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Joana Pinto July 2009

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Analyse the structures of Copper containing proteins and look for errors Keep making protein structures more and more accurate Relying on improved software and better knowledge of protein structure

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Difficulty in distinguish certain atoms Errors are kept because its not a curated database.

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Iron, Zinc, Calcium, Magnesium, Chlorine or Iodine, etc. Copper Two stable oxidation states [Ar] s 1 d 10 Copper proteins classification

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Blue Proteins 2 Histidines, 1 Cysteine and 1 Methionine There are about 50.000 proteins in this family CuN - Histidine S - Methionine Cysteine - S

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3 Histidines + Histidine or H 2 O or Methionine or Cysteine Colorless CuN - Histidine X

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Peptidylglycine - Hydroxylating Monooxygenase

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Cu A Cu B

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Binuclear centers: 3 Histidines and O 2 CuN - Histidine Cu O N - Histidine O

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*Deoxygenated state 4,6 *Oxygenated state 3,6 * Not validated by us

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REDUCED FORM - CUPROUS OXIDIZED FORM - CUPRIC [Ar] 4s 0 3d 10 Soft ion Soft ligands (S/P) C.N: 2, 3 and 4 [Ar] 4s 0 3d 9 Hard ion Hard ligands (N/O) C.N: 4, 5 and 6

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PDB Files: X-ray Crystallography Resolution better than 2.0 Cu + Cu 2+ Total Files 114562 Cu + Cu 2+ Used Files 35321

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WHAT IF: Calculations SKPNOO GRSION Distances plot YASARA: Pictures

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Cu + Total Files 114 Files Used 35 Num of ligands Num of Copper Ions 311 437 529 Geometry Num of Copper Ions Tetrahedral24 Other13 Ligands Num of Copper Ions 2 His, 1 Met, 1 Cys14 4 His7 Others3 Cu 2+ Total Files 562 Files Used 321 Num of ligands Num of Copper Ions 345 4384 5231 Geometry Num of Copper Ions Tetrahedral331 Square Planar8 Other45 Ligands Num of Copper Ions 2 His, 1 Met, 1 Cys210 3 His, 1 H 2 O69 Others52

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Geometry Num of Copper Ions Tetrahedral24 Regular tetrahedral14 Ligands Num of Copper Ions 2 His, 1 Met, 1 Cys13 3 Met, 1 SCN1 Geometry Num of Copper Ions Tetrahedral331 Regular tetrahedral215 Ligands Num of Copper Ions 2 His, 1 Met, 1 Cys149 3 His, 1 H 2 O43 Others23 13 Cu + ions (spread over 8 PDB files) and 149 Cu 2+ ions (spread over 94 PDB files) distances plot

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Average positions for all the intervenient residues in the regular tetrahedrals difference between the average positions is of 0.6 , being this value close to the error expected in the X-ray structure determination

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When superposed, 5PCY* and 6PCY* show no differences in its structures besides: a small change in a Proline residue, and a rotation of an Imidazole ring by 180 about C - C . * Guss, J.M., Harrowell, P.R., Murata, M., Norris, V.A., Freeman, H.C. (1986). Crystal Structure Analysis Of Reduced (Cu I) Poplar Plastocyanin At Six pH Values. J.Mol. Biol 192 (1986): 361-387

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No reason for the flipping of the Histidine At a resolution of both 5PCY (1.80 ) and 6PCY (1.90 ) the Pro puckering tends to be unobservable 5PCY6PCY

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Too few data Coordination Numbers: For Cu + and Cu 2+ : most often seen C.N. is 4, 5 and 3. Contradicting some older studies* No differences between the structures of Cu + and Cu 2+ Contradicting some older studies** *Belle, C., Rammal, W., Pierre, J.L., (2005). Sulfur Ligation in Copper Enzymes and Models. Journal of Inorganic Biochemistry 99: 1929-1936 **Taylor, M.K., Stevenson, D.E., Berlouis, L.E.A., Kennedy, A.R., Reglinski J. (2006). Modeling the Impact of Geometric Parameters On The Redox Potential Of Blue Copper Proteins. Journal of Inorganic Biochemistry 100: 250-259

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For coppers with C.N. 4, the most common geometry is tetrahedral Copper proteins classifications should be revised, for example, Type II classification is ambiguous. Some older publications should be reviewed

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What distinguishes between both oxidation states for copper? Are the differences too small to be detected? Are there any differences at all? One of the possibilities to be studied would be the protonation states for Histidine and Cysteine or other factors like protein bond

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Thank You Questions?