protein structure & modeling
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Protein Structure & Modeling. Biology 224 Instructor: Tom Peavy Nov 18 & 23, 2009. . Classical structural biology. Determine biochemical activity. Purify protein. Determine structure. - PowerPoint PPT PresentationTRANSCRIPT
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Protein Structure& Modeling
Biology 224Instructor: Tom Peavy
Nov 18 & 23, 2009
<Images adapted from Bioinformatics and Functional Genomics by Jonathan Pevsner>
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Classical structural biology
Determine biochemical activity
Purify protein
Determine structure
Understand mechanism, function
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Structural genomics
Determine genomic DNA sequence
Predict protein
Determine structure or analyze in silico
Understand mechanism, function
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Protein function and structure
Function is often assigned based on homology. However,homology based on sequence identity may be subtle.
Consider RBP and OBP: these are true homologs (they are both lipocalins, sharing the GXW motif).But they are distant relatives, and do not share significantamino acid identity in a pairwise alignment.
Protein structure evolves more slowlythan primary amino acid sequence. RBP and OBP sharehighly similar three dimensional structures.
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Principles of protein structure
Primary amino acid sequence
Secondary structure: helices, sheets
Tertiary structure: from X-ray, NMR
Quaternary structure: multiple subunits
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Protein secondary structure
Protein secondary structure is determined by the amino acid side chains.
Myoglobin is an example of a protein having many-helices. These are formed by amino acid stretches4-40 residues in length.
Thioredoxin from E. coli is an example of a proteinwith many b sheets, formed from strands composedof 5-10 residues. They are arranged in parallel orantiparallel orientations.
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Myoglobin(John Kendrew, 1958)
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Thioredoxin
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Secondary structure prediction
Chou and Fasman (1974) developed an algorithmbased on the frequencies of amino acids found in helices, -sheets, and turns.
Proline: occurs at turns, but not in helices.
GOR (Garnier, Osguthorpe, Robson): related algorithm
Modern algorithms: use multiple sequence alignmentsand achieve higher success rate (about 70-75%)
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Secondary structure prediction
Web servers:
GOR4JpredNNPREDICTPHDPredatorPredictProteinPSIPREDSAM-T99sec
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Tertiary protein structure: protein folding
Three main approaches:
[1] experimental determination (X-ray crystallography, NMR)
[2] Comparative modeling (based on homology)
[3] Ab initio (de novo) prediction
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Experimental approaches to protein structure
[1] X-ray crystallography-- Used to determine 80% of structures-- Requires high protein concentration-- Requires crystals-- Able to trace amino acid side chains-- Earliest structure solved was myoglobin
[2] NMR-- Magnetic field applied to proteins in solution-- Largest structures: 350 amino acids (40 kD)-- Does not require crystallization
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Access to PDB through NCBI
Molecular Modeling DataBase (MMDB)
Cn3D (“see in 3D” or three dimensions):structure visualization software
Vector Alignment Search Tool (VAST):view multiple structures
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Additional web-based sites to visualize structures
Swiss-PDB Viewer
Chime
RasMol
MICE
VRML
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Structural Classification of Proteins (SCOP)
SCOP describes protein structures using a hierarchical classification scheme:
ClassesFoldsSuperfamilies (likely evolutionary relationship)FamiliesDomainsIndividual PDB entries
http://scop.mrc.lmb.cam.ac.uk/scop/
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There are about >20,000 structures in PDB, andabout 1 million protein sequences in SwissProt/TrEMBL. For most proteins, structural modelsderive from computational biology approaches,rather than experimental methods.
The most reliable method of modeling and evaluatingnew structures is by comparison to previouslyknown structures. This is comparative modeling.
An alternative is ab initio modeling.
Approaches to predicting protein structures
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obtain sequence (target)
fold assignment
comparativemodeling
ab initiomodeling
build, assess model
Approaches to predicting protein structures
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[1] Perform fold assignment (e.g. BLAST, CATH, SCOP); identify structurally conserved regions
[2] Align the target (unknown protein) with the template. This is performed for >30% amino acid identity over a sufficient length
[3] Build a model
[4] Evaluate the model
Comparative modeling of protein structures
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Errors may occur for many reasons
[1] Errors in side-chain packing
[2] Distortions within correctly aligned regions
[3] Errors in regions of target that do not match template
[4] errors in sequence alignment
[5] use of incorrect templates
Errors in comparative modeling
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Many web servers offer comparative modeling services.
Examples areSWISS-MODEL (ExPASy)Predict Protein server (Columbia)WHAT IF (CMBI, Netherlands)
Comparative modeling
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Ab initio prediction can be performed when a proteinhas no detectable homologs.
Protein folding is modeled based on global free-energyminimum estimates.
The “Rosetta Stone” methods was applied to sequencefamilies lacking known structures. For 80 of 131 proteins, one of the top five ranked models successfullypredicted the structure within 6.0 Å RMSD (Bonneauet al., 2002).
Ab initio protein structure prediction