High-throughput stability screening for detergent-solubilized membrane proteins

Vadim Kotov,Thomas C Marlovits,Udaya K Tiruttani Subhramanyam,Isabel Moraes,Jörg Labahn,Katharina Veith,Maria M Garcia-Alai,Inokentijs Josts,Christian Günther,Henning Tidow,Kim Bartels,Christian Löw

doi:10.1038/s41598-019-46686-8

Abstract

Protein stability in detergent or membrane-like environments is the bottleneck for structural studies on integral membrane proteins (IMP). Irrespective of the method to study the structure of an IMP, detergent solubilization from the membrane is usually the first step in the workflow. Here, we establish a simple, high-throughput screening method to identify optimal detergent conditions for membrane protein stabilization. We apply differential scanning fluorimetry in combination with scattering upon thermal denaturation to study the unfolding of integral membrane proteins. Nine different prokaryotic and eukaryotic membrane proteins were used as test cases to benchmark our detergent screening method. Our results show that it is possible to measure the stability and solubility of IMPs by diluting them from their initial solubilization condition into different detergents. We were able to identify groups of detergents with characteristic stabilization and destabilization effects for selected targets. We further show that fos-choline and PEG family detergents may lead to membrane protein destabilization and unfolding. Finally, we determined thenmodynamic parameters that are important indicators of IMP stability. The described protocol allows the identification of conditions that are suitable for downstream handling of membrane proteins during purification.

Highlights

Numerous studies have addressed the gap between the large number of sequences representing integral membrane proteins (IMPs) in sequenced genomes[1] and the 863 unique known membrane protein structures reported up-to-date
Slotboom et al have addressed the behavior of IMPs in detergent micelles and lipid/detergent micelles in size exclusion chromatography (SEC)-LS analysis compared to sedimentation equilibrium centrifugation experiments[26]
Four IMPs belong to the major facilitator superfamily of transporters; DtpA, DgoT, and MdfA involved in nutrient and drug transport[33,34,35,36,37,38], and LacY is the well-characterised lactose permease[39]

Summary

Introduction

Numerous studies have addressed the gap between the large number of sequences representing integral membrane proteins (IMPs) in sequenced genomes (up to 30 percent)[1] and the 863 unique known membrane protein structures reported up-to-date (http://blanco.biomol.uci.edu/mpstruc/) This highlights the difficulties along the path to structure determination such as low expression levels, solubilization from the native lipid bilayer and stability in a new membrane mimetic[2,3,4]. Other studies have evaluated the stability of membrane proteins following the change of intrinsic fluorescence during thermal denaturation using a differential scanning fluorimetry device for the screening of lipid-like peptides[29] and ligands[30,31,32,33]. This will guide the selection of the appropriate detergent for further downstream processing of the IMP

Methods

Results

Conclusion