectroscopic and computational approaches, is to date not well-suited for identifying specific interactions in native membranes or in high-throughput formats, hampering any large-scale investigations. Thermofluor-based thermal-shift assays are high-throughput strategies widely used for screening for the binding of small molecule libraries[10,11] They cannot cope with unpurified samples, require large amounts of purified protein, and sometimes give uninterpretable results[12]. We compare the sensitivity of bacterial and eukaryotic membrane proteins to the presence of various physiologically relevant lipids and identify selectively stabilizing interactions

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