Rotary molecular motors are protein complexes which convert chemical or electrochemical energy from the environment into mechanical work in the form of rotary motion. The work in this thesis examines two of these motors: the F<sub>1</sub> portion of F<sub>1</sub>F<sub>O-</sub> ATP synthase, which is responsible for ATP production in bacteria and eukaryotes, and the bacterial flagellar motor (BFM), which rotates the flagella of a bacterium, enabling locomotion. The aim of these investigations was to measure the stepping dynamics of these motors, in order to further elucidate details of the stepping mechanism, the mechanism of rotation, and the mechanochemical cycle. A back-scattering laser dark field microscope of unprecedented resolution was designed and constructed to observe the rotation of gold nanoparticles attached to fixed motors. This micro- scope is capable of sub-nanometer and 20μs resolution. The protocols and algorithms to collect and analyze high resolution rotational data developed for these experiments have yielded novel discoveries for both F<sub>1</sub> and the BFM. While most of the previous single-molecule work has been done on F<sub>1</sub> from the thermophilic Bacilus PS3 (TF<sub>1</sub>), only mitochondrial F<sub>1</sub> has been well characterized by high-resolution crystal structures, and single-molecule studies of mesophilic F<sub>1</sub> are lacking. This thesis presents evidence that mesophilic F<sub>1</sub> from E. coli and wild type yeast F<sub>1</sub> from S. cerevisiae are governed by the same mechanism as TF<sub>1</sub> under laboratory conditions. Experiments with yeast F<sub>1</sub> mutants allow a direct comparison between single-molecule rotation studies and high resolution crystal structures. A data set of unprecedented size and resolution was acquired of high speed, low load BFM rotation, enabling the first observation of steps in the BFM under physiological conditions. Preliminary results from this analysis question previously published results of the dependence of speed on stator number at low load and provide novel hypotheses necessitating new models of BFM rotation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:640009 |
| Date | January 2014 |
| Creators | Nord, Ashley |
| Contributors | Berry, Richard |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:cc8fcaa9-50eb-4659-8e46-837894c64c89 |
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