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Theses Doctoral

Resonance-energy-transfer-based fluorescence imaging and free energy perturbation calculation

Xu, Fang

This thesis focuses on an important aspect of protein functionality – protein-protein interactions (PPI). Three physical chemistry techniques for or derived from protein-protein interaction investigation are discussed. First, in Chapter 2, we demonstrate a new fluorescent imaging technique that creates high-order nonlinear signals by harnessing the frustrated fluorescence resonance energy transfer (FRET) – energy transfer between certain proteins close in proximity which is commonly used in PPI studies. In Chapter 3, we combine fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET), two most commonly used approaches to monitor protein-protein interactions in vivo, to create a novel hybrid strategy, bioluminescence assisted switching and fluorescence imaging (BASFI), which integrates the advantages of FRET and BRET. We demonstrate BASFI with Dronpa-RLuc8 fusion constructs and drug-inducible intermolecular FKBP-FRB protein-protein interactions in live cells with high sensitivity, resolution, and specificity. Finally, in Chapter 4, we propose a systematic free energy perturbation (FEP) protocol to computationally calculate the binding affinities between proteins. We demonstrate our protocol with the gp120 envelope glycoprotein of HIV-1 and three broadly neutralizing antibodies (bNAbs) of the VRC01 class and analyze antibody residues’ contributions to the binding which further provides insights for antibody design.


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More About This Work

Academic Units
Thesis Advisors
Friesner, Richard A.
Ph.D., Columbia University
Published Here
January 19, 2018