My long term goal is to establish a successful academic career as an independently funded biophysicist applying single-molecule fluorescence. The K25 award proposed here will provide the mechanism for me to transfer into this line of study from my background of experimental physics. Specifically, I have proposed to determine the dynamic changes in conformation of a myosin cross-bridges as a result of Familial Hypertrophic Cardiomyopathy (FHC) via single molecule detection of polarized fluorescence so that these changes may be combatted. This is a very direct extension of my postdoctoral work in advanced fluorescence techniques and my collaborative projects in muscle function. Single molecule detection is of critical importance to this project, because the myosin cross-bridges rotate independently. Thus when polarized fluorescence is measured from an ensemble of cross-bridges, the signal becomes scrambled from the superposition of the individual signals of all labeled cross-bridges in the detection volume along with background contributions. The kinetics of a single myosin cross-bridge have been studied in vitro, but it is not at all obvious that the behavior would be the same ex vivo, when molecular crowding will be sure to have an effect. I propose to obtain ex vivo, single molecule detection of a myosin crossbridge in the following manner by (1) converting an existing, time-resolved, confocal microscope into a Stimulated Emission Depletion (STED), super-resolution microscope, (2) evaluating the use of Fluorescent Nanodiamonds (FNDs) for single molecule, ex vivo measurements in muscle, and finally (3) using the methods developed in the previous two stages to identify changes in kinetics induced by FHC. The STED technique is necessary to confine the focal volume of the confocal microscope to an area small enough that only one labeled myosin molecule is observed at any one time and the background contribution is minimized. Of the many super- resolution techniques currently under development or being employed, STED is the chosen technique, because it is the only one that can allow the study of fast dynamics on a scale that avoids smaller than the diffraction-limited point spread function of conventional microscope systems. However, single molecule observation of an organic fluorophore can be troublesome, as the molecules are prone to photoblinking as they oscillate between light and dark state. Even more troublesome, organic fluorophores are not very photostable-they are prone to photobleaching after only short periods of time. This effect is exacerbated by higher laser powers, which are necessary to obtain a sufficiently high signal from a single molecule. Thus we suggest the use of FNDs, which do not photoblink and have been shown to be photostable for hours. They will be attached to myosin by a new procedure developed in the course of this proposed project. With the microscopy and labeling concerns fully addressed, the third phase of this project will study the kinetics of a single myosin molecul in healthy and diseased heart tissue from transgenic mice. I received a Ph.D. in Condensed Matter Physics from Texas Christian University in 2011, and immediately transitioned to Postdoctoral appointment at the University of North Texas Health Science Center. Here I have been extremely productive in the development of advanced fluorescence techniques for the life sciences. My publishing record demonstrates my ability to translate my physics training into biologically relevant work and my extensively involvement in the investigation of muscle dynamics, if only in a technical role thus far. The K25 award will allow for necessary biological coursework, and mentored lab work to fully develop into a dedicated, independent biophysicist.
My project will improve our understanding of Familial Hypertrophic Cardiomyopathy (FHC) by identifying distinct changes in molecular motion during contraction of hypertrophic hearts. I will do this by tracking the motion of a single molecule usin the fluorescent nanodiamonds and stimulated emission depletion microscopy. I will develop measurement techniques that will be available to researchers in other fields of health-related studies.