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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM046683-02
Application #
3306124
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1992-01-01
Project End
1994-12-31
Budget Start
1993-01-01
Budget End
1993-12-31
Support Year
2
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Utah
Department
Type
Schools of Arts and Sciences
DUNS #
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Braun, T F; Blair, D F (2001) Targeted disulfide cross-linking of the MotB protein of Escherichia coli: evidence for two H(+) channels in the stator Complex. Biochemistry 40:13051-9
Kojima, S; Blair, D F (2001) Conformational change in the stator of the bacterial flagellar motor. Biochemistry 40:13041-50
Van Way, S M; Hosking, E R; Braun, T F et al. (2000) Mot protein assembly into the bacterial flagellum: a model based on mutational analysis of the motB gene. J Mol Biol 297:24-Jul
Braun, T F; Poulson, S; Gully, J B et al. (1999) Function of proline residues of MotA in torque generation by the flagellar motor of Escherichia coli. J Bacteriol 181:3542-51
Zhou, J; Lloyd, S A; Blair, D F (1998) Electrostatic interactions between rotor and stator in the bacterial flagellar motor. Proc Natl Acad Sci U S A 95:6436-41
Zhou, J; Sharp, L L; Tang, H L et al. (1998) Function of protonatable residues in the flagellar motor of Escherichia coli: a critical role for Asp 32 of MotB. J Bacteriol 180:2729-35
Zhou, J; Blair, D F (1997) Residues of the cytoplasmic domain of MotA essential for torque generation in the bacterial flagellar motor. J Mol Biol 273:428-39
Sharp, L L; Zhou, J; Blair, D F (1995) Features of MotA proton channel structure revealed by tryptophan-scanning mutagenesis. Proc Natl Acad Sci U S A 92:7946-50
Sharp, L L; Zhou, J; Blair, D F (1995) Tryptophan-scanning mutagenesis of MotB, an integral membrane protein essential for flagellar rotation in Escherichia coli. Biochemistry 34:9166-71
Zhou, J; Fazzio, R T; Blair, D F (1995) Membrane topology of the MotA protein of Escherichia coli. J Mol Biol 251:237-42