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- All Subjects: heliobacteria
- Creators: Ghirlanda, Giovanna
- Member of: Theses and Dissertations
- Status: Published
Heliobacteria are an anaerobic phototroph that require carbon sources such as pyruvate, <br/>lactate, or acetate for growth (Sattley, et. al. 2008). They are known for having one of the <br/>simplest phototrophic systems, the central component of which is a Type I reaction center (RC) <br/>that pumps protons to generate the electrochemical gradient for making ATP. Heliobacteria <br/>preform cyclic electron flow (CEF) with the RC in the light but can also grow chemotropically in <br/>the dark. Many anaerobes like heliobacteria, such as other members of the class Clostridia, <br/>possess the capability to produce hydrogen via a hydrogenase enzyme in the cell, as protons can <br/>serve as an electron acceptor in anaerobic metabolism. However, the species of heliobacteria <br/>studied here, H. modesticaldum have been seen to produce hydrogen via their nitrogenase <br/>enzyme but not when this enzyme is inactive. This study aimed to investigate if the reason for <br/>their lack of hydrogen production was due to a lack of an active hydrogenase enzyme, possibly <br/>indicating that the genes required for activity were lost by an H. modesticaldum ancestor. This <br/>was done by introducing genes encoding a clostridial [FeFe] hydrogenase from C. thermocellum<br/>via conjugation and measuring hydrogen production in the transformant cells. Transformant cells <br/>produced hydrogen and cells without the genes did not, meaning that the heliobacteria ferredoxin <br/>was capable of donating electrons to the foreign hydrogenase to make hydrogen. Because the <br/>[FeFe] hydrogenase must receive electrons from the cytosolic ferredoxin, it was hypothesized <br/>that hydrogen production in heliobacteria could be used to probe the redox state of the ferredoxin <br/>pool in conditions of varying electron availability. Results of this study showed that hydrogen <br/>production was affected by electron availability variations due to varying pyruvate <br/>concentrations in the media, light vs dark environment, use acetate as a carbon source, and being <br/>provided external electron donors. Hydrogen production, therefore, was predicted to be an <br/>effective indicator of electron availability in the reduced ferredoxin pool.
Non-canonical amino acids (NCAAs) can be used in protein chemistry to determine their structures. A common method for imaging proteins is cryo-electron microscopy (cryo-EM) which is ideal for imaging proteins that cannot be obtained in large quantities. Proteins with indistinguishable features are difficult to image using this method due to the large size requirements, therefore antibodies designed specifically for binding these proteins have been utilized to better identify the proteins. By using an existing antibody that binds to stilbene, NCAAs containing this molecule can be used as a linker between proteins and an antibody. Stilbene containing amino acids can be integrated into proteins to make this process more access able. In this paper, synthesis methods for various NCAAs containing stilbene were proposed. The resulting successfully synthesized NCAAs were E)-N6-(5-oxo-5-((4-styrylphenyl) amino) pentanoyl) lysine, (R,E)-2-amino-3-(5-oxo-5-((4-styrylphenyl)amino)pentanamido)propanoic acid, (E)-2-amino-5-(5-oxo-5-((4-styrylphenyl) amino) pentanamido) pentanoic acid. A synthesis for three more shorter amino acids, (R,E)-2-amino-3-(3-oxo-3-((4-styrylphenyl) amino) propanamido) propanoic acid, (E)-2-amino-5-(3-oxo-3-((4-styrylphenyl) amino) propanamido) pentanoic acid, and (E)-N6-(3-oxo-3-((4-styrylphenyl) amino) propanoyl) lysine, is also proposed.