Solvent-driven forces, such as hydrophobic packing, are the primary impetuses which determines protein structure, and protein structure determines much of protein function. Despite the known importance of protein-solvent interactions, global site-resolved measurement of solvent dynamics has not been performed.
The first aim of the proposed work is to determine the nature of structural waters in the protein interior and of hydration waters on the protein surface. High-resolution NMR will be used to measure protein-water dipole-dipole interactions, as manifested in the nuclear Overhauser effect (NOE). In bulk aqueous solution, solvent dynamics near the protein surface are too fast to measure large numbers of protein-solvent interactions on the protein surface. We will use reverse micelles to encapsulate the proteins for these experiments. Reverse micellar confinement slows solvent dynamics by up to two orders of magnitude, permitting measurement of tens of protein-water interactions on the protein surface, while maintaining the structural fidelity of the encapsulated protein. By measuring the protein-water NOEs for ubiquitin, cytochrome c, and flavodoxin in reverse micelles, we will be able to examine the effect of protein surface character, oxidation state, and ligand binding on the location and timescale of specific interactions between proteins and their solvating environment. In recent years, it has become clear that the dynamic motions of proteins are a vital aspect of their function. Our understanding of protein dynamic motions and their implications is in its infancy, and further elucidation of the fundamental aspects of such dynamic processes is needed. It is widely recognized that the confines of the cell present a dynamically altered and vastly more complex solvation environment than that of the bulk aqueous solutions. The effects of the altered solvation dynamics under nanoscale confinement on protein dynamics is thus of fundamental interest.
The second aim of the proposed research is to determine the impact of nanoconfinement on protein dynamics. Using reverse micelles as the confining medium, we will use high-resolution NMR measurements of backbone and methyl relaxation to evaluate the differences in protein dynamics as a result of nanoconfinement. Ubiquitin, cytochrome c, and flavodoxin will each be examined, allowing comparison of the effects of surface electrostatic character, oxidation state, and ligand binding on the interplay between solvent dynamics and protein dynamics. Explanation of the fundamental relationship between proteins and their solvating environment is crucial to improvements in the development of pharmaceutical therapeutics.
| Wand, A Joshua; Nucci, Nathaniel V (2015) Reply to Kitahara and Mulder: An ensemble view of protein stability best explains pressure effects in a T4 lysozyme cavity mutant. Proc Natl Acad Sci U S A 112:E924 |
| Valentine, Kathleen G; Mathies, Guinevere; Bédard, Sabrina et al. (2014) Reverse micelles as a platform for dynamic nuclear polarization in solution NMR of proteins. J Am Chem Soc 136:2800-7 |
| Nucci, Nathaniel V; Fuglestad, Brian; Athanasoula, Evangelia A et al. (2014) Role of cavities and hydration in the pressure unfolding of T4 lysozyme. Proc Natl Acad Sci U S A 111:13846-51 |
| Dodevski, Igor; Nucci, Nathaniel V; Valentine, Kathleen G et al. (2014) Optimized reverse micelle surfactant system for high-resolution NMR spectroscopy of encapsulated proteins and nucleic acids dissolved in low viscosity fluids. J Am Chem Soc 136:3465-74 |
| Marques, Bryan S; Nucci, Nathaniel V; Dodevski, Igor et al. (2014) Measurement and control of pH in the aqueous interior of reverse micelles. J Phys Chem B 118:2020-31 |
| Nucci, Nathaniel V; Marques, Bryan S; Bédard, Sabrina et al. (2011) Optimization of NMR spectroscopy of encapsulated proteins dissolved in low viscosity fluids. J Biomol NMR 50:421-30 |
| Peterson, Ronald W; Nucci, Nathaniel V; Wand, A Joshua (2011) Modification of encapsulation pressure of reverse micelles in liquid ethane. J Magn Reson 212:229-33 |
| Nucci, Nathaniel V; Pometun, Maxim S; Wand, A Joshua (2011) Mapping the hydration dynamics of ubiquitin. J Am Chem Soc 133:12326-9 |
| Nucci, Nathaniel V; Pometun, Maxim S; Wand, A Joshua (2011) Site-resolved measurement of water-protein interactions by solution NMR. Nat Struct Mol Biol 18:245-9 |
