Transcript No Slide Title

Protein Structure
& Modeling
Biology 224
Instructor: 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 significant
amino acid identity in a pairwise alignment.
Protein structure evolves more slowly
than primary amino acid sequence. RBP and OBP share
highly similar three dimensional structures.
Principles of protein structure
Primary amino acid sequence
Secondary structure: a helices, b 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
a-helices. These are formed by amino acid stretches
4-40 residues in length.
Thioredoxin from E. coli is an example of a protein
with many b sheets, formed from b strands composed
of 5-10 residues. They are arranged in parallel or
antiparallel orientations.
Myoglobin
(John Kendrew, 1958)
Thioredoxin
Secondary structure prediction
Chou and Fasman (1974) developed an algorithm
based on the frequencies of amino acids found in
a helices, b-sheets, and turns.
Proline: occurs at turns, but not in a helices.
GOR (Garnier, Osguthorpe, Robson): related algorithm
Modern algorithms: use multiple sequence alignments
and achieve higher success rate (about 70-75%)
Secondary structure prediction
Web servers:
GOR4
Jpred
NNPREDICT
PHD
Predator
PredictProtein
PSIPRED
SAM-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:
Classes
Folds
Superfamilies (likely evolutionary relationship)
Families
Domains
Individual PDB entries
http://scop.mrc.lmb.cam.ac.uk/scop/
Approaches to predicting protein structures
There are about >20,000 structures in PDB, and
about 1 million protein sequences in SwissProt/
TrEMBL. For most proteins, structural models
derive from computational biology approaches,
rather than experimental methods.
The most reliable method of modeling and evaluating
new structures is by comparison to previously
known structures. This is comparative modeling.
An alternative is ab initio modeling.
Approaches to predicting protein structures
obtain sequence (target)
fold assignment
comparative
modeling
ab initio
modeling
build, assess model
Comparative modeling of 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
Errors in comparative modeling
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
Comparative modeling
Many web servers offer comparative modeling services.
Examples are
SWISS-MODEL (ExPASy)
Predict Protein server (Columbia)
WHAT IF (CMBI, Netherlands)
Ab initio protein structure prediction
Ab initio prediction can be performed when a protein
has no detectable homologs.
Protein folding is modeled based on global free-energy
minimum estimates.
The “Rosetta Stone” methods was applied to sequence
families lacking known structures. For 80 of 131
proteins, one of the top five ranked models successfully
predicted the structure within 6.0 Å RMSD (Bonneau
et al., 2002).