This project aims to develop a method to "film" proteins, the tiny machines that enable all of life as we know it, as they perform their critical biological functions. Understanding how proteins function is one of the most exciting frontiers of science, and is necessary in order to address societal needs in fields as diverse as renewable energy and agriculture. The rich and intricate 3-dimensional shapes of more than 100,000 different kinds of proteins are now documented, thanks to revolutionary advances in X-ray spectroscopy, magnetic resonance, and other methods. However, while knowing the shape of a machine, for example, seeing a photograph of a sewing machine, might give one a clue as to how it works, a movie of a machine in action is far more revealing. The goal of this project is to develop new methods to make such "movies" of proteins, or stated more precisely, to develop methods to measure the time resolved conformational changes in protein structure (or structural dynamics). A team of graduate students and undergraduates, working at the interface between physics, chemistry, and biology, and working closely with international collaborators, will tackle this exciting problem, and will emerge well-positioned to become leaders in the nation's science, technology, engineering and mathematics (STEM) workforce.
"Filming" proteins in action requires a calibrated tool to measure distances of several nanometers with high throughput and, ideally, sub-millisecond time resolution. This tool must be compatible with the complex local environments in which proteins perform their functions; ideally, in aqueous solutions although many proteins continue to function as long as they are above about 215K. The primary goal of the proposed research is to use high-frequency (>200 GHz) electron paramagnetic resonance (EPR) combined with site-directed spin labeling with spin-7/2 Gd3+ moieties to measure time-resolved conformational changes of proteins. With prior NSF support, the PI and co-PI's collaboration has demonstrated that the simplest possible 240 GHz EPR measurements; measurements of the lineshapes of molecules containing a pair of Gd3+ spin labels; is capable of resolving distances greater than 3 nm even at room temperature. The proposed research will build on these key developments, focusing primarily on time-resolved distance measurements in the model protein Proteorhodopsin (PR), a photosynthetic trans-membrane proton pump that can be triggered with flashes of light to begin synchronized cascades of conformational changes on time scales ranging from less than 1 microsecond to more than 1 second. The main activities undertaken will be: (1) Developing a simple method based on measuring the lineshape of the Gd3+ EPR line to extract distances and distance distributions in the 1.5-4 nm range from PR doubly labeled with Gd3+. (2) Measuring conformational changes of PR at temperatures ranging from about 215K (where PR is frozen but active) to room temperature with sub-millisecond time resolution. (3) Pulsed EPR studies of Gd3+ to understand the spin physics of Gd3+ at very high frequencies.
