Open Access Dissertation
Date of Award
Biochemistry and Molecular Biology
The goal of my thesis is to develop an in-cell fluorescence technique that allows for measurement of the distances between fluorescence acceptors and donors within a protein or between two proteins inside the correct intracellular compartment of living cells. The successful achievement of this goal will allow us to obtain high-resolution structural information from a protein, one key step towards high-resolution structural biology of proteins inside the living cell.
To achieve this goal, we will apply the fluorescence resonance energy transfer (FRET) technique to the specifically labeled proteins inside the cells. Our rationale is to specifically label the protein(s) of interest in the test tube with a small molecule fluorophore and then deliver the labeled protein(s) into the correct intracellular compartment of living cells for in-cell FRET measurement. The QQ-protein delivery technique can specifically deliver a protein to its intracellular destiny based on its signal sequence. This will result in special mammalian cells that contain a fluorescence labeled target protein with unlabeled intracellular endogenous proteins as the background. The FRET measurement will be performed on this specifically labeled protein and the calculated FRET-distance will be between the donor and acceptor of the protein(s) of interest, thus, high-resolution structural information of a protein inside living cells can be obtained using this novel approach.
Murray, Victoria Lynn, "Development of an in-cell förster resonance energy transfer technique to study protein structure inside living cells" (2012). Wayne State University Dissertations. Paper 387.