Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Friday Speaker Abstracts

DNA INTERACTIONS WITH DRUGS AND SMALL MOLECULES INVESTIGATED BY SINGLE MOLECULE FORCE SPECTROSCOPY Márcio S Rocha 1 ; 1 Universidade Federal de Viçosa, Physics Department, Viçosa, Brazil In this talk we will present some recent studies from our group concerning the DNA interactions with drugs and other small relevant molecules. We will discuss how single molecule force spectroscopy measurements can be useful to determine the possible binding modes, the changes induced on the DNA structure and the physical chemistry of the DNA-ligand interactions. In particular, we present a recently developed quenched-disorder statistical model which allows one to extract the relevant physicochemical (binding) parameters of the interactions from pure mechanical (force-extension) measurements performed with the DNA-ligands complexes. Such a model in principle works well for any type of interaction that occurs between small ligand molecules and DNA, from intercalation to covalent binding, allowing a robust characterization of the interactions with a single experimental technique. THE POWER OF THE FORCE: MECHANO-PHYSIOLOGY OF THE GIANT TITIN J Andres Rivas Pardo 1,2 ; Zsolt Mártonfalvi 4 ; Yong Li 3 ; Wolfgang A Linke 3 ; Julio M Fernández 2 ; Jorge Alegre-Cebollada 5 ; 1 Universidad Mayor, Centro de Genómica y Bioinformática, Santiago, Chile 2 Columbia University, Department of Biological Sciences, New York, NY, USA 3 University of Muenster, Institute of Physiology II, Muenster, Germany 4 Semmelweis University, Department of Biophysics and Radiation Biology, Budapest, Hungary 5 Centro Nacional de Investigaciones Cardiovasculares , (CNIC), Madrid, Spain Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces are generated and sensed in biological tissues. However, testing these mechanical hypotheses on native molecules in their natural environment remains inaccessible to conventional genetics, biophysics and molecular biology tools. To overcome these limitations, here we demonstrate a genetically engineered knock-in mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness. Using our system, we have specifically severed the titin filament by digestion with TEV protease, and found that the response of muscle fibers to length changes requires mechanical transduction through titin’s intact polypeptide chain. HaloTag-based covalent tethering has enabled directed examination of the dynamics of native titin under physiological forces using recently developed magnetic tweezers. At physiological pulling forces lower than 10 pN, titin domains are readily recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Our results support an active role of titin in muscle contraction in coordination with actomyosin motors. Insertion of the HaloTag-TEV cassette in proteins with mechanical roles opens new grounds to explore the molecular basis of cellular force generation, mechanosensing and mechanotransduction.

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