Single-Molecule Kinetics Under Force: Probing Protein Folding and Enzymatic Activity with Optical Tweezers

Abstract

Weak non-covalent bonds between and within single molecules govern many aspects of biological structure and function (e.g. receptor-ligand binding, protein folding) In living systems, these interactions are often subject to mechanical forces, which can greatly alter their kinetics and activity. My group develops and applies single-molecule manipulation techniques to explore and quantify these force-dependent kinetics. We have developed a variety of optical tweezers techniques, such as high-resolution 3D position tracking using interference imaging (0.2 nm resolution in z, 1 nm in x-y) [1,2], active feedback for longterm stability in trap height and focus (1-2 nm stability) [2], and intensity modulation imaging for quantifying high-frequency fluctuation above the acquistion rate of a detector (power spectrum measurements above 100 kHz can be made with a slow camera) [3]. We have used these methods to quantify the force-dependent unfolding and refolding kinetics of proteins, including the cytoskeletal protein spectrin in collaboration with E. Evans [4], and the A2 domain of the von Willebrand factor blood clotting protein in collaboration with T. Springer [5]. Furthermore, we have studied the kinetics of the ADAMTS13 enzyme acting on a single A2 domain, and have shown that physiolgical forces in the circulation can act as a cofactor for enzymatic cleavage, regulating hemostatic activity [5].