[11] The identification of helical segments in the polypeptide chain of bacteriorhodopsin

This chapter summarizes the information that can be used to identify the helical regions and attempt to improve the assignments based on several approaches. An important source of information is provided by the action of proteolytic enzymes on the bacteriorhodopsin structure. The underlying assumption in such studies is that the soluble enzymes will act to cleave only polypeptide links that are exposed to the aqueous environment. Consequently, one expects that such cleavage sites will be in connecting loops between helices and can, therefore, be used to identify the spacer regions between helices. In addition to proteolysis, information from other kinds of modification may be useful. One important result comes from the application of lactoperoxidase-catalyzed iodination. Researchers have used an immobilized lactoperoxidase molecule to modify purple membrane fragments. Subsequent analysis showed 75% of the radioactive iodine to be located in the cyanogen bromide fragment from amino acid 118 to 145. In this region only two tyrosines are present, tyrosine 131 and 133. Consequently, the expectation is that one or both of these tyrosines must be exposed at the aqueous surface of the bactcriorhodopsin molecule. Another modification of interest is the derivitization of the membrane by a biotinyl reagent that reacts with lysine amino groups. In this study it is thought that a single lysine was modified by the reagent in such a way as to permit binding of an avidin-fcrritin complex. The use of electron microscopy resulted in a determination that only one side of the membrane, that part oriented toward the outside of the plasma membrane of the Halobacterium, is labeled.