Single-Molecule Optical Tweezers As a Tool for Delineating the Mechanisms of Protein-Processing Mechanoenzymes.

Mechanoenzymes convert chemical energy from the hydrolysis of nucleotide triphosphates to mechanical energy for carrying out cellular functions ranging from DNA unwinding to protein degradation. Protein-processing mechanoenzymes either remodel the protein structures or translocate them across cellular compartments in an energy-dependent manner. Optical-tweezer-based single-molecule force spectroscopy assays have divulged information on details of chemo-mechanical coupling, directed motion, as well as mechanical forces these enzymes are capable of generating. In this review, we introduce the working principles of optical tweezers as a single-molecule force spectroscopy tool and assays developed to decipher the properties such as unfolding kinetics, translocation velocities, and step sizes by protein remodeling mechanoenzymes. We focus on molecular motors involved in protein degradation and disaggregation, i.e., ClpXP, ClpAP, and ClpB, and insights provided by single-molecule assays on kinetics and stepping dynamics during protein unfolding and translocation. Cellular activities such as protein synthesis, folding, and translocation across membranes are also energy dependent, and the recent single-molecule studies decoding the role of mechanical forces on these processes have been discussed.