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Copper Binding to Premature Galactose Oxidase: Biogenesis of the Tyr-Cys Cofactor Alta Howells Dooley Group Chemistry and Biochemistry
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The Dooley Group Copper Metalloproteins NO 3 - NO 2 - NO N 2 O N 2 Nitrous Oxide Reductase RCH 2 NH 2 + O 2 RCHO + NH 3 + H 2 O 2 Amine Oxidase RCH 2 OH + O 2 RCHO + H 2 O 2 Galactose Oxidase
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Why Study Metalloproteins? 1/3 of all proteins require metals for their function –Fe, Cu, Co, Zn Cytochrome c Oxidase PDB: 1OCC A perfect example…
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Copper Proteins Produces reactive oxygen species H 2 O 2, Superoxide, Hydroxyl Genetic diseases Green house gases Biotechnology Galactose Oxidase RCH 2 OH + O 2 RCHO + H 2 O 2
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Implication of GO Secreted from Polyporus circinatus Fr. Believed to break down tree lignin Biomedical applications Sensor for colon cancer Disaccharide tumors Bioassays for D-Galactose and Lactose Synthesis of carbohydrates
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Structure-Function RelationshipStructure How the Structure is Generated The pieces that make it a whole Without those pieces LOSS OF FUNCTION
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H496 Y495 H581 Y272 C228 W290 H2OH2O Crystal Structure Essential Cofactors Cu Cu Tyr272-Cys228 Tyr272-Cys228 Crystal Structure and Active Site of Galactose Oxidase
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Maturation of Galactose Oxidase Four post-translational modification events: ?
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Previous Research Tyr-Cys crosslink is formed with Cu and oxygen or excess Cu(II) (Rogers) Cu(I) processes at a faster rate than Cu(II) (Whittaker) Rogers, Melanie. Biochemistry, 2008, 47, 39 Whittaker, Mei M. JBC, 2003, 278, 22090
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Previous Research Cu(II)SO 4 titration into Premat-GO in anaerobic condition 406 nm band increases over time and then decays Yellow complex is formed Rogers, Melanie. Biochemistry, 2008, 47 (39)
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Current Work on Cu(II) Binding Binding affinity of Cu(II) Titrate Cu(II)(Gly) 4 complex into Premat-GO in anaerobic conditions Use UV-Vis and CD spectroscopy Observe the change in abs (UV-Vis) and molar ellipticity (CD) as we are titrating in Cu(II) Rate of formation of certain intermediates
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Experiment 1: Cu(II)(Gly) 4 to Premat-GO in aerobic conditions
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Experiment 2: Cu(II)(Gly) 4 to premat-GO in anaerobic conditions 0.8 : 1 (Cu : protein) Issues: Signal to noise ratio Signal to noise ratio Protein precipitation Protein precipitation
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Experiment 3: Cu(II)(Gly) 4 complex to Premat- GO with anaerobic conditions425nm Peaks at a ratio of.5 mol Cu(II): 1 mol Premat-GO Issues: Signal to noise ratio Signal to noise ratio Incubation time Incubation time Temperature affecting kinetics Temperature affecting kinetics
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Experiment 4: Determining Incubation Time Incubation on ice (4 C) At 25 C Intermediate formation in 20 minutes Intermediate formation in 40 minutes Issues: O 2 contamination
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Current Research Summary What has been achieved… Cu(II)(Gly) 4 binds to premat-GO Yellow complex reaches maximum at 0.5:1 (Cu : Protein) Incubation time: 20 minutes at 25 C To obtain quality data… Reaction temperature Max protein concentration Maintain anaerobic condition
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Acknowledgements
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