INTRAMEMBRANE SUBSTITUTIONS IN HELIX-D OF BACTERIORHODOPSIN DISRUPT THE PURPLE MEMBRANE

The Halobacterium salinarium purple membrane is a two-dimensional crys talline lattice containing bacteriorhodopsin (BR) and lipid. To test w hether molecular packing within the lipid bilayer influences the struc tural stability of the lattice, BR mutants substituted on the membrane -embedded surface of the protein were expressed in H. salinarium. Latt ice stability was assessed by equilibrium density centrifugation of ce ll lysates containing similar amounts of BR, BR was distributed in low (1.12 to 1.15 g/ml) and high density (1.18 g/ml) membrane fractions. The high density fraction comprised 89% of the total BR in wild-type, but only 19% (G113L), 29% (I117A), 52% (G116L) and 79% (I117F) in the mutants. In each case, this fraction contained BR in a lattice form: i ts absorption maximum was blue-shifted by less than or equal to 4 nm r elative to the wild-type lattice, its light-dark difference spectrum w as normal, and its circular dichroism spectrum retained a bilobed feat ure characteristic of the lattice. Thus, the substitutions do not sign ificantly alter the tertiary structure of the protein. In the low dens ity fraction, the absorption maximum of BR was blue-shifted by 2 to 4 nm relative to the corresponding high density fraction, and the bilobe d circular dichroism feature was attenuated (I117F and G116L) or absen t (G113L and I117A). This suggests that the substitutions disrupt latt ice stability, causing an accumulation of BR monomers or small aggrega tes. These results support a model in which the BR lattice is stabiliz ed by hydrophobic packing at specific protein-protein and protein-lipi d interfaces within the membrane bilayer. (C) 1997 Academic Press Limi ted.