| Erika Allen | Kathleen Caldwell | Matthew Giordanengo |
| Megan Reilly | Lindsay Simon | Annie Watson |
Isolation and Characterization of an Acidic Flower Bud Protein from Brassica rapa
Erika Allen, 2006 (Advisor: Dr. Ann Kleinschmidt)
Peroxidases are enzymes found in bacteria, plants and animals. The majority of these enzymes contain a heme group and carry out the oxidation of organic compounds using the oxidizing agent hydrogen peroxide. Plant peroxidases have been categorized into three classes: class I, II and III. Class III, plant peroxidases, have many functions, not all of which are known. There are also enzymes that mimic peroxidase activity, such as annexins. The goal of this project was to further characterize a flower bud protein identified in Brassica rapa that has peroxidase activity. The protein was purified using a preparative agarose gel and a size exclusion column. Followed by separation by SDS-PAGE two separate protein bands were submitted for peptide analysis. One protein, approximately 42 kDa, was identified as showing close homology to annexin 1 and the other showed homology to a 14-3-3 protein. This data suggests that the presence of peroxidase activity may be due to the hypothesized peroxidase activity of annexin 1 in Arabidopsis thaliana.
Isolation and Characterization of an Acidic Peroxidase, BrPx6.7, from Brassica rapa Shoots
Kathleen Caldwell, 2006 (Advisor: Dr. Ann Kleinschmidt)
Peroxidases are enzymes found in all organisms whose primary role is to break down hydrogen peroxide. Plant peroxidases contain and iron heme and are divided into three separate classes. The Class III peroxidases are highly diversified in function using hydrogen peroxide to catalyze the oxidation of a wide variety of small organic compounds. In vitro substrate specificity of a partially purified peroxidase may provide insights for physiological peroxidase functions. Through prep gel, concentration, and electroelution techniques the acidic peroxidase BrPx6.7 was isolated from Brassica rapa shoot tissue. BrPx6.7 displayed a substrate preference for pyrogallol>ascorbate>o-dianisidine>o-phenylenediamine>guaiacol>IAA>ferulic acid. Inactivity with ferulic acid oxidation eliminated suberin synthesis as a possible function because ferulic acid is a precursor to suberin. However, BrPx6.7 did show some similar characteristics of ascorbate peroxidase and lignification also could not yet be eliminated as a possible role of BrPx6.7. The overall ability of this assay to analyze substrate preference for BrPx6.7 without performing multiple purification procedures was extremely important. The procedure could become widely utilized as an initial route to determining substrate preference inexpensively and efficiently.
EPR and kinetic analyses of cytosolic pea ascorbate peroxidase
Matthew Giordanengo, 2006 (Advisor: Dr. Ann Kleinschmidt)
Cytosolic pea ascorbate peroxidase is an important hydrogen peroxide scavenger in higher plants. It is characterized as a class I peroxidase, in which all peroxidases show 70-90% homology. One important motif is the cation binding pocket, which Cheek et al., have theorized focuses the radical in compound I on the porphyrin ring and not the near by trp radical as is seen in cytochrome c peroxidase. This study examines three mutants at N182 in this cation binding pocket using UV/Vis kinetics and electron paramagnetic resonance. The mutants showed a decrease in low spin epr signals, indicating a change to a 6-coordinate iron in the prosthetic group. Also, the data showed a decrease in the rates utilizing ascorbate and a phenolic substrate, o-dianisidine. The o-dianisidine rates decreased more so than with ascorbate. O-dianisidine binds to the delta edge of the heme, which is closer to the iron center than ascorbate, which binds on the gamma edge where two proprionate groups are situated. The increased field strength from a 6th ligand would hinder the binding of the o-dianisidine more than ascorbate. These data suggest that the mutations alter the orientation of the heme and push it closer to the distal histidine, his42 allowing it to associate, leaving a 6-coordinate, low spin iron in the heme.
