Date of Award

2017

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Microbiology and Immunology

Abstract

Single-molecule FRET (smFRET) is a method by which dynamic conformational changes can be monitored in a protein microscopically and in real time. smFRET relies on the creation of FRET (Förster Resonance Energy Transfer) between small molecule fluorophores conjugated to the biomolecules of interest. FRET efficiency allows calculation of interfluorophore distances. Changes in FRET efficiency represent changes in protein conformation which can inform further structural and molecular studies of the protein of interest. For example, in the Campbell Lab, we study the protein TRIM5α, an antiretroviral cellular protein which can cause premature dissociation of the HIV capsid core by an unknown mechanism. We can conjugate small fluorophores to specific sites chosen on TRIM5α or isolated TRIM5α domains and use smFRET to observe conformational changes in TRIM5α that may correlate to disruption of the viral core. Fluorophore conjugation can be done several ways, but we will focus on the use of cysteines to conjugate maleimide linked fluorophores to our protein of interest. For my thesis, I propose to adapt and optomise a protocol for smFRET usable by the Campbell Lab. To do this, I will first purify, label, and characterize peptides derived from the CC and L2 domains of TRIM5α that retain their secondary structure. I will then optimize methods for smFRET including instrument calibration, setting up the ability to track multiple fluorophores simultaneously, and analysis of smFRET data both to correctly align data and to render final data analyses.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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