Carboxyl groups contribute to the mechanism of proton translocation by a variety of proton pumps involved in metabolic energy transduction. The proposed experiments will explore the functional role of carboxyl groups in the bacteriorhodopsin proton pump within the framework of the previous identification of two sites which appears be important in the proton translocation mechanism, one at the purple membrane surface and the other in the interior. The latter site, Asp 115, reacts with dicyclohexyl carbodiimide (DCCD), while the former (probably Asp 96, Asp 102 and/or 104) reacts with water-soluble carbodiimides such as 1-ethyl-3-dimethylaminopropyl carbodiimide (EDC). The structures and properties of these modified membranes will be studied by vibrational and electronic spectroscopy, and by measuring proton pump activity. Cross polarization, magic angle spinning NMR spectroscopy has opened a new window on macromolecular function. It will now be possible to observe individual carboxyl groups at defined intermediate points in the proton pump cycle. Carboxyl protonation results in -4ppm upfield shift in the 13C NMR spectrum. This shift is sufficient, in principle, to permit observation of changes in proton binding to specific carboxyl groups in specific intermediate states of the pump cycle. 13C enrichment will be necessary to observe this effect. Biosynthetic incorporation of 13C will provide signals from all nine Asp side chain carboxyls. Specific modification reactions will be used to identify the signal due to Asp 115 and that due to Asp 96/102/104. This method may fail to sufficiently resolve the Asp signals or the changes in chemical shifts after modification may be too small. In that case an alternative approach will be taken. The two function related carboxyl sites are clustered within a carboxyl-rich region containing only one- eighth of bacteriorhodopsin's sequence but more than half of its Asp side chains. This part of the protein is easily obtained by chemical and enzymatic cleavage and therefore is prime target for incorporation of specifically 13C-labeled carboxyl side chains into bacteriorhodopsin by semisynthesis. We plan synthesize peptides containing Asp 115, 13C -Asp 104 13C - Asp 102, and 13C -Asp 96. We will recombine these fragments by established methods to form active bacteriorhodopsin. The NMR spectrum will then be examined for pump-dependent changes in proton binding to specific carboxyl group.
