Tryptophan-scanning mutagenesis of MotB, an integral membrane protein essential 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 transmembrane proton conduction. MotB has a single hydrophobic segment that spans the membrane. In order to determine which parts of the membrane-spanning segment can tolerate the introduction of a large, hydrophobic side chain, single Trp residues were substituted into many consecutive positions in the segment and the effects on function were measured. Trp residues were tolerated at positions near the periplasmic end of the MotB segment but not at positions near the cytoplasmic end. These results are different from what was seen in a similar mutational study of MotA, 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., & Blair, D. F. (1995) Proc. Natl. Acad. Sci. U.S.A. (in press)]. Those results suggested that the membrane-spanning segments of MotA are alpha-helices arranged in a bundle so that each has a face directed toward the lipid. The contrasting results seen with MotB indicate that its relationship to neighboring protein segments is different. Double-Trp substitutions, one each in MotA and MotB, also were studied. Many double substitutions had strongly synergistic effects which imply that the membrane segments of these proteins interact. (ABSTRACT TRUNCATED AT 250 WORDS)
Sharp
L L
LL
Department of Biology, University of Utah, Salt Lake City 84112, USA.
Zhou
J
J
Blair
D F
DF
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics* / Q000502:physiology; D005407:Flagella / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D011487:Protein Conformation; D011522:Protons; D012399:Rotation; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000235:genetics
D001426:Bacterial Proteins; D008565:Membrane Proteins; C063010:MotA protein, Bacteria; C064779:MotB protein, Bacteria; D011522:Protons; D014364:Tryptophan