Thesis: S.M., Harvard-MIT Program in Health Sciences and Technology, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 36-39). / The need for new antibiotics is great as bacterial strains with single and multiple drug resistance have continued to grow more prevalent since the 1980's. At the same time, the rate of approval of new antibiotics has dropped precipitously. Existing antibiotics commonly target the bacterial ribosome. A or cell wall synthetic pathways: two targets that are essential for bacterial survival. However, another option is to target a pathway which is more intimately connected to bacterial pathogenesis: protein secretion. In bacteria, most secreted polypeptides are pushed across the membrane, via the SecYEG channel, by the SecA ATPase. Relatively little is understood of how SecA couples ATP hydrolysis to polypeptide translocation. X-ray crystallography and many biochemical studies support a model in which the two-helix finger (2HF) of SecA pushes the polypeptide through the SecYEG channel, however some evidence is contradictory. We aim to directly measure conformational changes of the 2HF by utilizing single-molecule Fbrster resonance energy transfer (smFRET). Directly measuring conformational changes in an ATPase will also provide further insight into the guiding principles of ATPase function. First, we will build a smFRET microscope and assemble a software package to analyze the data it collects. We will then validate these tools by reproducing results currently in the literature from Holden et al. and McKinney et al.. Next, we will assess the potential limitations of current tools for smFRET data analysis, especially as applied to ATPases. We will propose a new approach that may be useful in these systems. Finally, we will use the smFRET microscope to measure ATP-dependent conformational dynamics of the 2HF. This evidence will help differentiate between three proposed models: the 2HF (1) is not directly involved in polypeptide translocation, (2) moves unidirectionally, directly driving translocation, or (3) moves back and forth but in a way that is coordinated by ATP hydrolysis with progress capture elsewhere in SecA. / by Jacob D. Sargent. / S.M.
| Identifer | oai:union.ndltd.org:MIT/oai:dspace.mit.edu:1721.1/98721 |
| Date | January 2015 |
| Creators | Sargent, Jacob D. (Jacob Daniel) |
| Contributors | Joseph J. Loparo., Harvard--MIT Program in Health Sciences and Technology., Harvard--MIT Program in Health Sciences and Technology. |
| Publisher | Massachusetts Institute of Technology |
| Source Sets | M.I.T. Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 39 pages, application/pdf |
| Rights | M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission., http://dspace.mit.edu/handle/1721.1/7582 |
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