Scientific Report 2006

Molecular Biology

Single-Molecule Biophysics

A.A. Deniz, S.Y. Berezhna, J.P. Clamme, A.C.M. Ferreon, Y. Gambin, E. Lemke, S. Mukhopadhyay, P. Zhu

We develop and use state-of-the-art single-molecule fluorescence methods to address key biological questions. Single-molecule and small-ensemble methods offer key advantages over traditional measurements, allowing us to directly observe the behavior of individual subpopulations in mixtures of molecules and to measure kinetics of structural transitions of stochastic processes under equilibrium conditions. We use these methods to study multiple structural states or reaction pathways during the folding and assembly of biomolecules.

A major goal is to apply single-molecule methods to studies of protein folding and aggregation. Using relatively simple model systems, we are addressing several fundamental questions about folding mechanisms. Partially folded or misfolded protein structures are also thought to play important cellular roles, and these states also can be studied by using single-molecule methods. In this context, we are examining the interplay between folding and aggregation of Sup35, a yeast prion protein, in collaboration with S.L. Lindquist, Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, and of α-synuclein, a protein implicated in the pathogenesis of Parkinson’s disease and other neurodegenerative diseases.

In addition, we have developed a single-molecule fluorescence quenching method that will be useful for measuring distances shorter than 30 Å in proteins and RNA, a scale at which the resolution of single-pair fluorescence resonance energy transfer (FRET) is low. This method is being used to monitor structural properties of Sup35 as a function of the aggregation process.

To better study the folding, assembly, and activity of larger and multicomponent biological complexes, we are developing new multicolor single-molecule FRET methods. As part of this continuing goal, we have been improving our recently developed diffusion 3-color single-molecule FRET method for simultaneously measuring more than a single intramolecular or intermolecular distance. In collaboration with J.R. Williamson, Department of Molecular Biology, we are using these novel methods to study the detailed mechanisms of assembly of fragments of the bacterial ribosome. Most recently, we began adding microfluidics capabilities to our experimental repertoire, to further facilitate studies of molecular structure, folding, and function.

Finally, using high-sensitivity fluorescence imaging, we are beginning to study and compare the pathways of nuclear and cytoplasmic RNA interference. In studies done in collaboration with P.G. Schultz, Department of Chemistry, our observations of the localization of small interfering RNA in live cells provide evidence for a yet-to-be-determined mechanism that directs the RNA to cellular compartments containing the target RNA.

Publications

Berezhna, S.Y., Supekova, L., Supek, F., Schultz, P.G., Deniz, A.A. siRNA in human cells selectively localizes to target RNA sites. Proc. Natl. Acad. Sci. U. S. A. 103:7682, 2006.

Zhu, P., Clamme, J.-P., Deniz, A.A. Fluorescence quenching by TEMPO: a sub-30 Å single-molecule ruler. Biophys. J. 89:L37, 2005.