The MotA protein of Escherichia coli is a proton-conducting membrane channel that is essential for flagellar rotation. It is anticipated that MotA will be the prototypical member of a large family of homologous proton channels, because a similar motility apparatus is found in many other species of bacteria, including motile pathogens. The structure of this novel channel, and the relation of its structure to its ion-conducting function, will be studied. The protein will be overexpressed and purified, and reconstituted into proteoliposomes. This will permit its intrinsic conductance to be measured. The effects on conductance of various factors such as pH, temperature, and driving force also will be examined. The membrane topology of MotA will be determined by introducing chemically reactive residues (cysteine) in defined locations by site-directed mutagenesis, and assessing their reactivity with membrane-impermeant probes. Its state of association will be determined by site-directed crosslinking using the same proteins produced for the topology study. Once the membrane-spanning segments have been defined, their structure will be examined more closely by introducing bulky, hydrophobic residues (tryptophan) into successive positions near the middle of each spanner. The disruptive effects of these substitutions should exhibit a pattern that reflects the secondary structure and packing of the membrane spanners. Finally, the role of the hydrophilic, presumably extramembranous domains will be examined, by deleting various parts of the mota gene that encode hydrophilic domains, and assessing the effects on conductance. The objective of these studies is to understand, at the molecular level, the mechanism of proton conduction by MotA. Proton conduction has a fundamental role in many biological energy conversions in mitochondria, chloroplasts, and bacteria. It is hoped that a detailed understanding of MotA will assist in understanding these other systems as well.