The Effects of Mutations of E165, K178, R365, and A375 on the Assembly and Maturation of Bacteriophage HK97 Capsids
Megan Reilly, 2006 (Advisor: Dr. Brandi Baros)
HK97 is a lambda-like bacteriophage consisting of 420 copies of capsid protein, gp5, portal protein, gp3, and viral protease, gp4 that associate to initiate the assembly and maturation process of HK97. Currently very little is known or understood about the assembly of this bacteriophage and thus this investigation, incorporating two experiments, was designed to study the effects of mutations at positions A375, E165, K178 and R365 on the assembly and maturation of HK97 capsids. The complementation assay was used to describe the efficiency of phage growth of each mutant when compared to the wild-type capsid protein. The SDS-PAGE and agarose gels, respectively indicated assembly stages of capsid proteins, whether or not cleavage and cross-linking occurred, and separated native proteins by size, shape and charge.
The results of these experiments indicated that A375 mutants function as wild type, despite side chain size differences and charge changes at this position. K178 mutations had drastic detrimental effects on capsid assembly and maturation processes. The complementation data suggested that K178 mutations could not mature or function as viable, wild type phage, and also caused dominant negative effects on the wild type phage during complementation. R365 and E165 mutants were able to mature to Head, but were not able to function as normal infectious phage because they were incapable of complementing 5- phage, suggesting that these mutants have roles interacting with other phage proteins. R365 mutants also had dominant negative effects during interactions with wild type phage. E165, K178 and R365 prove to be positions with important roles in normal assembly, maturation or function of the capsid protein of HK97.
Characterizations of mutations of the Hong Kong 97 capsid gene
Lindsay Simon, 2006 (Advisor: Dr. Brandi Baros)
HK97 is a dsDNA bacteriophage that infects Escherichia coli. Like many other bacteriophages, HK97 has a tail, portal, and capsid. The capsid, which holds the DNA of the phage, develops in four separate stages. In the first stage of HK97 capsid development, 420 copies of the gp5 capsid protein polymerize into an icosahedral shell known as Prohead I. This assembly is done without the aid of any scaffolding protein, in a process known as self-assembly. Then, the gp4 protease cleaves 102 residues from the gp5 polypeptide chain, known as the delta domain, in order to form Prohead II. After cleaving, the capsid can then expand to form Head I. Then, the 420 copies of gp5 protein form a chain mail configuration, known as cross-linking, leading to the mature capsid Head II.
The purpose of this study was to investigate the roles of eight specific amino acids in HK97 capsid development. These eight sites were mutated to K52Q, K75Q, N278L, N278R, E292F, E292Q, K289Q, K289E, D199K, D199L, A261T, or D231N. Each of these mutants was tested to see whether or not they could form viable phage and to what extent the capsids developed. All mutants under investigation were unable to form viable phage and showed varying hindrances in development. As such, each residue does play an integral role in HK97 capsid development.
Characterization of the Cytosolic Ascorbate Peroxidases from Brassica rapa and Pisum sativum
Annie Watson, 2006 (Advisor: Dr. Ann Kleinschmidt)
Class I ascorbate peroxidases, found in photosynthetic organisms, reduce the toxic accumulation of reactive oxygen species and use both organic and inorganic substrates. So far amino acid sequence analyses of the APX isoenzymes have been done between many organisms, but a direct biochemical comparison of peroxidases from two different families, Leguminosea and Brassicacea, has not. The goal of this project is to compare enzymatic characteristics of the cytosolic ascorbate peroxidases from Brassica rapa and Pissum sativum. These two APXs are 80% homologous and the amino acids most important for the enzymatic properties including Lys30, Cys32, His42, His163, Thr164, Arg172, Thr180, Asn182, and Asp187 are all conserved. Although these amino acids are conserved, the purpose of this project is to see if changes in the amino acid sequences at other sites lead to alteration in enzyme activity, that could have allowed for the evolution of new characteristics in plants. No biochemical results were obtained in this project, because the attempt to clone the cDNAs into the expression vector failed. However, to address possible outcomes had the cloning steps been successful, a discussion of outcomes for wild type and mutant versions of pea and soybean peroxidase will be provided.