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NUMBERS

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-dimensional (3D) structure (J. Kendrew, 1962) 1962-1985 (20 years) 200

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NUMBERS

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  1. 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-dimensional (3D) structure (J. Kendrew, 1962) 1962-1985 (20 years) 200 1986-1991 ( 5 years) 480 1992- 100/years

  2. 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)

  3. RECOGNITION PHENOMENA

  4. 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

  5. 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 -

  6. 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

  7. Strategies for determinations of 3D structures Experimental methods Theoretical methods Quantum chemistry: mechanics and dynamics Empirical calculations • X-ray diffraction • (Mioglobin, hemoglobin • Kendrew, Perutz, 1960) • crystal • time-intensive Relationship assay NMR Prediction of secondary structures (helix, b-turn) CD spectroscopy FT-IR spectroscopy Prediction of hydrophobic – hydrophilic regions

  8. 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

  9. Techniques for the detection of interaction/recognition phenomena 2. Cellular level • Bioassay (in vitro) • binding to cell • hemolysis • antibacterial effect • cytotoxicity

  10. Example 1Heparin 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 • E(1-191) E(192-299)

  11. Example 1Heparin 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 • Observation: ApoE and the two thrombin fragments bind heparin, indicating a minimum • of two heparin-binding domains ApoE High E(1-191) E(192-299) Low

  12. Example 1Heparin 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 HLRKLRKRLLRDADDLQKRLAVYQAGAREG • 144-169 LRKRLLRDADDLQKRLAVYQAGAREG • 148-169 LLRDADDLQKRLAVYQAGAREG • 141-155 LRKRLLRDADDL • -------------------------------------------------------------------------------------------------------------

  13. Example 1Heparin binding domains of Apolipoprotein E • Phase II • step Binding studies with 125I-labelled heparin. Dot-blot assay. • Observation: The critical residues for heparin binding resided between amino acid • 144 and 150 (Leu-Arg-Lys-Arg-Leu-Leu-Arg)

  14. Example 2Molecular 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.)

  15. Example 2Molecular 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

  16. Ag Ag Ag Ag Example 2Molecular 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

  17. Example 2Molecular 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))

  18. Example 3Localization 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

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

  20. Example 3Localization 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.

  21. Example 3Localization 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

  22. Example 3Localization 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)

  23. 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 kcal*mol-1 transfer from a hydrphobic to a hydrophilic phase. The scales do not all measure the same property

  24. Hydrophobicity scales for amino acid residues

  25. Example 4 Location of membrane-spanning regions of human intracellular adhesion molecule 1 (ICAM) • step Assignment • ITQRGRFDAPTPTPTGTQTPTTDASERTHILEPEAKNADGTREQWFY46…. • step Averaging • ITQRGRFDAPTPTPTG15TQTPTTDASERTHILEPEAKNADGTREQWFY46…. -1.8 -1.8 0.2 Hydrophilic/hydrophobic character 0.16 -0.69 1 15 • = (n1+n2+n3+ …… +n15) : 15 •  = (n2+n3+n4+ …… +n16) : 15  2 16  3 17 4 18 19 5

  26. Example 4 Location of membrane-spanning regions of human intracellular adhesion molecule 1 (ICAM) • step Evaluation

  27. 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

  28. Example 5 Localization of Domain Responsible for Hemolytic Activity of Melittin • step Peptide synthesis

  29. 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 2-26 3-26 4-26 5-26 6-26 Melittin 1-25 1-24 1-23 1-22 1-21 1-20 Melittin

  30. Example 5 Localization of Domain Responsible for Hemolytic Activity of Melittin Observation 2:The removal of amino acid residues from the N-terminal affect the amphipathic a-helix

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