Protein Engineering
Protein Engineering
Protein engineering :
structure prediction &
rational design
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Addapted from lectures of Sophie Bozonnet
3.1. Rationale for understanding protein structure and functions
3.2. Methods for determining protein structure
X-ray cristallography
NMR
Electron microscopy
3.3. Elements of protein 3D structure organization
3.4. Methods for predicting protein structure
Template-based modeling
Homology modeling
Fold recognition
Ab initio methods
3.5. CASP contest
3.6. Protein design
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3.1. Rationale for understanding protein structure and functions
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Protein folding
DNA …-CTA-AAA-GAA-GGT-GTT-AGC-AAG-GTT-…
one amino
acid
unfolded protein
mobile
inactive
spontaneous self-organisation (~1 second) expanded
irregular
native state
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Protein folding
DNA …-CTA-AAA-GAA-GGT-GTT-AGC-AAG-GTT-…
one amino
acid
unfolded protein
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Rationale for understanding protein structure and function
structure prediction
Protein sequence structure determination
large numbers of
sequences, including whole
genomes
Protein structure
-three dimensional
-complicated
? -mediates function
Protein function
-rational drug design and treatment of
homology
disease
rational mutagenesis
-protein and genetic engineering biochemical analysis
-build networks to model cellular pathways model studies
-study organism function and evolution
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Khoury et al., Trendsin Biotechnology, February2014, Vol. 32, No. 2
3.2. Methods for determining protein structure
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3.2. Methods for determining protein structure
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X-ray crystallography concept
• X-rays interact with electrons in protein molecules arranged in a crystal to produce
diffraction patterns.
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X-ray crystallography – steps of the procedure
• Preparation of protein crystals : proteins are organised in a precise crystallattice.
• Beam of x-rays on crystals : x-rays diffract intomany different directions.
Diffraction image
of a protein
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X-ray crystallography – steps of the procedure
• Preparation of protein crystals : proteins are organised in a precise crystal lattice.
• Beam of x-rays on crystals : x-ray diffract into many different directions.
• Phase determination and Intensities calculations.
• Fourier transform to provide maps of electron density.
• Interpret the map by fitting the polypeptide chain to the contours.
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X-ray crystallography – steps of the procedure
• Preparation of protein crystals : proteins are organised in a precise crystal lattice.
• Beam of x-rays on crystals : x-ray diffract into many different directions.
• Phase determination and Intensities calculations.
• Fourier transform to provide maps of electron density.
• Interpret the map by fitting the polypeptide chain to the contours.
• Refine the model by minimising the distance between the observed amplitudes and the
calculated amplitudes.
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X-ray crystallography – pros and cons
Advantages Disadvantages
Provide high-resolution information Require a protein crystal
Unlike solution NMR, does not require a Can not be used with amyloid fibrils
protein be soluble in a high-concentrated
solution Crystal contacts can distort protein
structure
Unlike solution NMR, can be applied to Can not be used with very flexible
proteins with MW > 200 kD molecules
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NMR spectroscopy - concept
• The magnetic-spin properties of atomic nuclei within a molecule are used to obtain a list
of distance constraints between atoms in the molecule, from which a three-dimensional
structure of the protein molecule can be obtained.
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NMR spectroscopy - concept
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NMR spectroscopy - concept
• The magnetic-spin properties of atomic nuclei within a molecule are used to obtain
a list of distance constraints between atoms in the molecule, from which a
three-dimensional structure of the protein molecule can be obtained.
Limited to
small
proteins:
<20 kDa
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NMR spectroscopy – pros and cons
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Cryo-Electron microscopy
Cryogenic electron microscopy (cryo-EM) is an electron microscopy (EM) technique
applied on samples cooled to cryogenic temperatures and embedded in an
environment of vitreous water.
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Cryo-Electron microscopy
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Cryo-Electron microscopy
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Computer representation of a protein structure
•Structures are stored in the protein data bank (PDB), a repository of most
experimental models based on X-ray crystallographic and NMR studies.
http://www.rcsb.org/pdb/home/home.do
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How to view protein structures?
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3.3. Elements of protein 3D structure organization
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Elements of protein 3D structure organization
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Specificity of the Peptide bond
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Polypeptide
Plane
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The Ramachandran plot
shows the phi-psi torsion
angles for all residues in the
structure (except those at the
chain termini).
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Ramachandran plots
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3.4. Methods for predicting protein structure
• comparative
modelling
• fold recognition
• ab initio prediction Comparative modeling, or homology modeling:
Construction of a 3D model of the target protein from its amino
acid sequence and an experimental three-dimensional structure of
a related homologous protein = the "template“.
• Template-
based Fold recognition, or protein threading:
modeling Method used to model proteins which have the same fold as proteins of
known structures, but do not have homologous proteins with known
structure.
It is used for proteins which do not have their homologous protein
structures deposited in the Protein Data Bank.
Ab initio prediction:
A protein tertiary structure is predicted only from its amino acid primary sequence.
requires vast computational resources, and thus, has only been carried out for small proteins.
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3.5. CASP contest : Critical Assessment of Structure Prediction
3 groups of participants:
• Experimentalists: before CASP, submit sequence of to-be-solved structure to
central repository;
• Predictors: download sequence and minimal information, make predictions in three
categories
• Assessors: programs and experts to evaluate predictions quality.
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3.6. Protein design
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From sequence to function and back…
Structure
Function
Sequence
KKAVINGEQIRSISDLHQTLKK
WELALPEYYGENLDALWDCLTG Evolution
VEYPLVLEWRQFEQSKQLTENG
AESVLQVFREAKAEGCDITI
ligand
Structure
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The inverse Protein Folding Problem
Target Fold
Model Protein
TestSequence
KKAVINGEQIRSISDLHQTLKK
WELALPEYYGENLDALWDCLTG
VEYPLVLEWRQFEQSKQLTENG
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Protein Sequence Design
Specific problems:
o Stability: the designed sequence must be stable for the target structure
o Specificity: the designed sequence must fold into the target structure,
and not into a competing fold.
Challenges:
o Search size: there are many possible sequences! 20N for a protein
of N residues