Protein arrangement in the bacterial flagellar motor probed by site directed cross-linking

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Publication Type thesis
School or College College of Science
Department Biology
Author Lowder, Bryan Jeffrey
Title Protein arrangement in the bacterial flagellar motor probed by site directed cross-linking
Date 2007-05-03
Description Bacteria swim through liquid with flagella; each flagellum is a rigid helix spun by a rotary motor at its base, powered by the transmembrane proton gradient (or Na+ gradient in some species). The motor is significant as a virulence factor, as a complex biochemical system with important evolutionary connections, and as a self-assembling molecular machine to inform nanoengineering efforts. Thoroughly understanding motor function demands detailed structural information. In Escherichia coli and Salmonella enterica serovar Typhimurium, the flagellar motor is ~45 nm in diameter and contains ~25 different proteins, some present in multiple copies. The rotating inner portion of the motor (the rotor) contains many copies of the proteins FliM, FliG, and FliN, all of which are essential to rotation and direction switching. Partial X-ray crystal structures are available for these rotor proteins, but spatial relationships between the proteins must also be understood. Electron microscopy provides structural information at a larger scale, but the resolution is not yet sufficient to dock crystal structures. To address the need for intermolecular structural data, I used the biochemical technique of site-directed cross-linking (SDCL) to find the rough relative placement between FliG and FliG, between FliM and FliM, and between FliM and FliG. Guided by crystal structures, I used site-directed mutagenesis to replace one or two surface residues with chemically reactive cysteine (Cys). Oxidizing or sulfhydryl-specific agents then induced cross-linking between those Cys residues close enough to react together. Assays of swimming ability controlled for disrupted motor assembly or function. Results indicate that adjacent FliG middle domains are close together with nothing intervening, and place constraints on the relative orientations of the domains. A study of FliM middle domains led to a similar conclusion about spacing and provide constraints for these portions. Results for FliG C-terminal domains imply larger relative spacing with significant conformation freedom. Preliminary results for FliG-FliM cross-linking are presented. Data on FliG-FliM binding are reviewed, along with a recent model (proposed by others) that FliG-FliM binding alternates depending on position. I propose a functional model based on this binding model and discuss considerations of the functional model.
Type Text
Publisher University of Utah
Subject Flagella; Bacterial proteins
Dissertation Institution University of Utah
Dissertation Name MS
Language eng
Relation is Version of Digital reproduction of "Protein arrangement in the bacterial flagellar motor probed by site directed cross-linking" J. Willard Marriott Library Special Collections QR6.5 2007 .L68
Rights Management © Bryan Jeffrey Lowder
Format Medium application/pdf
Format Extent 65,446 bytes
Identifier us-etd2,123717
Source Original: University of Utah J. Willard Marriott Library Special Collections
Conversion Specifications Original scanned on Epson GT-30000 as 400 dpi to pdf using ABBYY FineReader 9.0 Professional Edition.
ARK ark:/87278/s6qr5bpm
Setname ir_etd
Date Created 2012-04-23
Date Modified 2012-05-17
ID 193216
Reference URL