Stability of transmembrane regions in bacteriorhodopsin studied by progressive proteolysis.
Proteinase K digestions of bacteriorhodopsin were carried out with the aim of characterizing the membrane-embedded regions of the protein. Products of digestions for two, eight or 24 hours were separated by high-pressure liquid chromotography. A computerized search procedure was used to compare the amino acid analyses of peptide-containing peaks with segments of the bacteriorhodopsin sequence. Molecular weight distributions of the products were determined by sodium dodecylsulfate-urea polyacrylamide gel electrophoresis. The structural integrity of the protein after digestion was monitored through the visible absorption spectrum, by X-ray diffraction of partially dried membranes, and by following release of biosynthetically-incorporated 3H leucine from the digested membranes. During mild proteolysis, bacteriorhodopsin was cleaved near the amino and carboxyl termini and at two internal regions previously identified as being accessible to the aqueous medium. Longer digestion resulted in cleavage at new sites. Under conditions where no fragments of bacteriorhodopsin larger than 9000 mol wt were observed, a significant proportion of the digested membranes retained diffraction patterns similar to those of native purple membranes. The harshest digestion conditions led to complete loss of the X-ray diffraction patterns and optical absorption and to release of half the hydrophobic segments of the protein from the membrane in the form of small soluble peptides. Upon cleavage of aqueous loop regions of the protein, isolated transmembrane segments may experience motion in a direction perpendicular to the plane of the membrane, allowing them access to protease.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001436:Bacteriorhodopsins / Q000032:analysis* / Q000378:metabolism; D002338:Carotenoids / Q000032:analysis*; D002462:Cell Membrane; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D010450:Endopeptidases / Q000378:metabolism; D006217:Halobacterium / Q000648:ultrastructure; D010446:Peptide Fragments / Q000032:analysis; D012984:Software; D013997:Time Factors; D014961:X-Ray Diffraction
D000596:Amino Acids; D010446:Peptide Fragments; D002338:Carotenoids; D001436:Bacteriorhodopsins; D010450:Endopeptidases; D019286:Endopeptidase K