Single Molecule Fluorescence Analysis of Conformational Changes in Transmembrane Transporters

Project Collaborators:

Dr. Peter Maloney Johns Hopkins

Repeated FRET measurements on a single OxlT membrane protein.
Panel A: Fluorescence time trace of donor green and acceptor red.
Panel B: Corresponding energy transfer efficiency (ETE). The mean ETE is 0.315
(solid horizontal line) and the standard deviation is 0.086 (dashed horizontal lines).
The inset shows the homology model of OxlT with the donor and acceptor sites red features).

Most biochemical experiments are performed using huge numbers of molecules in bulk solution. In many cases, this leads to significant averaging over populations of molecules that may differ from each other. Working with the laboratory of Dr. Lukas Novotny, of the Institute of Optics at the University of Rochester, we are developing a technology for using fluorescence measurements on single molecules of membrane proteins to learn about the heterogeneity of conformations that such molecules adopt in the process of carrying out their molecular functions. Initial studies are focusing on a bacterial oxalate transporter, OxlT, that has been extensively studied in the laboratory of an additional collaborator, Dr. Peter Maloney, of The Johns Hopkins University. Fluorescent tags attached at specific positions in the protein are being used to monitor conformational changes in single molecules associated with the transport function of the protein and to probe the folding and unfolding of the protein. The measurements are facilitated by the application of a new technology for trapping individual molecules in fabricated nano-scale channels, allowing us to observe each molecule for much longer periods of time than would be possible if the molecules were freely diffusing.

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