DODA 2 Structure Elucidation and Kinetics Study

Jake Barras

Abstract

Betalins are water-soluble nitrogen-containing pigments that are responsible for the bright colors present in plants and fruits. The structural unit responsible for the formation of betalins, betalamic acid, has shown to have cancer preventative properties, as well as anti-gamma radiation properties. Betalamic acid can be produced by catalyzing L-DOPA, a precursor to dopamine, with a protein known as DODA. After this reaction takes place, the 4,5-seco-DOPA intermediate is formed. This step is followed by spontaneous cyclization of the intermediate to produce betalamic acid. It is currently unknown which part of the active site of DODA is responsible for this catalytic activity. Bettering our understanding of the structure of this protein will allow us to more effectively and efficiently model the protein and understand its biological role. Our strategy of structure determination began with genetic analysis and primer design. The goal of this analysis was to develop primers that were to be used in the PCR mutagenesis of the genes of our proteins. This technique works to introduce mutations in the plasmid DNA of a bacteria or protozoan. This is an important technique because it allows a researcher to modify the DNA primer that will match to the plasmid. In a protein-biochemistry perspective, this essentially allows a researcher to change the composition of a protein at a single amino acid level. In the context of this experiment, mutagenesis was done to change amino acids of suspected importance to an amino acid with different intermolecular forces and chemical properties to determine the function of the wild-type amino acid. From this point, DNA plasmids are transformed into bacterial cells and expressed through natural biological mechanisms of the bacteria. The proteins are then purified from the engineered and grown bacteria of the ladder portion of the experiment through a series of processes that serve to lyse the cell and separate our desired, manipulated DODA protein. The purified protein samples are then concentrated and allowed to crystalize in a series of buffers ranging in concentration and composition to ensure that the conditions of crystallization will be met. Crystallization is a vital step in this research because the final analytical technique, protein crystallography, requires a purified and crystalized sample to determine the tertiary structure. This technique works by blasting the crystalized sample with X-ray beams and measuring the angle and magnitude of refraction of the particles. This data is then converted into a three-dimensional model of the protein that can be used to make assumptions about the functionality of our manipulated protein samples.

Faculty Advisor: Dr. Soon Goo Lee

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Jake Barras

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Email: bjb1873@uncw.edu