SINPATH · Single protein folding Pathways

Final Activity Report Summary - SINPATH (Single protein folding pathways)

The Marie Curie IRG SINPATH project dealt with the development of a laser tweezer set-up and with the study of protein folding at the single molecule level. At the Department of Physics of the University of Modena and Reggio Emilia we have built a momentum-flux force sensor dual-beam laser trap that operates by direct measurement of light momentum. In this set-up individual proteins are manipulated by means of molecular handle, app. 500 bp DNA molecules, between two polystyrene beads; one bead is held in the force-measuring optical trap, while the other is held by suction at the end of a micropipette. The molecular handles connect specifically the protein to the beads and keep the attachment points at a distance at which unspecific interactions between the tethering surfaces are negligible. During the experiment, the molecule is unfolded and refolded by moving the pipette relative to the optical trap and the forces applied on the molecule determined by measuring directly the change in momentum flux of the light beams leaving the trap. Using this setup it is possible, among other things: i) to measure the strength of the forces that hold together tertiary and secondary protein structures; ii) to probe the deformability of the protein native state; iii) to monitor in real time fluctuations between different molecular conformations and characterise the kinetics and thermodynamics of these processes; iv) to investigate less probable folding trajectories, and v) to probe alternative regions of the protein's energy landscape to those explored in experiments with chemical and thermal denaturation. Using our laser tweezers set-up we were able to study folding trajectories of individual proteins such as RNase H, Azurin, and alpha-synuclein. Among the main results, we were able to determine the zero-force rate constants, the distances between the native states and the rate-limiting transition states along the direction of force, and characterise the dependence of the unfolding forces on the temperature. Moreover, molecular dynamic simulations were initiated to learn more about atomistic details of the folding reactions.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз