1. dia

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NUMBERS
Estimated number of proteins in the human body
100 000 Primary structure analysis (F. Sanger,
1953) 1953-1978 (25 years) 1081 1979-1991
(13 years) 16 000 1992- 1000/year Three-di
mensional (3D) structure (J. Kendrew,
1962) 1962-1985 (20 years) 200 1986-1991 (
5 years) 480 1992- 100/years
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CLASSIFICATION OF PROTEINS ACCORDING TO THEIR
FUNCTION

• Enzymatic catalysis (e.g. Ser proteases)
• Transport (e.g. transferrin for iron, serum
  albumin for fatty acids)
• Storage (e.g. ferrin for iron in liver, casein
  in milk)
• Protection
• toxins (e.g. ricin plant, diphteria bacteria)
• self and non-self discrimination, immune
  protection
• (e.g. antibodies, antigenes)
• Signal transduction (e.g. hormones, receptors)
• nerve impulses
• growth
• differentiation
• Cell to cell communication (e.g. adhesion,
  molecules factors, acceptors)
• Coordinated motion (e.g. muscle proteins)
• Mechanical support
• at cellular level (e.g. Membrane proteins)
• at tissue level (e.g. structural proteins, e.g.
  collagen in skin, bone)

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RECOGNITION PHENOMENA
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• METHODS FOR THE LOCALISATION OF
• FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
• Experimental methods

• Chemical modification
• side chain modification
• conjugation

• Chemical synthesis
• substituted analogs
• truncated/omitted analogs
• overlapping peptides
• peptide libraries

• Fragmentation
• enzymatic (e.g. trysin)
• chemical (e.g. BrCN)
• Separation
• centrifugation
• chromatography
• electrophoresis
• Identification
• amino acid analysis
• sequencing
• mass spectrometry

• Genetic engineering
• deletion
• chimeric proteins
• mutagenesis
• site directed
• random
• phage display libraries

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• METHODS FOR THE LOCALISATION OF
• FUNCTIONALLY RELEVANT DOMAINS IN PROTEINS
• Theoretical methods

• Quantum chemistry
• molecular mechanics
• molecular dynamics

• Predictions from the primary structure
• Probabilistic (statistical) 1970
• Physicochemical 1974
• Information theory 1974 -

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Approaches for the localiation of functionally
relevant domains in proteins
Protein primary structure
Know
Unknown
3D Structure
Isolation Fragmentation, separation Functional
assay with fragments Selection Structure
elucidation
Know
Unknown

• Prediction of
• secondary structure
• functionally relevant domain

Fragmentation, separation Functional assay with
fragments Selection 3D Structure elucidation
Chemical modification
Genetic engineering
Smallest functional domain
Chemical synthesis
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Strategies for determinations of 3D structures
Experimental methods
Theoretical methods

• X-ray diffraction
• (Mioglobin, hemoglobin
• Kendrew, Perutz, 1960)
• crystal
• time-intensive

Quantum chemistry mechanics and dynamics
Empirical calculations
Relationship assay
NMR
Prediction of secondary structures (helix,
b-turn)
CD spectroscopy FT-IR spectroscopy
Prediction of hydrophobic hydrophilic regions
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Techniques for the detection of
interaction/recognition phenomena
1. Molecular level
Detection with Separation
Detection without Separation

• Optical techniques
• absorption spectroscopy
• CD
• fluorescence spectroscopy
• IR and Raman spectroscopy

• Separation techniques
• equilibrium dialysis
• chromatography
• - gel filtration
• - affinity
• electrophoresis

• Resonance techniques
• NMR
• electron paramagnetic resonance (EPR)

• Detection techniqes
• spectroscopic
• radiochemical (125I, 35S, 3H, 14C)
• imunochemical
• - RIA/ELISA
• - blotting
• - immunprecipitation

• Scattering and Diffraction techniques
• X-ray crystallography
• neutron scattering
• electron microscopy

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Techniques for the detection of
interaction/recognition phenomena
2. Cellular level

• Bioassay (in vitro)
• binding to cell
• hemolysis
• antibacterial effect
• cytotoxicity

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Example 1 Heparin binding domains of
Apolipoprotein E

• Heparin glycosaminoglycan (GAG)
• Apolipoprotein E human plasma lipoprotein (ApoE)
• 299 amino acid, 3D structure unknown
• Aim Identification of primary GAG interaction
  sites (motifs), which can be
• used for prediction based on initial sequence
  inspection
• Phase I
• step Fragmentation of the lipid-free protein
• H----------------------------------Arg191-Ala192
  -----------------------------OH
• thrombin
• H ----------------------------------Arg191-OH
  H-Ala192-----------------------------OH

