Scientific Report 2005

Molecular Biology

Single-Molecule Biophysics

A.A. Deniz, S.Y. Berezhna, J.P. Clamme, A.C.M. Ferreon, E.A. Lemke, S. Mukhopadhyay, S. Stanford, 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 and stochastic dynamics during the folding and assembly of biomolecules.

One major goal is to apply single-molecule methods to studies of protein and RNA folding. 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 folding and aggregation of synuclein, a protein implicated in the pathogenesis of Parkinson’s disease and other neurodegenerative diseases.

We also continue to use single-pair fluorescence resonance energy transfer (FRET) to study the folding of RNA hairpin ribozymes, in collaboration with D.A. Millar, Department of Molecular Biology. In addition, we are developing a single-molecule fluorescence quenching method that will be useful for measuring distance changes of less than 30 Å in proteins and RNA, a scale at which the resolution of single-pair FRET is low.

To better study the folding, assembly, and activity of larger and multicomponent biological complexes, we are developing new multicolor single-molecule FRET methods. As a first step, we developed a diffusion 3-color single-molecule FRET method by which 2 or more intramolecular or intermolecular distances can be measured simultaneously. In collaboration with J.R. Williamson, Department of Molecular Biology, we are using these novel methods to study the detailed mechanisms of assembly of the bacterial ribosome. The small 30S subunit of the ribosome assembles from a large RNA and 21 small proteins through a complex process that involves several steps of binding and conformational changes. Initially, we are focusing on the conformational properties of small RNA fragments from the 30S subunit and on the interactions of the fragments with their protein partners. These studies are also being extended to the assembly of entire domains of the 30S subunit.

Finally, using a combination of high-sensitivity imaging and fluorescence correlation spectroscopy, we are beginning to study the lipid-mediated entry and intracellular delivery pathways of antisense oligodeoxynucleotides and small interfering RNA. An understanding of these mechanisms will be critical to improving the efficiencies of these important genetic tools.

Publications

Berezhna, S., Schaefer, S., Heintzmann, R., Jahnz, M., Boese, G., Deniz, A.A., Schwille, P. New effects in polynucleotide release from cationic lipid carriers revealed by confocal imaging, fluorescence cross-correlation spectroscopy and single particle tracking. Biochim. Biophys. Acta 1669:193, 2005.

Clamme, J.-P., Deniz, A.A. Three-color single-molecule fluorescence resonance energy transfer. Chemphyschem 6:74, 2005.

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