Presentation description

Elucidating physiological protein structure is challenging due to the complexity of attempting to recapitulate cellular conditions in vitro. Our research focuses on using Nuclear Magnetic Resonance (NMR) spectroscopy to better understand protein structure and dynamics in more relevant cell modeling contexts. To mimic cellular crowding, we encapsulated the model protein, ubiquitin, in reverse micelles (RMs), which consist of a water core that solvates the protein and is surrounded by a barrier of surfactant molecules. To ideally model cell environment, it is also important to form protein-containing RMs in a lower viscosity solvent, to allow protein movement through the solvent, and to ensure the generation of high-quality NMR spectra. Thus, to improve the protein encapsulation process for NMR, we focused on developing processes to define the water-to-surfactant ratio, or "water loading," of the RMs. Previous research showed that lowering temperature resulted in a shrinking of the RMs and a decrease in water loading: a process known as water shedding. We further explored this by varying the temperature to develop a refined process for generating and controlling RMs of a specific size. In addition, we overexpressed the well-characterized protein ubiquitin using Escherichia coli with uniform 15N-labeling. We purified the ubiquitin using immobilized metal affinity chromatography (IMAC) with a nickel column, followed by size exclusion chromatography. Purified ubiquitin was encapsulated in RMs using the surfactant dioctyl sodium sulfosuccinate (AOT) in deuterated pentane. Using these samples of RM-encapsulated ubiquitin, we utilized 1D high-resolution NMR to investigate micelle formation and the effects of temperature on RM size. By utilizing RM ubiquitin encapsulation, our work expands tools for investigating protein structure and dynamics in crowded, more cell-like environments.