Regeneration of native bacteriorhodopsin structure from fragments

The previously described chymotryptic fragment of bacteriorhodopsin, C-2 (amino acids 1-71), is cleaved by 70% formic acid to two fragments, A-1 (amino acids 1-36) and A-2 (amino acids 37-71), which have been separated by high pressure liquid chromatography. The fragments A-1 and A-2, separately or together, are not able to replace C-2 in forming a stable bacteriorhodopsin-like complex with the fragment C-1 (amino acids 72-248) and all-trans-retinal. A second set of bacteriorhodopsin fragments, B-1 (amino acids 1-155) and B-2 (amino acids 156-248), have been prepared by sodium borohydride cleavage of bacteriorhodopsin. Following denaturation, fragments B-1 and B-2 reassociate in the presence of retinal to regenerate the native bacteriorhodopsin chromophore (approximately 40%). Fragment B-1 also interacts with fragment C-1 and all-trans-retinal to form a complex with spectral properties and secondary structure similar to those of bacteriorhodopsin. Vesicles prepared from the reconstituted fragment complexes (B-1 + B-2) or (B-1 + C-1), show proton pumping activities comparable to the previously described activity fragments C-1 and C-2.