Rhodopsin Oligomerization in Synthetic Lipid Bilayers and Native Cellular Membranes as Studied by DEER of a Spin-labeled Retinal Analog

Abstract

Oligomerization of integral membrane proteins is often the final step of protein folding. This final step is likely to be affected by a number of factors including biophysical interactions with lipids and other proteins and so is the oligomers’ stability and their further organization into clusters/superlattices. Thus, the protein oligomers observed in different membrane mimetics including synthetic liposomes could be different from those formed in native membranes. Here we employed DEER spectroscopy to measure a distribution of long range (ca., 2-7 nm) constraints between specific protein sidechains labeled with paramagnetic tags. Spin-labeled derivative of retinal (4-[2,2,5,5-tetramethyl-1- pyrrolidinyloxy-3-carboxyl]-retinal) was synthesized and added to apo-rhodospsin for a tight non-covalent binding similar to the native retinal. Such an approach eliminated the need for covalent modification of proteins with conventional nitroxides. Further, the retinal binding is a useful probe for oligomerization of functional rhodopsins as the protein is known to lose the retinal upon denaturing. Thus, non-functional rhodopsins would be EPR-silent. Experimental DEER signals from such spin-labeled rhodopsin revealed significant differences in protein oligomerization in synthetic vs. native membranes. Specifically, while oligomers with well-defined distances were found to form in DMPC-DMPA, some shorter distances were determined to be present in the native cellular membranes. The latter distances were attributed to smaller oligomers (dimers, trimers, etc.) that could be intermediates to larger oligomers or incomplete oligomers which further oligomerization was terminated by the membrane defects and/or other protein components. The work at NCSU was supported by U.S. DOE Contract DE-FG02-02ER15354.