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Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

University of Florence

Laser beams have been demonstrated to be capable of exerting torque as well as forces on microparticles. Using a custom magneto-optic manipulator, we directly measured the torque exerted by laser light on absorbing microspheres as a result of the transfer of spin angular momentum. A general method for measuring torque has been developed, and the experimental apparatus has shown a sensitivity of approximately 1 pN/nm.

High-precision measurements of light-induced torque on absorbing microspheres.

We describe a dual-trap force-clamp configuration that applies constant loads between a binding protein and an intermittently interacting biological polymer. The method has a measurement delay of only âˆ¼10 Î¼s, allows detection of interactions as brief as âˆ¼100 Î¼s and probes sub-nanometer conformational changes with a time resolution of tens of microseconds. We tested our method on molecular motors and DNA-binding proteins. We could apply constant loads to a single motor domain of myosin before its working stroke was initiated (0.2-1 ms), thus directly measuring its load dependence. We found that, depending on the applied load, myosin weakly interacted (<1 ms) with actin without production of movement, fully developed its working stroke or prematurely detached (<5 ms), thus reducing the working stroke size with load. Our technique extends single-molecule force-clamp spectroscopy and opens new avenues for investigating the effects of forces on biological processes.

Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke.

The maintenance of intact genetic information, as well as the deployment of transcription for specific sets of genes, critically rely on a family of proteins interacting with DNA and recognizing specific sequences or features. The mechanisms by which these proteins search for target DNA are the subject of intense investigations employing a variety of methods in biology. A large interest in these processes stems from the faster-than-diffusion association rates, explained in current models by a combination of 3D and 1D diffusion. Here, we present a review of the single-molecule approaches at the forefront of the study of protein-DNA interaction dynamics and target search in vitro and in vivo. Flow stretch, optical and magnetic manipulation, single fluorophore detection and localization as well as combinations of different methods are described and the results obtained with these techniques are discussed in the framework of the current facilitated diffusion model.

Optical Methods to Study Protein-DNA Interactions in Vitro and in Living Cells at the Single-Molecule Level.

Single molecule force spectroscopy methods, such as optical and magnetic tweezers and atomic force microscopy, have opened up the possibility to study biological processes regulated by force, dynamics of structural conformations of proteins and nucleic acids, and load-dependent kinetics of molecular interactions. Among the various tools available today, optical tweezers have recently seen great progress in terms of spatial resolution, which now allows the measurement of atomic-scale conformational changes, and temporal resolution, which has reached the limit of the microsecond-scale relaxation times of biological molecules bound to a force probe. Here, we review different strategies and experimental configurations recently developed to apply and measure force using optical tweezers. We present the latest progress that has pushed optical tweezers' spatial and temporal resolution down to today's values, discussing the experimental variables and constraints that are influencing measurement resolution and how these can be optimized depending on the biological molecule under study.

Marco Capitanio

LENS - European Laboratory for Non-linear Spectroscopy,
Department of Physics and Astronomy

University of Florence

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

Marco Capitanio

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}LENS - European Laboratory for Non-linear Spectroscopy,Department of Physics and Astronomy

University of Florence

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

LENS - European Laboratory for Non-linear Spectroscopy,
Department of Physics and Astronomy