Protein Structure & Modeling

Protein Structure& Modeling

Biology 224Instructor: Tom Peavy

Nov 18 & 23, 2009

<Images adapted from Bioinformatics and Functional Genomics by Jonathan Pevsner>

Classical structural biology

Determine biochemical activity

Purify protein

Determine structure

Understand mechanism, function

Structural genomics

Determine genomic DNA sequence

Predict protein

Determine structure or analyze in silico

Understand mechanism, function

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.

Principles of protein structure

Primary amino acid sequence

Secondary structure: helices, sheets

Tertiary structure: from X-ray, NMR

Quaternary structure: multiple subunits

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.

Myoglobin(John Kendrew, 1958)

Thioredoxin

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%)

Secondary structure prediction

Web servers:

GOR4JpredNNPREDICTPHDPredatorPredictProteinPSIPREDSAM-T99sec

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

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

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

Additional web-based sites to visualize structures

Swiss-PDB Viewer

Chime

RasMol

MICE

VRML

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/

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

obtain sequence (target)

fold assignment

comparativemodeling

ab initiomodeling

build, assess model

Approaches to predicting protein structures

[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

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

Many web servers offer comparative modeling services.

Examples areSWISS-MODEL (ExPASy)Predict Protein server (Columbia)WHAT IF (CMBI, Netherlands)

Comparative modeling

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