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dc.contributor.advisorJoseph J. Loparo.en_US
dc.contributor.authorSargent, Jacob D. (Jacob Daniel)en_US
dc.contributor.otherHarvard--MIT Program in Health Sciences and Technology.en_US
dc.date.accessioned2015-09-17T19:07:24Z
dc.date.available2015-09-17T19:07:24Z
dc.date.copyright2015en_US
dc.date.issued2015en_US
dc.identifier.urihttp://hdl.handle.net/1721.1/98721
dc.descriptionThesis: S.M., Harvard-MIT Program in Health Sciences and Technology, 2015.en_US
dc.descriptionCataloged from PDF version of thesis.en_US
dc.descriptionIncludes bibliographical references (pages 36-39).en_US
dc.description.abstractThe 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.en_US
dc.description.statementofresponsibilityby Jacob D. Sargent.en_US
dc.format.extent39 pagesen_US
dc.language.isoengen_US
dc.publisherMassachusetts Institute of Technologyen_US
dc.rightsM.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.en_US
dc.rights.urihttp://dspace.mit.edu/handle/1721.1/7582en_US
dc.subjectHarvard--MIT Program in Health Sciences and Technology.en_US
dc.titleSingle-molecule visualization of conformational changes in the SecA ATPaseen_US
dc.typeThesisen_US
dc.description.degreeS.M.en_US
dc.contributor.departmentHarvard University--MIT Division of Health Sciences and Technology
dc.identifier.oclc920869141en_US


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