Monomer-oligomer equilibrium of bacteriorhodopsin in reconstituted proteoliposomes. A freeze-fracture electron microscope study.
An improved freeze-fracture electron microscope procedure has been developed and applied to the study of the association of bacteriorhodopsin in large proteoliposomes reconstituted by reverse-phase evaporation with egg lecithin. Due to the improved accuracy and resolution of this procedure, intramembrane particles, the diameter of which (4.5 nm) closely matched that of bacteriorhodopsin monomer, could be observed at high lipid to protein ratios (greater than or equal to 40 w/w). At lower lipid to protein ratios, larger particles (diameter 7.5 nm) progressively appeared, resulting in bimodal particle size distributions up to a lipid to protein ratio of 1, where the large particles were the sole species present. These large particles were interpreted as corresponding to bacteriorhodopsin oligomers. Because of the large size and homogeneity of proteoliposomes, accurate particle density measurements could be performed. These confirmed the occurrence of a lipid to protein ratio-dependent bacteriorhodopsin monomer-oligomer equilibrium and further allowed us to identify the oligomer as a trimer or a tetramer. In complementary experiments, it was found that the bacteriorhodopsin monomer and oligomer had identical visible CD spectra and light-induced proton pumping rates. However, a large increase of the proton passive leak rate of proteoliposomes was found to be associated with oligomer formation. The appearance of these oligomers may be important as the first step in the formation of two-dimensional crystals of bacteriorhodopsin.
Gulik-Krzywicki
T
T
Centre de Génétique Moléculaire du Centre National de la Recherche Scientifique, Gif sur Yvette, France.
Seigneuret
M
M
Rigaud
J L
JL
D001436:Bacteriorhodopsins; D002942:Circular Dichroism; D005614:Freeze Fracturing; D008563:Membrane Lipids; D008565:Membrane Proteins; D008854:Microscopy, Electron; D011510:Proteolipids
D008563:Membrane Lipids; D008565:Membrane Proteins; D011510:Proteolipids; C020485:proteoliposomes; D001436:Bacteriorhodopsins