Abstract

We propose to study localized phenomena in single protein attached to polystyrene beads, which are trapped in a coupled optical tweezers setup. For this purpose, high speed video microscopy with sub-nanometer resolution will be implemented. There are several challenges to achieve this resolution: high speed feedback control mechanism; synchronized video microscopy of trapped particles; three- dimensional tracking of trapped particles; increasing tether’s stiffness. We would like to remark that in this proposal, in order to achieve sub-nanometer spatial resolution, we plan to improve upon the current state of the art of coupled optical tweezers by Bustamante and coworkers, by considering three-dimensional motion tracking by means of sub-pixel video-microscopy at high acquisition rates, due to transversal (x-y) sampling neglects longitudinal fluctuations by projecting them into the two-dimensional sampling plane. The goal is to represent the Brownian fluctuations of the trapped micro-beads in the complete three- dimensional space for a detailed comprehension of the mechanical cycles of the biological targets under study. This new setup will be compared with motion tracking using quadrant photodetectors. In particular, we will use the coupled optical tweezers setup to study, for instance, the effect of the substrate binding and catalysis on the thermodynamic and mechanical stability of adenylate kinase at the single protein level. Besides, it is a milestone of this proposal to test the use of synthetic handles or UV-radiated DNA handles in the biological setup protocol to increase tether’s stiffness for resolution improvement.