Refolding of bacteriorhodopsin. Protease V8 fragmentation and chromophore reconstitution from proteolytic V8 fragments.
Staphylococcus aureus protease V8 cleaves bacteriorhodopsin to two main fragments, V-1 and V-2. Proteolytic digestion of the purple membrane integrated protein is carried out in the presence of limited amounts of sodium dodecyl sulfate (0.5 g detergent/g bacteriorhodopsin). The fragment V-1 includes the arylisothiocyanate binding site (Lys41). The V-2 fragment comprises the two C-terminal transmembrane segments of bacteriorhodopsin. Improved renaturation of bacteriorhodopsin and the ternary complex, reformed from its V8 proteolytic fragments, is attained by peptide extraction in chloroform/methanol/0.1 M ammonium acetate and subsequent incorporation into phospholipid/detergent micelles. In the presence of retinal, V8 fragments reform chromophoric ternary complexes. Light-adapted reconstituted chromophores absorb incident light at 560 nm. Protein secondary structures are partially conserved in the course of solvent extraction and are restored in the reconstituted system. Vesicles prepared from the reconstituted complexes show light-dependent proton translocation activity.
Sigrist
H
H
Institute of Biochemistry, University of Berne, Switzerland.
Wenger
R H
RH
Kislig
E
E
Wüthrich
M
M
D001436:Bacteriorhodopsins / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D008027:Light; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism; D011489:Protein Denaturation; D012172:Retinaldehyde / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology; D014178:Translocation, Genetic
D010446:Peptide Fragments; D001436:Bacteriorhodopsins; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D012172:Retinaldehyde