Purification and characterization of recombinant pyruvate dehydrogenase kinases from pea and soybean plants [abstract]

Abstract only availableFaculty Mentor: Dr. Douglas Randall, BiochemistryThe pyruvate dehydrogenase complex (PDC) is a large multienzyme complex catalyzing the oxidative decarboxylation of pyruvate and concomitant reduction of NAD to yield acetyl-CoA and NADH. The plant PDCs have vital roles in catabolic and anabolic metabolism. The plant complexes contain three primary components: pyruvate dehydrogenase (E1), dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3). Additionally, mitochondrial PDC (mtPDC) contains two associated regulatory enzymes: pyruvate dehydrogenase kinase (PDK) and phospho-pyruvate dehydrogenase phosphatase. PDK catalyzes phosphorylation on the subunit of E1, resulting in inactivation of the complex. We have cloned two PDKs from soybean and recently we have cloned three PDKs from pea. cDNAs encoding soybean PDK 1 and 2 and pea PDK 1, 2 and 3 were subcloned into pET expression vector and E. coli BL21 (DE3) cells were transformed with each pET-28-H6-PDK construct. Recombinant proteins were expressed and purified by Ni-NTA agarose column chromatography to approximately 95% homogeneity. Biochemical characterization of these proteins is underway.The pyruvate dehydrogenase complex (PDC) is a large multienzyme complex catalyzing the oxidative decarboxylation of pyruvate and concomitant reduction of NAD to yield acetyl-CoA and NADH. The plant PDCs have vital roles in catabolic and anabolic metabolism. The plant complexes contain three primary components: pyruvate dehydrogenase (E1), dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3). Additionally, mitochondrial PDC (mtPDC) contains two associated regulatory enzymes: pyruvate dehydrogenase kinase (PDK) and phospho-pyruvate dehydrogenase phosphatase. PDK catalyzes phosphorylation on the a subunit of E1, resulting in inactivation of the complex. We have cloned two PDKs from soybean and recently we have cloned three PDKs from pea. cDNAs encoding soybean PDK 1 and 2 and pea PDK 1, 2 and 3 were subcloned into pET expression vector and E. coli BL21 (DE3) cells were transformed with each pET-28-H6-PDK construct. Recombinant proteins were expressed and purified by Ni-NTA agarose column chromatography to approximately 95% homogeneity. Biochemical characterization of these proteins is underway

Subcellular localization of the Arabidopsis thaliana atDjC37 molecular chaperone protein

Abstract only availableThere are 94 genes encoding J-domain molecular chaperone proteins in the Arabidopsis thaliana genome. These genes have been grouped into 51 families (Miernyk 2001 Cell Stress Chaperones 6: 209-218). Family 4 consists of two proteins, atDjC6 and atDjC37. It has been previously determined that atDjC6 is nuclear localized (Suo & Miernyk 2004 Protoplasma 224: 79-89). We now wish to determine the subcellular localization of atDjC37. In silico analysis of the atDjC37 deduced amino acid sequence (http://maple.bioc.columbia.edu/predictNLS/) yielded the prediction that residues -R253RSSKKS- comprise a nuclear localization signal (NLS) sequence. Our experimental strategy has been to construct plasmids that encode full-length atDjC37 protein and a C-terminal truncated version that lacks the NLS sequence, fused to the red fluorescent protein. These proteins will be transiently expressed in biolistically-transformed tobacco BY2 cells, and localized using laser-scanning confocal microscopy. The transformed cells will be simultaneously incubated with a fluorescent nuclear stain to test for signal coincidence. Four nuclear stains are being evaluated for their utility; propidium iodide (PI), DAPI, SYTO Green, and Hoechst 33342. The SYTO and Hoechst 33342 stains are considered cell-permeant, while DAPI is "semi-permeant" and PI is impermeant. The PI and DAPI stains are UV blue-fluorescent, while PI is red and SYTO Green is, naturally, green.Plant Genomics Internship @ M

Analysis of family 24 of the Arabidopsis thaliana J-domain chaperone proteins [abstract]

Abstract only availableFaculty Mentor: Dr. Jan Miernyk, Plant BiochemistryThe Arabidopsis thaliana genome includes an unexpectedly large and diverse group of J-domain chaperone proteins. The 93 A. thaliana J-domain protein sequences have been grouped into 51 families, many of which do not have any well-studied counterpart in microbes or mammalian cells. Based upon the results of silico analyses, three proteins, atDjC43, atDjC48, and atDjC49, were assigned to Family 24. Homologous proteins are present in maize, rice, and soybean, and in a variety of animals, but none has been characterized. Members of Family 24 have approximately 300 amino acid residues; besides the J-domain the only prominent structural feature is a predicted transmembrane helix near the C-terminus. Preliminary experiments were conducted to try to understand the multiplicity of plant J-domain proteins, and to address the question of redundancy versus specialization. In order to determine where and when these three J-domain proteins are expressed, primers were designed for semi-quantitative RT-PCR. Total RNA was isolated from the rosette leaves and roots of 4-week old A. thaliana ecotype Columbia plants, and from flowers and green siliques. The results establish a pattern of organ-specific expression. We are also using T-DNA insertion knockout plants for our analyses. We currently have homozygous KO plants for atDjC48, and are in the final stages of screening for atDjC43 knockouts. It is difficult to express eukaryotic integral membrane proteins in bacteria. The sequence encoding the C-terminal transmembrane helix was deleted from the atDjC48 reading frame. The atDjC48���C sequence was then cloned into the pCal-n vector for expression in Escherichia coli as a chimera with the CaM-Binding-Peptide. The recombinant protein will be assayed for activity in vitro. Using these diverse strategies, we hope to gain insight into the roles of this Family of molecular chaperone proteins.University of Missouri--Columbia. Office of Undergraduate ResearchPlant Genomics Internship @ M