Internal protein motion has long been thought to be dependent on the solvation environment. Though the detailed interactions between solvent water and protein have been extensively studied computationally, experimentally derived site-resolved information on this critical interaction is severely lacking. This research project seeks to help fill this void. The first objective is to directly detect and localize long-lived waters of hydration of proteins. Reverse micelles will be used to encapsulate individual protein molecules. Reverse micelles are spontaneously organized assemblies of surfactant and water molecules that form nanoscale sequestered water pools. Proteins can now be readily incorporated into this water pool with high structural fidelity. High-resolution NMR will then be employed to directly measure protein-water interactions. Encapsulation of proteins greatly simplifies the interpretation of the results. The second objective is to determine the effect of confinement and the concomitant slowed water dynamics on the internal motion of the proteins. The protein backbone and side chain dynamics under nano-confinement will be characterized using NMR relaxation methods. Over the past decade, the necessary technology and expertise have been developed to make encapsulation of proteins for high resolution NMR characterization possible. These systems thus serve as a mimic of the confined spaces of the cell and are exploited here to answer several pressing questions including the degree and type of coupling between solvent motion, which is slowed in the reverse micelle, and internal protein motion.
The work will be carried out at the University of Pennsylvania with the participation of postdoctoral, graduate and undergraduate students and high school students from the region. This laboratory has a significant history of involvement in cutting edge research by undergraduate and high school students. In addition, the postdoctoral associate affiliated with this project is actively engaged in the iPRAXIS program in Philadelphia designed to nurture the participation of under-represented minorities in the sciences. Both he and the PI are continuing this interaction during the current research effort. The results of this work will be disseminated in high profile journals and presented at international and national meetings providing significant exposure not only for the scientific product itself but also for those who have generated it. Indeed, postdoctoral associates, graduate students and undergraduates participating in this research have traveled to major international meetings to present their work. In addition, the PI has in the recent past organized international meetings that resonate with the central themes of this research program and will do so in the future. Finally, this work rests on an emerging technology - NMR spectroscopy of encapsulated proteins in low viscosity fluids. This research effort will more widely illustrate the utility and capabilities of this approach and thereby continue to bring a new and innovative technology to the biophysical community.
