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Discovering critical residues in glutathione reductasehttp://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
● 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.)
Multiple sequence alignments
Multiple sequence alignments
Multiple sequence alignments
Multiple sequence alignmentsClustalW Dialign-T Muscle ProbCons
● “Probabilistic consistency”● Pair-HMM based● Three-way alignment consistency● Parameters derived from training● Maximized accuracy
ProbCons
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
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
ConSurf
● Calculates evolutionary conservation (Bayesian)
● Maps onto protein structure● Input flexibility
– PDB -> seq. -> PSI-BLAST -> MSA -> NJ -> CS● Can't yet analyze subfamilies
NADPH environment
Disulfide environment
Catalytic: H467+D472
Structure without function?
Surface: F354+D22
Surface: D316+T321
FAD binding
Stabilizing the phosphate
Structural stability
What next?
● Check for validity of tree model● Tree-determinant residues● Experimental functional determination
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
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).