Structural studies on Halobacterium halobium bacteriorhodopsin.
Bacteriorhodopsin, isolated from Halobacterium halobium, is an intrinsic membrane protein containing covalently attached retinal and driven by a cyclic light-driven protonmotive force. In order to extend three-dimensional analyses already in progress and to define the covalent features of such proteins, the determination of the primary structure of bacteriorhodopsin has been initiated, using techniques that can be applied to the sequence analyses of other intrinsic membrane proteins as well. The amino acid composition of bacteriorhodopsin prepared by new procedures compares well to that previously published. Various delipidation procedures have been examined for their effectiveness in yielding protein in a physical state that is susceptible to fragmentation by a number of chemical and proteolytic agents. Organic extraction, particularly in acetone:ammonium hydroxide, has proved most useful. Detergent extraction was effective in delipidating but rendered the protein inert to attack by various cleavage reagents. Sequence information was obtained from fragments isolated from several hydrolytic preparations of low percent cleavage. The most complete fragmentation was obtained from hydrolysis of bacteriorhodopsin previously modified with succinic or maleic anhydride. Both solubility and fragmentation susceptibility were enhanced by these reactions and several other derivatives of bacteriorhodopsin are currently being investigated. The behavior of bacteriorhodopsin under the conditions examined, as well as the sequence information obtained, suggest that bacteriorhodopsin is mostly contained within the lipid milieu of the membrane and may represent a class of membrane proteins that do not contain appreciable hydrophilic domains.
D000596:Amino Acids / Q000032:analysis; D001436:Bacteriorhodopsins / Q000302:isolation & purification / Q000378:metabolism; D002338:Carotenoids / Q000378:metabolism; D003488:Cyanogen Bromide; D006217:Halobacterium / Q000378:metabolism*; D006358:Hot Temperature; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis
D000596:Amino Acids; D010446:Peptide Fragments; D002338:Carotenoids; D001436:Bacteriorhodopsins; D003488:Cyanogen Bromide