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ELIZABETH RHOADES

My research interests are in using optical techniques for the biophysical characterization of systems of biological and biochemical interest at the molecular and cellular level, including protein folding, protein-protein interactions, and protein-membrane interactions. I am pursuing two main paths, one focused on the development and use of single molecule techniques to study protein conformations and the other on the characterization protein-membrane interactions, with an interest in amyloid-forming proteins motivating both pathways. My research approach will involve using fluorescence-based spectroscopy and microscopy to characterize the proteins of interest in environments of multiple levels of complexity: in solution, associated with model membrane systems, and in cells.

Single molecule studies of protein conformation
In the past decade, advances in laser and detection technologies have lead to rapid development of single molecule spectroscopies and their application to biological systems. One major advantage to studying biological molecules on the single molecule level is that it eliminates ensemble averaging and thus allows for characterization of the underlying heterogeneity of processes that are often described by single rate constants or pathways via ensemble methods. Single molecule fluorescence techniques have been used to characterize fluctuations in substrate binding and enzymatic activity and to identify conformational subpopulations of proteins.

Single molecule techniques will be adapted for studies of protein conformational changes over a range of time scales. Proteins diffusing freely in solution have a typical residence time in the focal volume of ~500 µs, while proteins encapsulated in immobilized vesicles or in gels remain in the focal volume indefinitely. Highly sensitive fluorescent techniques (FRET, FCS) will be used to determine distributions of conformations of proteins in various solution conditions as well as to observe and quantify actual conformational fluctuations and transitions between structures.

Protein-membrane interactions
The interaction of amyloid-forming proteins with cell membranes has been proposed as the common mechanism of cytotoxicity for many amyloid diseases, and thus characterization of interactions of amyloid proteins with membranes is of great interest. Freely suspended model membranes of giant unilamellar vesicles (GUVs) provide a membrane model that closely mimics the properties of biological bilayer membranes and may serve as a physiologically relevant model of protein/membrane interactions. One feature of particular interest is that GUVs can be induced to form optically resolvable ordered and disordered domains, which may be considered to be a good model for protein partitioning in cell membranes. This model system will allow for the selective addition of lipid components to mimic various cell membranes such as synaptic vesicles, mitochondria, and the plasma membrane.

Confocal, multiphoton, and TIRF microscopy will be used to directly visualize and quantify the binding of the fluorescently labeled proteins to the membranes. We will study co-localization of the protein with various membrane components as well as the effects of lipid phase on protein partitioning (and the reverse).

Selected Publications
Rhoades, E. and Gafni, A. Detection of micelles formed by an amyloidogenic fragment of human islet amyloid polypeptide. Biophysical Journal 84, 3480-3487 (2003)

Rhoades, E., Gussakovsky, E. and Haran, G. Watching proteins fold one molecule at a time. Proc. Natl. Acad. Sci. U.S.A. 100, 3197 (2003)

Rhoades, E., Cohen, M., Schuler, B. and Haran, G. Two-state folding observed in individual protein molecules. Journal of the American Chemical Society 126, 14686-14687 (2004)

Park, H. Y., Qiu, E., Rhoades, E., Korlach, J., Kwok, L. W., Zipfel, W. R., Webb, W. W. and Pollack, L. Achieving uniform mixing in a microfluidic device: hydrodynamic focusing prior to mixing. Analytical Chemistry 78, 4465-4473 (2006)

Rhoades, E., Webb, W. W. and Eliezer, D. Quantification of alpha-synuclein binding to lipid vesicles using fluorescence correlation spectroscopy. Biophysical Journal 90, 4692-4700 (2006)

Last Updated 12-18-06