Intramembrane substitutions in helix D of bacteriorhodopsin disrupt the purple membrane.
The Halobacterium salinarium purple membrane is a two-dimensional crystalline lattice containing bacteriorhodopsin (BR) and lipid. To test whether molecular packing within the lipid bilayer influences the structural stability of the lattice, BR mutants substituted on the membrane-embedded surface of the protein were expressed in H. salinarium. Lattice stability was assessed by equilibrium density centrifugation of cell 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: its absorption maximum was blue-shifted by < or = 4 nm relative to the wild-type lattice, its light-dark difference spectrum was normal, and its circular dichroism spectrum retained a bilobed feature characteristic of the lattice. Thus, the substitutions do not significantly alter the tertiary structure of the protein. In the low density fraction, the absorption maximum of BR was blue-shifted by 2 to 4 nm relative to the corresponding high density fraction, and the bilobed circular dichroism feature was attenuated (I117F and G116L) or absent (G113L and I117A). This suggests that the substitutions disrupt lattice stability, causing an accumulation of BR monomers or small aggregates. These results support a model in which the BR lattice is stabilized by hydrophobic packing at specific protein-protein and protein-lipid interfaces within the membrane bilayer.
Krebs
M P
MP
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison 53706-1532, USA.
Li
W
W
Halambeck
T P
TP
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D008051:Lipid Bilayers / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D018717:Purple Membrane / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D014961:X-Ray Diffraction
D008051:Lipid Bilayers; D001436:Bacteriorhodopsins