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Proteins

Proteins. Protein Function. Catalysis Structure Movement Defense Regulation Transport Antibodies. Monomers—Amino Acids. R-groups Hydrophilic Hydrophobic Uncharged Charged Large Small Confer unique chemical properties on each aa. PROTEIN LEVELS OF STRUCTURE. PRIMARY STRUCTURE.

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Proteins

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  1. Proteins

  2. Protein Function • Catalysis • Structure • Movement • Defense • Regulation • Transport • Antibodies

  3. Monomers—Amino Acids • R-groups • Hydrophilic • Hydrophobic • Uncharged • Charged • Large • Small • Confer unique chemical properties on each aa

  4. PROTEIN LEVELS OF STRUCTURE

  5. PRIMARY STRUCTURE • Is a unique characteristic of every protein • Is encoded by the nucleotide sequence of DNA • Is thus a form of genetic information • Is read from the amino terminus to the carboxyl terminus

  6. Nature of Protein Sequences • Sequences and composition reflect the function of the protein: • Membrane proteins have more hydrophobic residues. • Homologous proteins from different organisms have similar sequences. • e.g., cytochrome c is highly conserved

  7. Cytochrome c

  8. SECONDARY STRUCTURE I: THE -HELIX

  9. a Helix If N-terminus is at bottom, then all peptide N-H bonds point “down” and all peptide C=O bonds point “up”. N-H of residue n is H-bonded to C=O of residue n+4.

  10. Secondary Structure II: The -Strand approx. 3.4 A Severalb-strands assemble into a b-sheet (a tertiary structural element)

  11. TERTIARY STRUCTURE • 3-D structure. • Form follows function!! • Native vs denatured • Determinants of tertiary structure • Amino acid sequence • Environment in which the protein resides

  12. Stabilizing Interactions • Hydrogen Bonds • Electrostatic interactions (“salt-bridges” or ion pairs) • van der Waals interactions (dipole-dipole and dispersion) • Hydrophobic interactions • Disulfide bridges

  13. Protein Denaturation • Denaturants--Anything that can disrupt stabilizing interactions • Heat • Salts • pH • Organic solvents

  14. Quaternary Structure

  15. FLUORESCEIN – a hapten ANTIBODIES • Extremely specific • Definitions: • Antigen • Epitope (antigenic determinant) • Hapten

  16. Antigen binding site Antigen binding site V V V V Constant Constant Constant Constant SS SS Light Chain Light Chain SS SS Antibody Structure Heavy Chains

  17. Antibody Structure

  18. Antibody Structure

  19. Recognition and Binding The N-terminal region of antibody light chains and heavy chains form the antigen binding site The variability in amino acid sequence provides the structural basis for the diversity of antigen-binding sites

  20. Antigen 3 Variable Heavy Variable Light Antigen Binding Antigen 1 Antigen 2

  21. Polyclonal vs Monoclonal Abs • 107-109 genetically distinct lymphocytes, each producing a single type of Ab. • Polyclonal—normal immune response. Several Abs, recognition of various epitopes with varying affinities. • Monoclonal

  22. Monoclonal Ab Production • Given: • Normal cells—Mortal • Transformed cells—Immortal • Two Pathways of DNA Synthesis • Major • Salvage—Requires HGPRT • 8-azaguanine—HGPRT poison. • Aminopterin---Interferes w/ major pathway • PEG---promotes cell fusion

  23. HAT Selection • Select HGPRT- mutant myeloma by treatment with 8-azaguanine • Fuse HGPRT- mutant myeloma with normal cells using PEG • Select with aminopterin • Normal? • Myeloma? • Hybridoma? • Screen for desired monoclonal.

  24. MAbs in the Lab • Macs extremely useful in molecular biology and medicine • Applications • Affinity columns • Western blots • ELISA (Enzyme Linked ImmunoSorbent Assay)

  25. Back

  26. The Future? • Single Chain Antibodies • Catalytic antibodies • Bifunctional antibodies • Etc.

  27. Features of a single-chain antibody (sFv). Consists of the variable light (VL) chain of an antibody joined via a linker to the variable heavy (VH) domain. The linker typically consists of a flexible/soluble peptide (for example, [GGGGS]6) The sFv maintains the antigen binding specificity (but not always the affinity) of the parent antibody. N VL Linker C VH CL VL VH CH1 CH2 CH3 CH1 CH2 CH3 VH VL CL Back

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