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Construction Blueprint of a Biomolecular Rotary Motor

Renate Lux* and Shahid Khan

Department of Physiology & Biophysics, Albert Einstein College of Medicine

This is an abstract for a presentation given at the
Sixth Foresight Conference on Molecular Nanotechnology.
There will be a link from here to the full article when it is available on the web.

 

A key issue in the formation of supra-biomolecular assemblies is whether an initial core structure serves as assembly template. This issue is being addressed with regard to the rotary motor of bacterial flagella, utilizing metal replication electron microscopy and single particle image processing. This machine is part of the flagellar basal body and consists of a set of ring structures organized around a scaffold. The scaffold is a discoid particle, the MS-ring, that inserts spontaneously into the cytoplasmic membrane. A ring, the C-ring, attached to the cytoplasmic face of this scaffold, forms the rotor. The stator consists of a ring of intramembrane particles, thought to be bolted to the cell wall that cluster around the MS-ring. We wish to determine whether the MS-ring serves as organizing template, as well as a scaffold. Interestingly the various ring modules have different subunit stoichiometries.

There are 26 subunits in the MS-ring that consists of multiple copies of a single protein, FliF (Ueno et al. 1992). The C-ring has a 2-ring architecture, a design feature thought to be essential for switching of motor rotation sense. In each ring 33-34 subunits were identified that are most likely built by the proteins FliG, FliM, and FliN (Khan et al. 1998). The MS-ring is surrounded by 10 12 intramembrane ring particles that are thought to be formed by the MotA MotB proteins (reviewed in Khan 1997). If the MS-ring serves as a template, the organizing principle cannot be a simple match-up of subunits.

In order to fully explore the idea of template-based assembly, constructs overexpressing various combinations of the component proteins have been made. We seek to determine whether subunit stoichiometries are fixed or vary as a function of subunit concentration. Issues of this nature may be resolved by analysis of the periodicity of the resulting structures. Further, abundant expression of components may allow isolation of rare assembly intermediates. For example association of the distal C-ring with the MS-ring, has never been observed when the proximal ring is absent in native preparations. However, this may simply be because such intermediates are rare.

4 proteins, FliF, FliG, FliM and FliN, were inferred from previous work to be involved in formation of the initial core structure. They were overexpressed in a strain lacking all motility proteins, ca. 40 in total. C-ring structures were formed that are indistinguishable from those found in the native hook-basal-body complex as visualized in negative stain. Electrophoretic gel analysis revealed that all four proteins were present in purified preparations of these structures. This proves that other motility proteins are neither components of the C-ring nor required for their assembly onto the MS-ring template.

This overexpression approach is also being used to study interactions of the transmembrane stator, MotA MotB ring particles, with the cytoplasmic C-ring rotor structure. The Mot proteins can be overexpressed with different combinations of the 4 MS-/C-ring components. The minimal unit needed for morphological integrity and protein transport function is being defined by freeze-fracture electron microscopy and fluorescence quenching of pH indicator dyes respectively.

 

Khan, S (1997) Rotary chemiosmotic machines, Biochim. Biophys. Acta 1322, 86-105.

Khan, S., Zhao, R., and Reese, T.S. (1998) Architectural features of the Salmonella typhimurium flagellar motor switch revealed by disrupted C-rings, J. Struct. Biol. 122, 311-319.

Ueno, T., Oosawa, K., and Aizawa, S-I. (1992) M-ring, S-ring and proximal rod of the flagellar basal body of Salmonella typhimurium are composed of subunits of a single protein, FliF, J. Mol.Biol. 227, 672-677.


*Corresponding Address:
Dr. Renate Lux
Department of Physiology & Biophysics, Albert Einstein College of Medicine
1300 Morris Park Avenue, Bronx NY 10461
Phone: 718-430-4046; Fax: 718-430-8819
email: lux@aecom.yu.edu



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