Partial primary structure of bacteriorhodopsin: sequencing methods for membrane proteins.

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Abstract
The sequence of 102 amino acid residues from the NH2 terminus and that of 39 amino acid residues from the COOH terminus of bacteriorhodopsin [from Halobacterium halobium] were determined. These results are in agreement with those recently published. Chymotryptic cleavage of bacteriorhodopsin produced 2 fragments, C-1 (MW 19,000) and C-2 (MW 6900), the latter containing the blocked NH2 terminus (pyroglutamic acid). Further fragmentation with CNBr gave mostly hydrophobic fragments, which were separated by gel permeation and reverse-phase high-pressure liquid chromatography in formic acid/ethanol/water mixtures. The fragments were sequenced by a judicious combination of mass spectrometric peptide sequencing and automated Edman degradation. The C-2 fragments were ordered on the basis of methionine-containing peptides identified by gas chromatographic mass spectrometry, while C-1 and C-2 were arranged by analysis of an overlapping CNBr fragment.