G protein-coupled membrane receptors (GPCR) are key molecules in signal transduction pathways that transmit signals of a variety of ligands such as hormones and neurotransmitters to the cell interior. They are also a major target of pharmacological intervention to aid recovery from disease. Receptor function is strongly modulated by homo-oligomerization of GPCR, hetero-oligomerization between different GPCR, as well as by interaction of GPCR with other membrane-associated proteins. The protonated GPCR bovine rhodopsin was reconstituted into proteoliposomes composed of perdeuterated lipids extracted from E. coli bacteria grown on fully deuterated medium. It was observed that intensity of small-angle neutron scattering (SANS) from entire proteoliposomes in 98% D2O buffer, measured at angles q&lt;0.008 Å-1 reached a minimum at a protein/deuterated lipid molar ratio near 1/500, while yielding maximal scattering contrast for incorporated opsin (bleached rhodopsin) over the wider angular range of 0.008 Å-1&lt;q&lt;0.3 Å-1. A Guinier analysis of the SANS spectrum gave a radius of gyration of opsin in proteoliposomes of 30.8±2.0 Å and a molecular weight of 86.5±0.9 kDA, both consistent with formation of opsin dimers. Incoherent scattering of neutrons was very low due to overall low proton content of samples. This enabled determination of a structure factor of rhodopsin dimers at good resolution. The dimer shape fits a parallelepiped with the dimensions 108.1±2.6 Å by 25.6±1.6 Å by 61.8±1.8 Å. Reconstitution of membrane proteins into liposomes that match neutron scattering length density of D2O makes SANS a powerful method for studying oligomerization.

Dimerization of a GPCR in membranes investigated by SANS

253rd National Meeting (2017)

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