Polymer-based nanodiscs for studying structure and dynamics of G-protein-coupled receptors and other clinically relevant membrane proteins

• Applicant: Dr. Jana Bröcker
• Subject Area: Structural Biology
• Term: from 2014 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 269790842

Integral membrane proteins require membrane mimetics to remain soluble in aqueous solutions under in vitro conditions. Most often, this involves the use of detergents during both the solubilization of proteins from biological membranes as well as their subsequent biochemical, biophysical, or structural characterization. Unfortunately, many pharmacologically relevant but notoriously labile membrane proteins, such as G protein coupled receptors (GPCRs), are unstable and inactive in detergent micelles. This interferes with the determination of physiologically relevant high-resolution structures and limits our understanding of the inner workings of these proteins. Recently, a copolymer synthesized from styrene and maleic anhydride has been shown to solubilize membrane proteins without the need of detergents. In doing so, the polymer forms nanometer-sized bilayer discs by wrapping around the protein and its native lipid environment. In this proposal, we use this polymer technology for the detergent free solubilization, stabilization, and crystallization of unmodified GPCRs and other pharmacologically relevant but labile membrane proteins. We explore the potential of this strategy, thereby avoiding extensive protein modifications, which otherwise are often used to enhance stability but may entail poorly predictable structural and functional consequences. As a proof-of-principle, we have demonstrated that these polymers efficiently solubilize the GPCR bovine rhodopsin from native membranes and that the polymer nanodiscs provide a more membrane like environment for rhodopsin than detergent micelles do. Here, we examine the ability of different types of polymers to form stable and functional nanodiscs with well characterized model proteins as well as their suitability for crystallization in lipid cubic phases. Then, we expand the technique to analyze more challenging membrane proteins, which are typically unstable in detergent micelles under in vitro conditions and have thus far evaded a thorough functional and structural examination. To this end, we chose the GPCR human peripheral cannabinoid receptor CB2 and the ABC transporter human cystic fibrosis transmembrane conductance regulator (CFTR), which are involved in the origin of multiple sclerosis and cystic fibrosis, respectively. Following a combined biochemical and biophysical approach including, among others, X ray crystallography and electron paramagnetic resonance spectroscopy, we deduce biophysical and structural information on these crucial proteins. This improves our understanding of their signaling mechanisms initiated by ligand binding, protein protein interactions, and membrane thickness or composition. The availability of native-state structures of membrane proteins will have far reaching implications for the discovery of drugs with higher selectivity and affinity.

• DFG Programme: Research Fellowships
• International Connection: Canada
• Host: Professor Dr. Oliver-Peter Ernst