"Visualizing Protein Conformation and Dynamics Using Single Molecule FRET Spectroscopy"

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Date/Time:Monday, 11 Feb 2013 from 3:10 pm to 4:00 pm
Location:Physics 0005
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Hoi Sung Chung (National Institutes of Health)


Single molecule spectroscopy is a powerful tool for characterizing a heterogeneous system by probing individual molecular states and events. My broad research interests encompass gaining deep understanding of diverse conformations and folding processes of proteins by further advancing this technique. I will present the development of novel methods that utilize single molecule Förster Resonance Energy Transfer (FRET) spectroscopy and statistical analysis tools and their applications to the characterization of the transition path of protein folding and the determination of the conformational distribution of a membrane-binding protein complex.

Protein folding is a very complex process. Even for proteins that populate only two-states ��" folded and unfolded ��" there are many pathways that connect the conformational ensembles of the two states. To understand the mechanism of protein folding, therefore, it is necessary to characterize the individual pathways, called transition paths. The transition path corresponds to the rare molecular trajectory that crosses the barrier between the two states. This barrier-crossing event, which is a unique single molecule property and invisible to ensemble measurements, has never been observed for any molecular system in the condensed phase. By developing a statistical method based on the photon-by-photon maximum likelihood analysis and carrying out a collective analysis of a large number of transitions, I could dramatically improve the time resolution of single molecule FRET spectroscopy. This development allowed for the determination of a transition-path time of ~ 2 microseconds for the WW domain (fast folding protein) and an upper bound of ~ 10 microseconds for protein GB1 (slow folding protein). Surprisingly, the transition-path times for the two proteins differ by less than 5-fold while the folding rates differ by a factor of 10,000. This result shows that a slow-folding protein can fold almost as fast as a fast-folding protein when folding actually occurs.

The structure determination of large, complex, and dynamic proteins such as the endosomal sorting complex required for transport (ESCRT) is challenging because the protein complex does not crystallize and is too large for the structure determination using NMR. Single molecule spectroscopy combined with other experimental and computational techniques can be an alternative method to determine the protein structure. I will present a structure refinement technique that generates a large conformational ensemble by computer simulation and selects the conformations consistent with diverse experimental results including single molecule FRET. This refinement revealed a broad conformational distribution of the ESCRT-I and -II complex ranging from compact to highly extended, which are required for the membrane deformation and budding process.