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Local temperature jumps for single Protein Molecules: a route to study dynamics from Microseconds to hours (LTJSPM)
Start date: 01 Aug 2007, End date: 31 Jul 2009 PROJECT  FINISHED 

We propose to physically trap a single bio-molecule into successive conformational sub-states by rapidly freezing and thawing it with its immediate surroundings. After many successive freeze-thaw cycles, we will reconstruct the dynamics of the same single molecule from the series of frozen snapshots, probed by the fluorescence of optical labels. Fast cooling and heating will be achieved by maintaining the molecule in a focused laser spot within a cold cryostat. We will control the local temperature by modulating the power of the heating beam. The time response, given by heat diffusion out of the diffraction limited hot spot is as short as a few microseconds. In the past several years unique know-how and infrastructure have been developed and feasibility of quick freeze-thawing cycles has been demonstrated in Huygens Laboratory, Leiden University. In the coming years, we want to extend the measurements first to oligopeptides and small proteins, then to larger proteins in physiological conditions. We expect this project to open a completely new window on protein dynamics, by enabling us to observe the same single molecule over large time intervals, at high-time resolution. The series of snapshots will give information on relaxation processes down to microsecond timescales, inaccessible to other single-molecule methods, and make it possible to map out the topography of the energy landscape, i.e., not only the accessible conformational sub-states, but also the favored pathways connecting them.
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