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Protein Engineering

protein engineering

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12 views45 pages

Protein Engineering

protein engineering

Uploaded by

nhinb.22bi13350
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
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Lecture 3:

Protein engineering :
structure prediction &
rational design

1
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

2
3.1. Rationale for understanding protein structure and functions

3
Protein folding
DNA …-CTA-AAA-GAA-GGT-GTT-AGC-AAG-GTT-…

protein sequence …-L-K-E-C-V-S-K-D-…

one amino
acid

unfolded protein

mobile
inactive
spontaneous self-organisation (~1 second) expanded
irregular

native state

4
Protein folding
DNA …-CTA-AAA-GAA-GGT-GTT-AGC-AAG-GTT-…

protein sequence …-L-K-E-C-V-S-K-D-…

one amino
acid

unfolded protein

spontaneous self-organisation (~1 second)


unique shape precisely
ordered stable/functional
globular/compact helices
native state and sheets

5
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
6
7
Khoury et al., Trendsin Biotechnology, February2014, Vol. 32, No. 2
3.2. Methods for determining protein structure

❖ X-ray cristallography : X-ray diffraction pattern


❖ NMR spectroscopy: information on the local conformation and distance between atoms
❖ Cryo-Electron microscopy: image of the overall shape of the molecule

experimental knowledge many pieces of


data about the about the information to
structure of the molecular create the final
molecule structure atomic model

9
3.2. Methods for determining protein structure

❖ X-ray cristallography : X-ray diffraction pattern


❖ NMR spectroscopy: information on the local conformation and distance between atoms
❖ Cryo-Electron microscopy: image of the overall shape of the molecule

experimental knowledge many pieces of


data about the about the information to
structure of the molecular create the final
molecule structure atomic model

experimental the preferred


information is not geometry of atoms in
sufficient to build a typical protein: the
an atomic model bond lengths and
from scratch bond angles

10
X-ray crystallography concept
• X-rays interact with electrons in protein molecules arranged in a crystal to produce
diffraction patterns.

Diffraction pattern of an hemoglobin crystal. 11


X-ray crystallography concept

• The diffraction patterns of the x-rays


can be used to determine the three-
dimensional structure of proteins.

Provides a “static” picture


12
From <http://info.bio.cmu.edu/courses/03231/LecF01/Lec25/lec25.html>
X-ray crystallography – steps of the procedure
•Preparation of protein crystals : proteins are
forced to organise in a precise crystal lattice.

•Method: mixing of protein solution with a


solution containing a precipitating agent (salts,
PEG, volatile organic compounds (VOCs),....)

13
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.

measuring the angles and


intensities of these
diffracted beams lead to a
three-dimensional picture
of the density of electrons
within the crystal.

Diffraction image
of a protein
14
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.

15
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.

16
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

17
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.

18
NMR spectroscopy - concept

• NMR involves the quantum


mechanical properties of the
central core ("nucleus") of the
atom. These properties
depend on the local molecular
environment, and their
measurement provides a map
of how the atoms are linked
chemically, how close they are
in space, and how rapidly they
move with respect to each
other.

19
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.

• Provides a “dynamic” picture.

Limited to
small
proteins:
<20 kDa

NK-lysin (1nkl) S1 RNA binding domain (1sro)

20
NMR spectroscopy – pros and cons

21
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.

22
Cryo-Electron microscopy

23
Cryo-Electron microscopy

24
25
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

26
How to view protein structures?

27
3.3. Elements of protein 3D structure organization

28
Elements of protein 3D structure organization

Specificity of the Peptide bond


The peptide bond is planar: The bond between the
carbonyl carbon and the
nitrogen has a partial
double bond character:
rotation around this bond
is restricted.

Resonance interactions between


electrons in the C=O bond and
the C–N bond restricts the
number of conformations in
proteins .

29
Specificity of the Peptide bond

• Two conformations cis or trans


• cis conformation is very rare due to local steric hindrance
• Proportion of trans conformation > 98-99%.

30
Polypeptide

Plane

The rotation between two C-N bond planes is


defined by two dihedral angles (torsion angles):
❖  (N-C) and
❖  (C-C)
Torsion angle

31
32
33
34
35
36
The Ramachandran plot
shows the phi-psi torsion
angles for all residues in the
structure (except those at the
chain termini).

Red regions : Allowed


torsion angles avoiding steric
hindrance

Pink regions: Allowed if


some small steric collisions
are allowed

37
Ramachandran plots

38
39
40
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.
42
3.5. CASP contest : Critical Assessment of Structure Prediction

3D Protein Structure Prediction


• Biannual meeting since 1994 (last, may 2016).
• Worldwide event aiming at objectively testing structure prediction methods
and delivering an independent assessment of the state of the art in protein
structure modeling.
World championship of protein 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.

43
3.6. Protein design

44
From sequence to function and back…
Structure

Function
Sequence

KKAVINGEQIRSISDLHQTLKK
WELALPEYYGENLDALWDCLTG Evolution
VEYPLVLEWRQFEQSKQLTENG
AESVLQVFREAKAEGCDITI

ligand

Structure
45
The inverse Protein Folding Problem
Target Fold

Model Protein

TestSequence
KKAVINGEQIRSISDLHQTLKK
WELALPEYYGENLDALWDCLTG
VEYPLVLEWRQFEQSKQLTENG

46
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

o Energy: We need to evaluate the free energy ΔG = G(fold) – G(unfold)


for a sequence:
• What is the structure of the protein? We know the backbone, need
to predict sidechains
• How do we model the unfolded state?
• Need to evaluate energy fast (so many sequences!)
47

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