Project Details
Projekt
Lipid interactions of the human G-protein-coupled receptor chemokine receptor CXCR1 in asymmetric vesicles
Applicant
Dr. Sebastian Fiedler
Subject Area
Biophysics
Term
from 2014 to 2017
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 262245765
Most biological membranes are highly asymmetric. For example, the outer leaflets of mammalian cellular membranes contain mainly phosphatidylcholine and sphingomyelin, whereas phosphatidylethanolamine and phosphatidylserine predominate in the inner leaflet. In addition to modulating the structure of membranes, lipid asymmetry affects the orientation of membrane proteins, the formation of nanodomains, the binding of proteins to the membrane, and many other vital processes. However, until recently, no straightforward approach has been available to create asymmetric lipid vesicles, which are indispensable systems for in vitro studies of protein-lipid interactions. In the proposed project, we utilize a novel assay based on lipid transfer by methyl-beta-cyclodextrin (MbetaCD) to generate asymmetric proteoliposomes containing a human G-protein-coupled receptor (GPCR). We systematically alter the lipid components of the outer monolayers of proteoliposomes such as to resemble that of eukaryotic cellular membranes. Thereby, we analyze the influence on ligand binding and GPCR oligomerization. In addition, we explore how the receptor influences the propensity of different types of lipids to partition into the outer leaflet of lipid bilayers. As a model protein, we study the chemokine receptor CXCR1, a major mediator of immune and inflammatory responses. CXCR1 selectively binds interleukin-8 (IL-8), which is suggested to directly involve the lipid-water interface, as the receptor N-terminus dissociates from the membrane upon binding of IL-8. To shed more light on the molecular determinants of IL-8-CXCR1 binding, we apply isothermal titration calorimetry (ITC) to obtain thermodynamic profiles of the binding reaction in different types of asymmetric proteoliposomes. Such profiles provide key parameters for drug-discovery studies including binding affinity, stoichiometry, enthalpy, and entropy. Notably, no thermodynamic profiles currently exist for any ligand-GPCR interaction in a lipid-bilayer environment. To address how membrane asymmetry affects CXCR1 oligomerization, we monitor the supramolecular organization of ligand-bound CXCR1 with the help of electron paramagnetic resonance (EPR) spectroscopy. To this end, we acquire pulsed EPR spectra using double electron-electron resonance (DEER) to measure interspin distances of spin-labeled IL-8 protomers bound to CXCR1. Moreover, we analyze how CXCR1 influences the propensity of different types of lipids to partition into the outer leaflet of bilayer membranes utilizing an ITC-based lipid-exchange assay. From both ITC and ESR spectroscopy, we deduce how different lipid micro-environments affect supramolecular receptor assemblies, which has far-reaching implications for the formation of functional nanodomains in living cells.
DFG Programme
Research Fellowships
International Connection
Canada
