Browsing by Subject "ssNMR"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Biophysical Characterization of Interactions Between Two Membrane Proteins: SERCA and Sarcolipin
    (2017-05) Dicke, Alysha
    Understanding the structures and interactions of proteins that interact with membranes has many implications. Membrane proteins play roles in the transfer of necessary materials and information between cells and their environments as well as within cells (e.g., between the cytosol and organelles). As such, they currently constitute more than half of all drug targets, and some peptides, such as antimicrobial peptides (AMPs), are also being investigated for their therapeutic use in treating bacterial infections for humans. However, studying the structures of membrane proteins has proven more challenging compared to soluble proteins. This is due to the necessity of including the membrane or a good membrane mimic to ensure the integrity of the membrane protein remains intact, as poor mimics or no membrane can detrimentally affect membrane protein structure and function. Some proteins, like the AMP chionodracine, are highly amenable to study with methods such as solution NMR spectroscopy (Chapter 2), but larger membrane proteins prove challenging or impossible to measure in solution due to the molecular weight limitations and frequently do not crystallize easily either. Solid-state NMR spectroscopy (ssNMR) has helped to overcome these obstacles and more methodology continues to be developed expanding the application of ssNMR. For example, Chapters 3 and 4 of this thesis describe new ssNMR methods using the sarco(endo)plasmic reticulum (SERCA) and sarcolipin (SLN), respectively. SERCA and SLN form a crucial complex in the membrane of the sarcoplasmic reticulum in skeletal muscles cells. Muscle relaxation is largely controlled by SERCA pumping calcium out of the cytosol using energy from ATP hydrolysis, and SLN inhibits SERCA as well as uncouples the ATP hydrolysis and calcium transport. SLN’s effect on SERCA leads to more heat production, which may be important to thermogenesis in mammals as well as an additional mechanism to control energy expenditure. Chapter 5 primarily uses ssNMR with the goal to better understand the mechanism by which SLN inhibits and uncouples SERCA. Overall, gaining a better understanding of how SERCA is regulated will aid in developing therapies for diseases resulting from improper calcium cycling.
  • Thumbnail Image
    Item
    Investigation and Characterization of Flavor to Food Matrix Interactions using Solid State Nuclear Magnetic Resonance
    (2016-09) Jilek, Margaret
    Intermolecular interactions between flavor molecules and food matrices are known to influence flavor release. However, the mechanistic interactions themselves remain largely uncharacterized and the dynamics unstudied. The overall goal of this research project is to develop a research platform which can characterize and observe the dynamics of flavor to matrix interactions. Solid state nuclear magnetic resonance (ssNMR) was applied to characterize molecular interaction dynamics at the atomic scale in flavored gum matrix model systems. Preferential interactions between flavor compounds (Melonal and ethyl propionate) and model polymers were detected and quantified. Chemical shift perturbances were calculated for Melonal in model matrices providing site-specific information. This methodology studied flavor to matrix interactions in situ, determined if preferential binding behavior exists and indentified which functional groups were involved in the interactions. Next, isotopically enriched model polymer matrices were utilized to assess differences in flavor to matrix interactions as physical properties of the matrix changed. Short molecular weight (MW), long MW and mixtures of the short and long MW matrices were mixed with carbon-13 labeled acetophenone. The interaction dynamics were studied using ssNMR. Acetophenone was shown to be more mobile, and therefore available to release, in mixtures of short and long MW matrices. Acetophenone release profiles also showed greater release from mixed short and long MW matrices. Quantification confirmed greater acetophenone release from blended short and long MW matrices. These effects were overlooked by a traditional thermodynamic based prediction method. Finally, the impact of polymer type and MW on the strength of interactions of a flavor compound was examined. Interactions between acetophenone and model matrices were further characterized by two-dimensional nuclear Overhauser effect spectroscopy (NOESY) combined with INEPT. These interactions were reported to occur between the aromatic carbons of acetophenone and the hydrocarbon backbone and side chains of D block. The strength of these interactions was affected by MW changes. In summary, the implementation and refinement of ssNMR techniques was able to provide deep insight into the flavor to matrix interactions which govern flavor release. This technology and approach has to ability to make significant gains toward understanding the mechanisms involved in flavor release.