Angstrom-Resolution Optical Tweezers: Seeing Structure and Function of Biological Molecular Machines in Real Time | Kent State University

Angstrom-Resolution Optical Tweezers: Seeing Structure and Function of Biological Molecular Machines in Real Time

Fundamental processes of life are carried out within cells by nm-scale molecular machines. For example, there are protein motor molecules that burn chemical fuel to exert forces and move along RNA tracks in order to modify molecular structures.  Single molecule biophysics provides powerful experimental methods to investigate such systems, allowing us to observe the action of individual molecules in real time.  I will present new methods that combine two of the most powerful techniques: angstrom-resolution optical tweezers and single molecule fluorescence microscopy (Comstock et al., Nature Methods 2011 and Science 2015).  I will then present two areas of biomolecular investigation in my lab. First, I will present high-resolution real-time measurements of RNA unwinding by two contrasting types of RNA helicase: a ‘Moving’ motor, Mtr4p (Comstock et al., Nature Chemical Biology 2017) and a ‘stationary’ machine, Ded1p. For each we can resolve the individual base pair scale stepwise activity of each machine in order to understand their mechanisms. Second, we investigate how molecular machines are built, and I will present high resolution measurements of single protein molecules folding and unforlding under force. We can trade high spatial resolution for high time resolution, down to 10’s of microseconds. I will present investigations of two small model proteins: the B1 domain of protein G (PG) and the human Yes-associated protein (hYap). For PG, the usual out-of-equilibrium force ramp measurements suggest complex multiple folding pathways.  Long duration high resolution equilibrium measurements directly reveal folding and unfolding reaction rate constants vs force and confirm this complexity. For hYap, we are able to observe sub-millisecond folding and unfolding at low forces in force ramp experiments.