Chapter 14
Solubilization and Stabilization of Native Membrane
Proteins for Drug Discovery
Vincent Corvest and Anass Jawhari
Abstract
Membrane proteins (MPs) are stable in their native lipid environment. To enable structural and functional
investigations, MPs need to be extracted from the membrane. This is a critical step that represents the main
obstacle for MP biochemistry and structural biology. Here we describe detergent solubilization screening of
MPs using dot-blot and Western-blot analyses. Good solubilization conditions are ranked for their best
capacity to stabilize MPs using thermal shift assay. The protein functionality is evaluated by radioligand
binding (for G-protein-coupled receptor) and ATPase activity (ABC Transporter) and finally the aggregation status as well as protein homogeneity are assessed by Native-polyacrylamide gel, chemical cross-linking,
and size exclusion chromatography.
Key words Membrane proteins, Solubilization, Stabilization, Detergent, Native-PAGE, Size exclusion chromatography, Chemical-crosslinking, Function
1 Introduction
Membrane proteins (MP) represent ~30% of human proteins
[1]. They are crucial for cellular physiology as they are directly
involved in cell adhesion, cell–cell communication, signal transduction, and transport [1]. This is why large majority (~70%) of
therapeutic targets are membrane proteins (MPs) [2]. To better
understand MP function and to enable drug development using
structure-based drug design or fragment-based drug discovery,
MPs need to be extracted from their membrane environment
[3]. MPs extraction and solubilization represent the most critical
step of MPs isolation [4], since depending on the used surfactant/
detergent, they can go through significant structural constraints
which may significantly compromise their structural and functional
integrities. To date, no detergent has been reported which tackles
membrane protein solubilization and stabilization universally.
Therefore, it is essential to screen for the most suitable detergent
[5]. Here we show that solubilization efficiency of different
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_14, © Springer Science+Business Media, LLC, part of Springer Nature 2021
257
Solubilization and Stabilization of Native Membrane
Proteins for Drug Discovery
Vincent Corvest and Anass Jawhari
Abstract
Membrane proteins (MPs) are stable in their native lipid environment. To enable structural and functional
investigations, MPs need to be extracted from the membrane. This is a critical step that represents the main
obstacle for MP biochemistry and structural biology. Here we describe detergent solubilization screening of
MPs using dot-blot and Western-blot analyses. Good solubilization conditions are ranked for their best
capacity to stabilize MPs using thermal shift assay. The protein functionality is evaluated by radioligand
binding (for G-protein-coupled receptor) and ATPase activity (ABC Transporter) and finally the aggregation status as well as protein homogeneity are assessed by Native-polyacrylamide gel, chemical cross-linking,
and size exclusion chromatography.
Key words Membrane proteins, Solubilization, Stabilization, Detergent, Native-PAGE, Size exclusion chromatography, Chemical-crosslinking, Function
1 Introduction
Membrane proteins (MP) represent ~30% of human proteins
[1]. They are crucial for cellular physiology as they are directly
involved in cell adhesion, cell–cell communication, signal transduction, and transport [1]. This is why large majority (~70%) of
therapeutic targets are membrane proteins (MPs) [2]. To better
understand MP function and to enable drug development using
structure-based drug design or fragment-based drug discovery,
MPs need to be extracted from their membrane environment
[3]. MPs extraction and solubilization represent the most critical
step of MPs isolation [4], since depending on the used surfactant/
detergent, they can go through significant structural constraints
which may significantly compromise their structural and functional
integrities. To date, no detergent has been reported which tackles
membrane protein solubilization and stabilization universally.
Therefore, it is essential to screen for the most suitable detergent
[5]. Here we show that solubilization efficiency of different
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_14, © Springer Science+Business Media, LLC, part of Springer Nature 2021
257