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).