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Example 1 Heparin binding domains of
Apolipoprotein E

• Phase I
• step Separation of the fragments
• step Binding studies with 125I-labelled heparin
• Method
• Transfer of fragments to nitrocellulose by
  blotting
• Incubation of nitrocellulose with labelled
  heparin
• Radioautoradigraphy

ApoE
High
E(1-191)
E(192-299)
Low
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Example 1 Heparin binding domains of
Apolipoprotein E

• Phase II
• step Synthetic peptide with successive deletions
  from the amino- and carboxyl-terminal
• ends were prepared by SPPS
• Residue Amino acid sequence
• --------------------------------------------------
  --------------------------------------------------
  ---------
• 129-169 STEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREG
  
• 139-169 HLRKLRKRLLRDADDLQKRLAVYQAGAR
  EG
• 144-169 LRKRLLRDADDLQKRLAVYQAGAREG
• 148-169 LLRDADDLQKRLAVYQAGAREG
• 141-155 LRKRLLRDADDL
• --------------------------------------------------
  --------------------------------------------------
  ---------

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Example 1 Heparin binding domains of
Apolipoprotein E

• Phase II
• step Binding studies with 125I-labelled heparin.
  Dot-blot assay.

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Example 2 Molecular Mapping of Immunogenic
Determinants of Human CD4 Using Chimeric
Interspecies Molecules and Antibodies
CD4/L3T4 lymphocyte antigen, expressed on helper
T-cells and macrophages, primary and 3D
structure knonw for CD4 (human), primary
structure for L3T4 (mose) Antibodies 37 human
and mouse monoclonal antibodies recognising
CD4/L3T4 positive cells Interaction Antibody
antigen interactions Aim Identification of
epitop regions recognized by anti-CD4
antibodies. (Lack of binding of certain
antibodies to overlapping peptides
corresponding to CD4 indicated the presence of
discontinuous conformational epitopes.)
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Example 2 Molecular Mapping of Immunogenic
Determinants of Human CD4 Using Chimeric
Interspecies Molecules and Antibodies

• step Preparation of chimeric CD4 cDNA molecules
  using human CD4 and mouse L3T4 cDNA clones.
• Method Recombinant DNA technique (Bacterial
  homologous recombination system)
• step Expression of chimeric CD4 cDNA molecules
• Method Transfection into an L3T4 negative
  variant of the murine T-cell line, EL-4
• Observation 9 chimeric L3T4/CD4 (mouse amino
  terminal) and CD4/L3T4 (human amino terminal)
• molecules with cross-over in the extracellular
  region of the mature protein were generated

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Example 2 Molecular mapping of immunogenic
determinants of Human CD4 using chimeric
interspecies molecules and antibodies

• step Binding studies with purified CD4 or L3T4
  specific antibodies on EL-4 cells
• expressing chimeric CD4 in 3D form.
• Method
• Staining cells with CD4/L3T4 antibodies
• Incubation of stained cells with FITC-labeled
  goat anti-mouse Ig
• Flow cytometry on FACS

Antigen molecule or cells

Ag-specific antibody
Fluorochrome or enzyme-labeled Ag-specific
antibody
Fluorochrome or enzyme-labeled anti-Ig
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Example 2 Molecular mapping of immunogenic
determinants of Human CD4 using chimeric
interspecies molecules and antibodies

• step
• Observation All chimeric molecules analys ??? in
  transfectants detectable with
• human and/or mouse specific anti-CD4
  antibodies.
• Using the chimerics, it was possible to
  localise most of the CD4
• epitopes to specific region of the CD4
  protein.
• NB. 1. CD4/L3T4 recognise antigen in the
  context of class II MHC antigens.
• 2. As expected from their functional
  similarities, the human and the mouse
• CD4 molecules are highly homologous at both
  the DNA (70) and
• amino acid (54) levels.
• Reference P. Estess et al. Current Research in
  Protein Chemistry (ED. J. J. Villafranca,
  Academic Press,
• San Diego, p. 499 (1990))

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Example 3 Localization of Immunogenic
Determinants (Epitopes) of human epithelial
mucin glycoprotein, MUC-1 Using synthetic
Peptides and MUC-1 specific Antibodies

