Date of Award
Winter 12-15-2015
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Solid-state NMR was used to study the structures of phorbol diacetate and bryostatin bound to protein kinase Cδ C1b domain in lipid bilayers. The interaction of protein kinase C (PKC) with native physiological ligands drives fundamental cellular signal transductions, and aberrant PKC signaling is associated with cancer, cardiovascular disease, neurological disorders, stroke, pain, etc. Bryostatin modulates PKC and exhibits remarkable potential in treating cancer, Alzheimer's disease, and HIV/AIDS. However, the structural information and dynamics of this modulation, which are crucial for better understanding of PKC-ligand interactions in the membrane microenvironment to drive the development of new drugs, remain elusive. REDOR NMR was used to measure the distances between spin pairs in [13C, 2H3]phorbol diacetate and [19F, 13C, 2H3]bryostatin bound to PKCδ C1b domain in lipid bilayers to report on the bio-active bound conformation of the ligands. A single distance was obtained between the 13C and CD3 group in phorbol diacetate, indicating a single conformation, whereas Gaussian distributions of distances were obtained for the three spin pairs in bryostatin, indicating a distribution of conformations. The REDOR results suggest that the binding site of the PKC C1b domain complexed with bryostatin but not with phorbol diacetate is probably dynamic in solution, and the lyophilized sample therefore contains a distribution of trapped conformations and a distribution of distances. Solid-state NMR experiments also revealed ~60% of phorbol diacetate is peptide bound, and the rest ~40% is lipid associated, whereas almost all bryostatin is peptide bound. This observation is consistent with the fact that bryostatin has a much higher binding affinity to PKC than phorbol diacetate.
Solid-state NMR was also used to study the tertiary structure of the peptidoglycan of Enterococcus faecalis. Enterococci have emerged as leading hospital-acquired pathogens in recent years. They are intrinsically resistant to several commonly used antibiotics, and are able to acquire resistance to almost all currently available antibiotics, including the drug of last resort, vancomycin. The E. faecalis bacterial cell wall is crucial to maintaining the integrity of the cell structure, and is an important target of antibiotics. Solid-state NMR study revealed ~ 50% cross-linking of E. faecalis peptidoglycan and the presence of an active carboxypeptidase that modifies uncross-linked stems. NMR results support the conclusion that the peptidoglycan of E. faecalis has the same short-bridge architecture as that of the FemA mutant of S. aureus.
Solid-state NMR was used to study the reversible CO2 capture mechanism of a humidity-swing polymeric sorbent. The CO2 concentration in the atmosphere has increased from ~280 ppm before the industrial revolution to over 400 ppm in 2015, and is projected to increase to 550 ppm to 900 ppm by the end of this century. This rapid atmospheric CO2 concentration increase causes global warming and climate change. Together, they pose unprecedented challenges to humans and our environment, calling for our immediate actions to capture and sequester CO2 from the atmosphere. The sorbent, containing quaternary ammonium cations, has been shown to absorb CO2 selectively from dry air and release CO2 when exposed to humidified air. This could be an inexpensive and recyclable means to capture atmospheric CO2. The solid-state NMR investigation here unambiguously reveals the humidity-driven CO2 absorption and release processes, and suggests that CO32− is formed upon CO2 absorption and replaced by OH- upon CO2 release when exposed to humidified air. Based on the NMR evidence, a capture mechanism is proposed which involves electrostatic interactions between the quaternary ammonium cations and CO32−. This mechanism relies on chemical equilibria to explain why humidity is the driving force for CO2 absorption and release.
Language
English (en)
Chair and Committee
Jacob Schaefer
Committee Members
Sophia Hayes, Richard Loomis, Garland Marshall, Jay Ponder
Recommended Citation
Yang, Hao, "Solid-State NMR Study of the Tertiary Structure of the Peptidoglycan of Enterococcus faecalis and the Structures of Phorbol Diacetate and Bryostatin Bound to Protein Kinase Cδ C1b Domain" (2015). Arts & Sciences Electronic Theses and Dissertations. 693.
https://openscholarship.wustl.edu/art_sci_etds/693
Comments
Permanent URL: https://doi.org/10.7936/K7833Q9M