Synchrotron Radiation Circular Dichroism (SRCD) Spectroscopy Investigations of the Structure and Orientation of Membrane Proteins in Oriented Lipid Bilayers

Abstract

Oriented SRCD spectroscopy is a challenging but revealing tool for investigating the structure of membrane proteins in lipid bilayers. Using the B23 beamtime at Diamond Light Source, Oxfordshire, UK, we have been developing methods for the preparation and measurement of oriented SRCD samples to characterise the structure of integral membrane proteins and their orientation with respect to the lipid bilayer. In the work presented here we outline two methods for the preparation of oriented membrane proteins. The first relies on mechanical orientation obtained by macroscopically aligning bilayers on a Suprasil quartz support. The second exploits magnetic fields to align bicelle membrane mimetics with the membrane director either parallel or perpendicular to the direction of propagation of the light beam. The merits and drawbacks of these two methods for the determination of membrane protein orientation will be discussed.As a model membrane protein for these measurements we are using the transmembrane domain (TMD) of a putative glycosyltransferase Fukutin-I (FkI), whose mislocalization has been linked to the onset of Fukuyama Muscular Dytrophy. The localisation of Fk-I in the late ER/Golgi is thought to be mediated through changes in oligomeric state and structure induced by the surrounding lipids. Using oriented samples for both SRCD and solid-state NMR we are beginning to understand how the distinct lipid composition within the late ER/Golgi may govern the retention of this protein in these compartments.