Our approaches include biochemical, biophysical and structural studies of actin, myosin, and associated proteins from eukaryotic cells. We have designed and developed in vitro assays for ATP-dependent movement of purified myosin on filaments reconstituted from purified actin. We have taken this assay to the single molecule level, using laser traps, total internal reflection fluorescence microscopy, and gold nanoparticle tracking. Myosin cloning and expression of mutagenized forms that are analyzed for altered functions is routine in our laboratory.
Loaded in vitro motility assay
A key assay for determining changes in power output due to HCM and DCM mutations is the loaded in vitro motility assay. The yellow actin filament is being driven in the direction of the arrow by the red myosin heads that are attached to the surface by the blue anchor molecules. The green actin-binding proteins serve as a load on the actin.
Single molecule analysis
A dual beam laser trap allows measurements of nanometer displacements and piconewton forces produced by myosin interaction with actin.