Ultrafast force-clamp spectroscopy to probe lac repressor-DNA interactions

Carina Monico; Marco Capitanio; Gionata Belcastro; Francesco Vanzi; Francesco S. Pavone

doi:10.1117/12.2032542

17 June 2013 Ultrafast force-clamp spectroscopy to probe lac repressor-DNA interactions

Carina Monico, Marco Capitanio, Gionata Belcastro, Francesco Vanzi, Francesco S. Pavone

Proceedings Volume 8797, Advanced Microscopy Techniques III; 87970F (2013) https://doi.org/10.1117/12.2032542
Event: European Conferences on Biomedical Optics, 2013, Munich, Germany

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.

Citation Download Citation

Carina Monico, Marco Capitanio, Gionata Belcastro, Francesco Vanzi, and Francesco S. Pavone "Ultrafast force-clamp spectroscopy to probe lac repressor-DNA interactions", Proc. SPIE 8797, Advanced Microscopy Techniques III, 87970F (17 June 2013); https://doi.org/10.1117/12.2032542

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