Abstract
Membrane proteins have a range of crucial biological functions and are the target of about 60% of all prescribed drugs. For most studies, they need to be extracted out of the lipid-bilayer, e.g. by detergent solubilisation, leading to the loss of native lipids, which may disturb important protein-lipid/bilayer interactions and thus functional and structural integrity. Relipidation of membrane proteins has proven extremely successful for studying challenging targets, but the identification of suitable lipids can be expensive and laborious. Therefore, we developed a screen to aid the high-throughput identification of beneficial lipids. The screen covers a large lipid space and was designed to be suitable for a range of stability assessment methods. Here, we demonstrate its use as a tool for identifying stabilising lipids for three membrane proteins: a bacterial pyrophosphatase (Tm-PPase), a fungal purine transporter (UapA) and a human GPCR (A2AR). A2AR is stabilised by cholesteryl hemisuccinate, a lipid well known to stabilise GPCRs, validating the approach. Additionally, our screen also identified a range of new lipids which stabilised our test proteins, providing a starting point for further investigation and demonstrating its value as a novel tool for membrane protein research. The pre-dispensed screen will be made commercially available to the scientific community in future and has a number of potential applications in the field.
Highlights
Integral membrane proteins are intimately associated with biological, lipid-based membranes
The lipidic environment of the biological membrane acts as the scaffold in which membrane proteins are embedded but can affect protein stability and correct folding as well as oligomerisation and function
It was designed to be a versatile tool for lipid screening and can be used for stability testing, functional assays and structural characterisation for example via protein crystallisation using vapour diffusion or high lipid-detergent (HiLiDe) methods [42]
Summary
Integral membrane proteins are intimately associated with biological, lipid-based membranes. They are involved in a variety of cellular processes crucial for organism survival such as catalysis, signal transduction and transport of ions or small biomolecules in and out of the cell. The RAMP lipid screen salaries for authors (CN, EP, JH, ST) and provided both the lipids used in the screen as well as the screen plates themselves but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section
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