Reverse micelle encapsulation as a biophysical technique to study the effects of confinement and low temperature on water and protein

Reverse micelle encapsulation is a powerful biophysical approach capable of resolving fundamental and long-standing questions. Reverse micelles are thermodynamically stable assemblies of surfactant molecules organized around an aqueous core (nanopool) that spontaneously form transparent solutions in low polarity liquids; proteins, nucleic acids, and organic molecules can be encapsulated within the water pool. Reverse micelles provide a unique environment that allows complete control over pH, ionic strength, and hydration of a biomacromolecule. The focus of this dissertation is the characterization of reverse micelle parameters for the investigation of both confinement and low-temperature effects on water and protein using solution Nuclear Magnetic Resonance (NMR) spectroscopy, a technique that allows atomic level investigation of both structure and dynamics. The areas of protein cold denaturation and confinement are traditionally difficult to examine, and reverse micelles are proving to be a valuable tool for these fields. First, a novel pulse sequence was created that allows pulsed field gradient NMR experiments to be conducted on reverse micelles in order to examine the radius of hydration and polydispersity of the particles. Next, studies were conducted to show that high ionic strength is necessary for the stability of reverse micelles at subzero temperatures. A new phenomenon, termed water shedding, was characterized, and multiple populations of water were seen to exist under water shedding conditions, illustrating the ability of solution NMR to detect different types of confined water. Subsequently, the protein ubiquitin was encapsulated and cold denaturation studies were conducted under high salt conditions. The protein was seen to unfold in a globally cooperative manner under hydrating conditions. Conversely, under dehydrating conditions, significant protein-reverse micelle interactions were observed and a method for minimizing proteins-micelle interactions was subsequently developed. Finally, the protein ubiquitin was encapsulated and NMR relaxation studies were conducted on the protein, which supported the investigation of site-specific dynamic information of the protein backbone. Overall, this indicates the that the fast motion dynamics of encapsulated ubiquitin are very similar to those characterized for the free solution form of the protein, with only subtle changes in the flexibility of loop regions and the beginning of secondary structures.