Seeing structure and function of biological molecular machines in real-time: angstrom-resolution optical trapping and single molecule fluorescence.

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 DNA tracks in order to modify molecular structures (e.g., helicases ripping open the DNA double helix) or copy information (e.g., polymerases transcribing DNA into messenger RNA). Single molecule biophysics provides powerful experimental methods to gain understanding of such molecular machine systems, allowing us to observe the action of individual molecular systems in real time. However, biological molecular systems are multi-component, highly coordinated, multi-degree-of-freedom systems with fundamental activity at the angstrom scale (the scale of individual bases of DNA). Current single molecular measurement techniques suffer from being able to measure only one degree-of-freedom or molecular component at a time or lack angstrom spatial resolution. I will present a new instrument that we have constructed that overcomes this limitation by combining into one two of the most powerful techniques of single molecule biophysics: angstrom-resolution optical trapping and single molecule fluorescence microscopy. The optical tweezers portion of the new instrument is based on a timeshared dual optical trap design and is interlaced with a confocal fluorescence microscope. I will present experiments involving different aspects of DNA metabolism that demonstrate the capability of our new instrument. I will first show measurements of the dynamics of individual strands of DNA hybridizing (binding) and melting (unbinding). I will then present high resolution measurements of DNA unwinding by the UvrD helicase molecular motor. Here we are able to obtain an unprecedented detailed view of motor dynamics by simultaneously measuring single base pair scale unwinding (via the optical tweezers) and molecular conformation and configuration (via FRET fluorescence). We can directly see how molecular configuration and conformation corresponds to motor activity.