• MUC-1 high molecular weight, MUC-1 gene related
  glycoprotein,
• associated with human breast and ovarian
  carcinoma,
• primary structure is known
• Antibodies mouse monoclonal antibodies
  recognizing MUC-1 glycoprotein
• HMFG-1, C595, B55, etc.
• Interaction Antibody antigen interactions
• Aim Identification of epitopes recognized by
  anti-MUC-1 antibodies
• Phase I
• step Analysis of the primary structure of MUC-1
  glycoprotein.
• Method Prediction of B-cell epitopes using
  various algorithms searching for
• hydrophilic region and
• b-turn secondary structure

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Example 3 Localization of Immunogenic
Determinants (Epitopes) of human epithelial
mucin glycoprotein, MUC-1 Using synthetic
Peptides and MUC-1 specific Antibodies
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Example 3 Localization of Immunogenic
Determinants (Epitopes) of human epithelial
mucin glycoprotein, MUC-1 Using synthetic
Peptides and MUC-1 specific Antibodies

• Phase I
• step Synthesis of overlapping heptapeptide
  covering the repeat of an antigenic
• 20 amino acid sequence of MUC-1
• Method Solid phase synthesis of peptides on
  polyethylene pin support.
• (Peptides were not removed from the pin
  during testing.)
• step Binding studies with purified MUC-1 specific
  antibodies on immobilized
• synthetic overlapping peptides.
• Method
• Incubation of peptides with antibody HMFG-1.
• Incubation with peroxidase-labeled rabbit
  anti-mouseIg.
• Development of colour reaction by the addition of
  substrate solution
• (azino-di-3-ethyl-benzothiazoline-sulphonate)
  and hydrogen peroxidase.
• Reading of absorbance at 405 nm.

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Example 3 Localization of Immunogenic
Determinants (Epitopes) of human epithelial
mucin glycoprotein, MUC-1 Using synthetic
Peptides and MUC-1 specific Antibodies
Observation Mucin specific MoAb, HMFG-1 binds
to heptapeptides containing PDTR sequence
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Example 3 Localization of Immunogenic
Determinants (Epitopes) of human epithelial
mucin glycoprotein, MUC-1 Using synthetic
Peptides and MUC-1 specific Antibodies
Phase I Determination of 3D structure of epitope
region containing PDTR sequence. Methods 2D
NMR (1H HOHAHA) Reference M.R.
Price, F. Hudecz et al. Mol. Immunol. 62 795
(1900) S.J.B. Tendler Biochem. J. 267 733
(1900)
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Hydrophobicity scales for amino acid residues1
1The order is by decreasing hydrophobicity on the
consensus scale. The magnitudes for all but the
two scales on the right may be considered Roughly
in kcalmol-1 transfer from a hydrphobic to a
hydrophilic phase. The scales do not all measure
the same property
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Hydrophobicity scales for amino acid residues
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Example 4 Location of membrane-spanning regions
of human intracellular adhesion molecule 1
(ICAM)

• step Assignment
• ITQRGRFDAPTPTPTGTQTPTTDASERTHILEPEAKNADGTREQWFY46
  .
• step Averaging
• ITQRGRFDAPTPTPTG15TQTPTTDASERTHILEPEAKNADGTREQWFY
  46.

-1.8
-1.8
0.2
Hydrophilic/hydrophobic character
0.16
-0.69
1
15

• (n1n2n3 n15) 15
• ? (n2n3n4 n16) 15

?
2
16
?
3
17
4
18
19
5
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Example 4 Location of membrane-spanning regions
of human intracellular adhesion molecule 1
(ICAM)

• step Evaluation

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Example 5 Localization of Domain Responsible
for Hemolytic Activity of Melittin
Melittin Amphipathic 26-residue
peptide primary and 3D srtucture are
known Target Human red blood cells (RBC) 1.8
x 107 binding sites per erythrocyte Kd 10-7
3x10-8 M Aim Identification of the smallest
fragment, which can induce hemolysis at
melittin level
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Example 5 Localization of Domain Responsible
for Hemolytic Activity of Melittin

• step Peptide synthesis

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Example 5 Localization of Domain Responsible
for Hemolytic Activity of Melittin

• step Binding studies with human red blood cells.
  Hemolytic assay
• Method
• Incubation of peptides at equimolar ratio with
  washed and counted
• RBC for 1h at 37 oC
• Centrifugation.
• Measurement of absorbance at 414 nm e 14.7x10-4.

Observation 1 The critical residues For
hemolytic activity of melittin are Between amino
acid 2 and 22
Melittin
2-26 3-26 4-26 5-26 6-26
1-25 1-24 1-23 1-22 1-21 1-20
Melittin
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Example 5 Localization of Domain Responsible
for Hemolytic Activity of Melittin
Observation 2The removal of amino acid residues
from the N-terminal affect the amphipathic
a-helix