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Biophysics and Quantitative Biology Seminar: "Watching complexes and invisible states of proteins by high-precision FRET in vitro and in vivo"

Event Type
Center for the Physics of Living Cells
B102 CLSL (Chemical and Life Sciences Laboratory)
Feb 13, 2014   2:00 pm  
Dr. Claus Seidel, Chair of Molecular Physical Chemistry, Heinrich Heine University, Germany
Angi Meharry
Originating Calendar
Physics - Physics of Living Cells Seminar

So far our view of protein function is formed, to a significant extent, by traditional structure determination showing many beautiful specific snapshots of static protein structures. Recent experiments by single-molecule and other techniques have shown the heterogeneity and flexibility of biomolecular structures and questioned the idea that proteins and other biomolecules are static structures. The visualization of transiently populated conformational states and the identification of exchange pathways are key steps to understand enzyme function.

I will present a comprehensive toolkit for Förster resonance energy transfer (FRET)-restrained modeling of proteins and their complexes for quantitative applications in structural biology [1-3] and cell biology [4, 5]. The experiments are performed by multi-parameter fluorescence detection on the single-molecule level and for complexes imaged in live cells. A dramatic improvement in the precision of FRET-derived structures is achieved by explicitly considering spatial distributions of dye positions, which greatly reduces uncertainties due to flexible dye linkers. The precision and confidence levels of the models are calculated by rigorous error estimation. The accuracy of this approach is demonstrated by docking a DNA primer-template to HIV-1 reverse transcriptase. The derived model agrees with the known X-ray structure with a root mean square deviation of 0.5 Å. Furthermore, we introduce FRET-guided “screening” of a large structural ensemble created by computer simulations. Moreover we use filtered fluorescence correlation spectroscopy to characterize enzyme function and introduce a state matrix of conformational and enzyme states that assigns a functional role to conformational fluctuations. Hybrid studies of T4 Lysozyme, DNA polymerase I and the large GTPase hGPB1 using FRET, SAXS and EPR will be presented.  

[1] Sisamakis, E., et al.; Methods in Enzymology 475, 455-514 (2010)

[2] Sindbert, S., et al.; J. Am. Chem. Soc. 133, 2463-2480 (2011)

[3] Kalinin et al. Nat. Methods 9, 1218-1225 (2012).

[4] Stahl, Y., et al. Current Biology 23, 362–371 (2013).

[5] Kravets, E., et. al. J. Biol. Chem. 287, 27452–27466 (2012).

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