Structural Biology

Biography

Biography: Marco Capitanio

Abstract

We developed a constant-force laser trap that allows us to investigate molecular interactions and sub-nanometer conformational changes occurring on a time scale of few tens of microseconds. [Capitanio et al., Nature Methods 9, 1013-1019 (2012)]. The method is effective in studying the sequence-dependent affinity of DNA-binding proteins along a single DNA molecule. The improvement in time resolution provides important means of investigation on the long-puzzled mechanism of target search on DNA. In fact, one poorly understood issue in the field of protein-DNA interaction is how proteins weakly interact with non-cognate DNA sequences and how they efficiently find the sequence of interest among an extremely large amount of non-specific sequences. Using our technique, we could discriminate sequence and conformational -dependent interactions of a single Lac repressor protein (LacI) on DNA at physiological salt concentrations. The lac operon is a well-known example of gene expression regulation, based on the specific interaction of LacI with its cognate DNA sequence (operator). We observed LacI switching between different interaction modalities on DNA (weak, strong, sliding), depending on the molecule conformation and DNA sequence. We provide a method for measuring 1D-diffusion constants of DNA-binding proteins along DNA with a spatial resolution of about 30 base pairs, observing a broad distribution of 1D-diffusion constants of LacI and sequence-dependent diffusion constants. Our measurements provide a model of target-search and molecular switching mechanism of Lac repressor.

References:

1. Gardini, L., Capitanio, M., Pavone F.S., “3D tracking of single nanoparticles and quantum dots in living cells by out-of-focus imaging with diffraction pattern recognition”, Sci. Rep. 5, 16088; doi: 10.1038/srep16088 (2015).
2. Monico, C., Belcastro, G., Vanzi, F., Pavone, F.S. and M. Capitanio, “Combining single-molecule manipulation and imaging for the study of protein-DNA interactions”, J. Vis. Exp. 90, e51446, doi:10.3791/51446 (2014).
3. Capitanio, M and Pavone, F.S. “Interrogating biology with force: single-molecule high-resolution measurements with optical tweezers”, Biophys. J. 105, 1293-1303 (2013).
4. Monico, C., Capitanio, M., Belcastro, G., Vanzi, F., Pavone, F.S., “Optical Methods to Study Protein-DNA Interactions in Vitro and in Living Cells at the Single-Molecule Level”, Int. J. Mol. Sci. 14, 3961-3992 (2013).
5. Capitanio, M., Canepari, M., Maffei, M., Beneventi, D., Monico, C., Vanzi, F., Bottinelli, R. and Pavone F.S. “Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke”, Nature Methods, 9, 1013–1019 (2012).