Matching Items (4)

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Incorporation, Expression, and Retained Enzymatic Activity of Six Tryptophan Analogues in Dihydrofolate Reductase at Positions 30 and 47

Description

Due to a continued interest in the fundamental properties of dihydrofolate reductase (DHFR) and its enzymatic activities, this study employed the use of six fluorescent tryptophan derivatives, for single site

Due to a continued interest in the fundamental properties of dihydrofolate reductase (DHFR) and its enzymatic activities, this study employed the use of six fluorescent tryptophan derivatives, for single site amino acid replacements. The two positions 30 and 47 within DHFR were studied to discover the rate at which these larger tryptophan analogues may be incorporated. Additionally, it was to be determined how much activity the mutated DHFR’s could retain when compared to their wild type counterpart. Through a review of literature, it was shown that previous studies have illustrated successful incorporation and toleration of unnatural amino acids.
Each of the six analogues A through F were relatively efficiently incorporated into the enzyme and well tolerated. Each maintained at least a third of their catalytic activity, measured through the consumption of β-nicotinamide adenine dinucleotide phosphate. Primarily, derivatives B, C, and D were able to retain the highest amount of activity in each position; B and D were the most tolerated in positions 30 and 47 with respective values of 68 ± 6.1 and 80 ± 12. The findings in this study illustrate that single tryptophan derivatives are able to be incorporated into Escherichia coli DHFR while still allowing the maintenance of a significant portion of its enzymatic activity.

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  • 2015-05

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The Effects of the PsaL Calcium Binding on the Oligomeric and Spectroscopic Properties in Synechocystis sp. PCC 6803

Description

In oxygenic photosynthesis, conversion of solar energy to chemical energy is catalyzed by the<br/>pigment-protein complexes Photosystem II (PSII) and Photosystem I (PSI) embedded within the<br/>thylakoid membrane of photoautotrophs. The function

In oxygenic photosynthesis, conversion of solar energy to chemical energy is catalyzed by the<br/>pigment-protein complexes Photosystem II (PSII) and Photosystem I (PSI) embedded within the<br/>thylakoid membrane of photoautotrophs. The function of these pigment-protein complexes are<br/>conserved between all photoautotrophs, however, the oligomeric structure, as well as the<br/>spectroscopic properties of the PSI complex, differ. In early evolving photoautotrophs, PSI<br/>exists in a trimeric organization, but in later evolving species this was lost and PSI exists solely<br/>as a monomer. While the reasons for a change in oligomerization are not fully understood, one<br/>of the 11 subunits within cyanobacterial PSI, PsaL, is thought to be involved in trimerization<br/>through the coordination of a calcium ion in an adjacent monomer. Recently published<br/>structures have demonstrated that PSI complexes are capable of trimerization without<br/>coordinating the calcium ion within PsaL.<br/>5 Here we explore the role the calcium ion plays in both<br/>the oligomeric and spectroscopic properties in PSI isolated from Synechocystis sp. PCC 6803.

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  • 2021-05

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Validating a Model for Catalytic Function in 9°N Polymerase Based on Structural Conservation

Description

Nucleic acids encode the information required to create life, and polymerases are the gatekeepers charged with maintaining the storage and flow of this genetic information. Synthetic biologists utilize this universal

Nucleic acids encode the information required to create life, and polymerases are the gatekeepers charged with maintaining the storage and flow of this genetic information. Synthetic biologists utilize this universal property to modify organisms and other systems to create unique traits or improve the function of others. One of the many realms in synthetic biology involves the study of biopolymers that do not exist naturally, which is known as xenobiology. Although life depends on two biopolymers for genetic storage, it may be possible that alternative molecules (xenonucleic acids – XNAs), could be used in their place in either a living or non-living system. However, implementation of an XNA based system requires the development of polymerases that can encode and decode information stored in these artificial polymers. A strategy called directed evolution is used to modify or alter the function of a protein of interest, but identifying mutations that can modify polymerase function is made problematic by their size and overall complexity. To reduce the amount of sequence space that needs to be samples when attempting to identify polymerase variants, we can try to make informed decisions about which amino acid residues may have functional roles in catalysis. An analysis of Family B polymerases has shown that residues which are involved in substrate specificity are often highly conserved both at the sequence and structure level. In order to validate the hypothesis that a strong correlation exists between structural conservation and catalytic activity, we have selected and mutated residues in the 9°N polymerase using a loss of function mutagenesis strategy based on a computational analysis of several homologues from a diverse range of taxa. Improvement of these models will hopefully lead to quicker identification of loci which are ideal engineering targets.

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  • 2015-05

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An Infusion Approach to Optimizing the Mutagenesis of Rhodobacter sphaeroides

Description

Photosynthesis is the process by which plants, algae, and bacteria use light energy to synthesize organic compounds to use as energy. Among these organisms are a kind of purple photosynthetic

Photosynthesis is the process by which plants, algae, and bacteria use light energy to synthesize organic compounds to use as energy. Among these organisms are a kind of purple photosynthetic bacteria called Rhodobacter sphaeroides, a non-sulfur purple bacteria that grows aerobically in the dark by respiration. There have been many contributions throughout the history of this group of bacteria. Rhodobacter sphaeroides is metabolically very diverse as it has many different ways to obtain energy--aerobic respiration and anoxygenic photosynthesis being just a couple of the ways to do so. This project is part of a larger ongoing project to study different mutant strains of Rhodobacter and the different ways in which carries out electron transfer/photosynthesis. This thesis focused on the improvements made to protocol (standard procedure of site directed mutagenesis) through a more efficient technique known as infusion.

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  • 2014-05