Discovering critical residues in glutathione reductase http://dev.gentoo.org/~spyderous/ bioinformatics_GR_presentation.ppt

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Discovering critical residues in glutathione
reductasehttp//dev.gentoo.org/spyderous/bioinf
ormatics_GR_presentation.ppt
Donnie Berkholz
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What and How

• Role
• Reduced thiols
• Oxidative stress
• DNA precursors
• H transport
• Mechanism
• Flavoprotein
• NADPH
• Disulfide

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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.)

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Multiple sequence alignments
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Multiple sequence alignments
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Multiple sequence alignments
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Multiple sequence alignments
ClustalW
Dialign-T
Muscle
ProbCons
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ProbCons

• Probabilistic consistency
• Pair-HMM based
• Three-way alignment consistency
• Parameters derived from training
• Maximized accuracy

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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

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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 -gt WAG
• Try variable-rate models

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ConSurf

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

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NADPH environment
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Disulfide environment
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Catalytic H467D472
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Structure without function?
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Surface F354D22
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Surface D316T321
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FAD binding
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Stabilizing the phosphate
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Structural stability
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What next?

• Check for validity of tree model
• Tree-determinant residues
• Experimental functional determination

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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? F354D22, D316T321
• Binding I26, R218
• Structure H434 etc

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References
ClustalW Chenna et al. NAR 31 3497
(2003). Muscle Edgar. NAR 32 1792
(2004). Dialign-T Morgenstern. NAR 32 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. NAR 33 W299 (2005). PyMol DeLano.
www.pymol.org (2005).