Characterization of phospholipid compositions and physical properties of DMPC/bacteriorhodopsin vesicles produced by a detergent-free method

Abstract

Homogeneous complexes of bacteriorhodopsin (BR) from Halobacterium halobium purple membrane (PM) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) have been produced by a detergent-free process in which bovine liver non-specific phospholipid transfer protein (nsTP) promotes net transfer of DMPC from small unilamellar vesicles directly into PM. The number of DMPC molecules incorporated per BR monomer follows a close to linear dependence with the relative proportions of DMPC and PM added to the initial mixture over the ranges studied. The resulting complexes, with total lipid phosphate/BR contents of between 31:1 and 152:1 (mole/mole), were purified free from any remaining unincorporated DMPC by sucrose density gradient centrifugation. Broad line 31P-NMR spectra and partitioning studies with the nitroxide spin label, Tempo, confirm that the BR and DMPC coexist in bilayer complexes. Quantitative analysis of high resolution 31P-NMR spectra from complexes after solubilization in 4% SDS revealed 74-84% of the major PM phospholipid to be retained in the complexes.