1. Proteins

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

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

4. PROTEIN LEVELS OF STRUCTURE

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

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

7. Cytochrome c

8. SECONDARY STRUCTURE I: THE  -HELIX

9.  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 Several  -strands assemble into a  -sheet (a tertiary structural element)

11. TERTIARY STRUCTURE 3-D structure. 3-D structure. Form follows function!! Form follows function!! Native vs denatured Native vs denatured Determinants of tertiary structure Determinants of tertiary structure Amino acid sequence Amino acid sequence Environment in which the protein resides 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 Denaturants--Anything that can disrupt stabilizing interactions Heat Heat Salts Salts pH pH Organic solvents Organic solvents

14. Quaternary Structure

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

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

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

21. Polyclonal vs Monoclonal Abs 10 7 -10 9 genetically distinct lymphocytes, each producing a single type of Ab. 10 7 -10 9 genetically distinct lymphocytes, each producing a single type of Ab. Polyclonal—normal immune response. Several Abs, recognition of various epitopes with varying affinities. Polyclonal—normal immune response. Several Abs, recognition of various epitopes with varying affinities. Monoclonal Monoclonal

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

23. HAT Selection 1) Select HGPRT - mutant myeloma by treatment with 8-azaguanine 2) Fuse HGPRT - mutant myeloma with normal cells using PEG 3) Select with aminopterin 1) Normal? 2) Myeloma? 3) Hybridoma? 4) Screen for desired monoclonal.

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

25. Back

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

27. VL VH N C VL VH VL VH Features of a single-chain antibody (sFv). Linker 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. CH2 CH3 CH1 CL Back