TRYPTOPHAN-SCANNING MUTAGENESIS OF MOTB, AN INTEGRAL MEMBRANE-PROTEINESSENTIAL FOR FLAGELLAR ROTATION IN ESCHERICHIA-COLI

The MotB protein of Escherichia coli is an essential component of the flagella that functions together with the MotA protein in transmembran e proton conduction. MotB has a single hydrophobic segment that spans the membrane. In order to determine which parts of the membrane-spanni ng segment can tolerate the introduction of a large, hydrophobic side chain, single Trp residues were substituted into many consecutive posi tions in the segment and the effects on function were measured. Trp re sidues were tolerated at positions near the periplasmic end of the Mot B segment but not at positions near the cytoplasmic end. These results are different from what was seen in a similar mutational study of Mot A, in that protein Trp residues were tolerated at positions that would be clustered together on one face of each hydrophobic segment if they are alpha-helices [Sharp, L. L., Zhou, J., and Blah, D. F. (1995) Pro c. Natl. Acad. Sci. U.S.A. (in press)]. Those results suggested that t he membrane-spanning segments of MotA are alpha-helices arranged in a bundle so that each has a face directed toward the lipid. The contrast ing results seen with MotB indicate that its relationship to neighbori ng protein segments is different. Double-Trp substitutions, one each i n MotA and MotB also were studied. Many double substitutions had stron gly synergistic effects which imply that the membrane segments of thes e proteins interact. Together, the results suggest a hypothesis for th e structure of the MotA/MotB channel in which the membrane-spanning se gment of MotB is associated with those of MotA but is tilted relative to them so that its cytoplasmic end is embedded in the complex and its periplasmic end is relatively exposed to the lipid.