Intellectual Merit: Proteins perform the majority of functional tasks in living systems and to do so they must be dynamic. Internal protein motion is well known to be dependent on the solvation environment. Though the interaction between solvent water and protein has been extensively studied computationally, site-resolved experimentally derived information on this critical interaction is severely lacking. This project will help to fill this void by quantitatively describing the hydration and dynamics of model proteins under nanoconfined conditions. Both specific water-protein interactions and bulk solvation properties have critical effects on protein structure and function. Solvation conditions under nanoconfinement are now known to differ considerably from bulk solvation, raising the question of how the altered solvation environment in the tight spaces of the cell affects protein behavior. Reverse micelles have become a primary model for studying physicochemical properties of nanoconfined species. Over the past decade, reverse micelle encapsulation has also been adapted for the structural study of proteins using high-resolution NMR. This project will take advantage of the favorable properties of the reverse micelle system to answer fundamental questions of how confinement alters protein hydration and dynamics. Advanced NMR methods will be employed to characterize the effects of confinement on the picosecond-nanosecond-microsecond-millisecond time scales. Hydration waters will be detected and characterized also using NMR-based methods. The data yielded by these studies will permit analysis of the correlations between protein structural character, hydration properties, and protein motion. Such data will help fill the gap between theory and experiment in the exploration of the protein-solvent dynamic landscape.
Broader Impacts: The project will be carried out in a laboratory that has a long history of involving undergraduate, graduate and postdoctoral trainees in cutting edge research. In addition, personnel affiliated with this project are engaged in the NSF SPARK program in Philadelphia. SPARK is designed to nurture the participation of under-represented minorities in the sciences. The results of the research project will be disseminated in high profile journals providing significant exposure not only for the scientific product itself but also for those who have generated it. Finally, the project rests on an emerging technology NMR spectroscopy of encapsulated proteins in low viscosity fluids - and will illustrate the utility and capabilities of this approach thereby continuing to bring a new and innovative technology to the biophysical community. This project is jointly supported by the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences and the Experimental Physical Chemistry Program in the Chemistry Division.
Proteins perform the majority of functional tasks in living systems and to do so they must be dynamic. 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. A schematic illustration of a reverse micelle system is shown in the Figure. Reverse micelles are spontaneously organized assemblies of surfactant and water molecules that form nanoscale sequestered water pools. Through techniques that we have developed, proteins can now be readily incorporated into this water pool with high structural fidelity. High-resolution NMR techniques have been adapted to the reverse micelle system to directly measure protein-water interactions. Encapsulation of proteins greatly simplifies the interpretation of the results through several advantageous properties: the residence time of the water molecules at the surface of the protein are greatly increased, the serves to enhance the basic signal that we wish to measure; the chemistry of hydrogen exchange, which confounds the interpretation of the data, is greatly suppressed; and, the ability to control the effective molecular tumbling time of the protein by changing the bulk solvent permits tuning of the experimental parameters to allow more difficult types of experiments to be undertaken. Using this approach we have able to successfully characterize the hydration of the small protein ubiquitin. We have observed a remarkable clustering of water dynamics its surface that correlates with the regions of the protein that are involved in protein-protein interactions. This has several important implications with respect to how the interaction of proteins with their partners can be influenced by the behavior of the hydration water. We are pursuing this new avenue vigorously. The second objective of this project was 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 nanoconfinement 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. Final results from this line of inquiry are just now being summarized for formal publication. Briefly, we find that the effects of confinement within a bath of highly viscous water have subtle but very interesting effects on the internal main chain and methyl-bearing side chain dynamics of the protein. The work has been 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. The results of this work have been disseminated in high profile journals and have been 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, Professor Wand 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.
