Methodological development of reverse micelle applications in biophysics and structural biology.

摘要

Reverse micelles are nanoparticles comprised of surfactants that surround an aqueous core and are solubilized in apolar solvents. The reverse micelle water core is easily tuned to host or encapsulate a variety of biologically relevant macromolecules. Coupling reverse micelle encapsulation with nuclear magnetic resonance spectroscopy (NMR) gives a unique tool that is well suited to investigate fundamental processes in biophysics and resolve important issues in structural biology. NMR is naturally paired with reverse micelle technology because it is routinely used in atomic resolution examination of biomacromolecular structure and dynamics. However, NMR based studies of encapsulated proteins are still in relatively infant stages. This dissertation focuses on methodological development of NMR based reverse micelle applications to encapsulated proteins in biophysics and structural biology—specifically protein cold denaturation, confinement of water and proteins, and reverse micelle encapsulation of membrane proteins. First, low temperature studies of encapsulated proteins developed proper methodologies for examination of protein cold denaturation. During these studies, a new phenomenon called water shedding was characterized. When reverse micelles are exposed to low temperatures, water is expelled from the reverse micelle and aggregates as ice in the bottom of the sample. In depth characterization of water shedding revealed the potential for of this system for exploration of confined water. Multiple populations of relatively long-lived water were detected in reverse micelles effectively, illustrating the promise of this technique. Because the amount of water in a reverse micelle generally dictates the size of the particle, and water shedding causes a loss of water to the surroundings, a novel method was developed which uses reverse micelles and water shedding to investigate the effects of confinement and excluded volume on proteins. The results of variable reverse micelle confinement were compared to an analogous crowded solution using bovine serum albumin as a crowding agent. It was verified that confinement and crowding are fundamentally similar, demonstrating the inherent utility of the combination of reverse micelles and NMR. Lastly, inroads were made to the important field of membrane protein structural biology. It was shown that reverse micelles are inherently compatible with membrane protein structural studies.