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Donnie Berkholz

Discovering critical residues in glutathione reductase http://dev.gentoo.org/~spyderous/ bioinformatics_GR_presentation.ppt. Donnie Berkholz. What and How. Role Reduced thiols Oxidative stress DNA precursors H + transport Mechanism Flavoprotein NADPH Disulfide. Goals.

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Donnie Berkholz

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  1. Discovering critical residues in glutathione reductasehttp://dev.gentoo.org/~spyderous/bioinformatics_GR_presentation.ppt Donnie Berkholz

  2. What and How • Role • Reduced thiols • Oxidative stress • DNA precursors • H+ transport • Mechanism • Flavoprotein • NADPH • Disulfide

  3. Goals • Figure out the best programs and methods for this analysis • Search for unknown critical residues • Verify whether residues already thought critical are actually conserved • Check for potential differences in function and specificity among subfamilies (Podar et al.)

  4. Multiple sequence alignments

  5. Multiple sequence alignments

  6. Multiple sequence alignments

  7. Multiple sequence alignments ClustalW Dialign-T Muscle ProbCons

  8. ProbCons • “Probabilistic consistency” • Pair-HMM based • Three-way alignment consistency • Parameters derived from training • Maximized accuracy

  9. How to find important residues? • Principal component analysis (PCA) • Each sequence becomes a vector • Successive dimensions grow less significant • Evolutionary trace and friends • Divide tree into groups, then check them • So, first we need trees

  10. Trees • Maximum likelihood • ProML (PHYLIP) • Gamma distribution + invariant sites • Approximate with 5 rate categories • Bayesian • MrBayes • Gamma distribution + invariant sites • MCMC: Markov chain Monte Carlo • Mixed: sample with probability -> WAG • Try variable-rate models

  11. ConSurf • Calculates evolutionary conservation (Bayesian) • Maps onto protein structure • Input flexibility • PDB -> seq. -> PSI-BLAST -> MSA -> NJ -> CS • Can't yet analyze subfamilies

  12. NADPH environment

  13. Disulfide environment

  14. Catalytic: H467+D472

  15. Structure without function?

  16. Surface: F354+D22

  17. Surface: D316+T321

  18. FAD binding

  19. Stabilizing the phosphate

  20. Structural stability

  21. What next? • Check for validity of tree model • Tree-determinant residues • Experimental functional determination

  22. Summary • ProbCons is great for MSA's • Bayesian trees take forever, but they provide confidence values (no bootstrap!) • ConSurf maps sequence conservationonto protein structures • Supports catalytic hypothesis • New putative functional roles: • Interactions? F354+D22, D316+T321 • Binding: I26, R218 • Structure: H434 etc

  23. References ClustalW: Chenna et al. NAR31: 3497 (2003). Muscle: Edgar. NAR32: 1792 (2004). Dialign-T: Morgenstern. NAR32: W33 (2004). ProbCons: Chuong et al. Genome Res.15: 330 (2005) Jalview: Clamp et al. Bioinform.12: 426 (2004). PHYLIP: Felsenstein. Distributed by author (2005). MrBayes: Ronquist and Huelsenbeck. Bioinform.19: 1572 (2003). ConSurf: Landau et al. NAR33: W299 (2005). PyMol: DeLano. www.pymol.org (2005).

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