Single Molecule Biophysics
Developing first combined single molecule force and fluorescence microscope to control and monitor macromolecular function:
Conventional single molecule techniques like Fluorescence Resonance Energy Transfer (FRET) and force measurements with the Atomic Force Microscope (AFM) have facilitated much progress in single molecule biophysics. Single molecule FRET can report on the conformational state and the dynamics of biomolecules while the AFM can be used to mechanically manipulate molecules and measure unbinding forces with pN force resolution. However each of these techniques suffers from limitations that can be overcome by the use of a combined single molecule AFM-FRET approach. For example, with a stand-alone AFM it is difficult to determine precisely where force exerts its effect within a macromolecular complex. This location can however be identified by attaching fluorescent probes to molecular sub-domains and monitoring their motion using single molecule FRET. Similarly, molecular binding events observed by fluorescence can be related to force induced structural changes to determine the coupling between applied load and biochemistry. Our laboratory is building the first instrument that successfully combines a single molecule Atomic Force Microscope (AFM) with Fluorescence Resonance Energy Transfer (FRET) to apply forces on individual biological molecules and simultaneously monitor their conformational dynamics.