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Proceedings Paper

Ultrafast force-clamp spectroscopy to probe lac repressor-DNA interactions
Author(s): Carina Monico; Marco Capitanio; Gionata Belcastro; Francesco Vanzi; Francesco S. Pavone
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Paper Abstract

We recently developed an ultrafast force-clamp laser trap capable to probe, under controlled force, bimolecular interactions with unprecedented temporal resolution. Here we present the technique in the framework of protein-DNA interactions, specifically on Lactose repressor protein (LacI). The high temporal resolution of the method reveals the kinetics of both short- and long-lived interactions of LacI along the DNA template (from ∼100 μs to tens of seconds), as well the dependence on force of such interaction kinetics. The two kinetically well-distinct populations of interactions observed clearly represent specific interactions with the operator sequences and a fast scanning of LacI along non-cognate DNA. These results demonstrate the effectiveness of the method to study the sequence-dependent affinity of DNA-binding proteins along the DNA and the effects of force on a wide range of interaction durations, including μs time scales not accessible to other single-molecule methods. This improvement in time resolution provides also important means of investigation on the long-puzzled mechanism of target search on DNA and possible protein conformational changes occurring upon target recognition.

Paper Details

Date Published: 17 June 2013
PDF: 8 pages
Proc. SPIE 8797, Advanced Microscopy Techniques III, 87970F (17 June 2013); doi: 10.1117/12.2032542
Show Author Affiliations
Carina Monico, LENS, European Lab. for Non-linear Spectroscopy (Italy)
Marco Capitanio, LENS, European Lab. for Non-linear Spectroscopy (Italy)
Univ. of Florence (Italy)
Gionata Belcastro, LENS, European Lab. for Non-linear Spectroscopy (Italy)
Francesco Vanzi, LENS, European Lab. for Non-linear Spectroscopy (Italy)
Univ. of Florence (Italy)
Francesco S. Pavone, LENS, European Lab. for Non-linear Spectroscopy (Italy)
Univ. of Florence and National Institute of Optics, National Research council, Florence (Italy)
International Ctr. Of Computational Neurophotonics (Italy)

Published in SPIE Proceedings Vol. 8797:
Advanced Microscopy Techniques III
Emmanuel Beaurepaire; Peter T. C. So, Editor(s)

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