Interpreting Effects of Allosteric Agents

1. 100 Shift to R state Fractional Saturation Shift to T state 50 pO2 Interpreting Effects of Allosteric Agents Bind O2 95 80 O2 OH- CO2 Release O2 -Hb BPG 10 H+

2. 4 RULES Governing Allosteric Proteins RULE: Allostery is a property of proteins that contain multiple subunits or a single subunit with multiple binding sites. RULE: Allosteric proteins must be able to bind more than one ligand. RULE: A ligand has the capacity to induce a change in the shape of an allosteric protein. RULE: By changing the protein’s shape, a ligand can facilitate (positive) or deter (negative) the binding of additional ligands to the protein.

3. MODELS OF ALLOSTERY See Strategies p.120 CONCERTED: (ALL OR NONE) Subunits exist in two conformational states The two states are in equilibrium All subunits have the same conformation; no hybrids Ligand binds to any subunit in either conformation Conservation of symmetry is the driving force

4. Problems with Concerted Model • Symmetry is not preserved in most oligomeric proteins • Some allosteric oligomeric proteins do not have identical subunits • Cannot explain in a two state model how negative and positive cooperativity occur in the same protein

5. Sequential Model S S S S S S S S S S S S S S Ligand binding induces change in subunit Change is progressive Symmetry is not preserved Mechanical coupling between subunits may be weak

6. Sickle Cell Anemia(a genetic disease) One Amino acid in a protein can make a difference between life and death Distortion Is it a membrane protein? Is it a cytosolic protein? Is it something other than a protein?

7. Hb (alpha) N- Val-Leu-Ser-Pro-Ala-Asp-Lys-Thr…….. Glu Hb (beta) N- Val-His-Leu-Thr-Pro- -Glu-Lys……. Val Sickle Cell Glu6 Val6 (each beta chain) Glu GAA or GAG Val GUA or GUG Transposition A U replaces an A in the codon (a pyrimidine/purine exchange)

8. Val Polymerized deoxy Hb Aggregates  Insoluble fibers Distorts the overall shape of hemoglobin Distorts the overall shape of an erythrocyte Distorted erythrocytes cannot pass through capillaries

9. Antibodies Ancient Greeks: Thucydides (460-400 B.C.) Speaking of the plague of Athens, Yet, it was in those that had recovered from the disease that the sick and dying found most compassion. These knew what it was from experience, and had now no fear for themselves; for the same man was never attacked twice-never at least fatally.

10. Early Theories Pauling 1940: The “Instructive” Theory Antigens act like templates that direct the folding of a nascent antibody chain MacFarlane Burnet: The Selective or clonal theory The combining site on an antibody molecule is completely determined before it encounters an antigen

11. Bone Marrow Precursor lymphocytes leukocytes macrophages B-lymphocytes T-lymphocytes (T-cells) Antibodies Cytotoxic T cells (TC cells) Humoral Helper T cells (TH cells) Cell-Mediated Cytokines

12. Essential Features • Each antibody-producing B-cell makes a single kind of antibody…no antigen is needed. • Specificity is determined by amino acid sequence…each cell’s DNA is distinctive. • Immature cells that make antibodies are destroyed early in life…self tolerance • Mature cells make and display antibodies on their surface • Interaction with antigen triggers cell to divide, making large amounts of a particular antibody that persists until antigen is gone

13. Antibodies are large complex molecules Composition: Antigen-binding site Constant Variable Hinge region 2 heavy, 2 light chains Disulfide bonds

14. 5 Classes of Antibodies (Immunoglobulins, Ig) • IgA…external secretions, tears,saliva, bronchial and intestinal mucous • IgG…principal antibody in the serum, originally called gamma globulin • IgD…least understood • IgE…no clear function, has receptor on mast cells and stimulates production of histamine and is linked to allergic responses • IgM…first class to appear, highest combining sites, effective against bacteria

15. Mechanism • Preexisting B cell synthesizes IgM first • IgG, IgA, IgD, IgE of same specificity are made later • Light chain is unchanged during switch • Variable region of heavy chain is unchanged during switch • Only the Constant region of heavy chain changes…(class switching or CH switching) • In the mouse, switching uses the appropriate Constant region gene, [C, C, C, C, C] for the antibody class

16. Cell-Mediated Immunity Major histocompatibility complex (MHC) Function: In conjunction with T-cell surveillance, MHC is a series of polymorphic proteins designed to display digested peptide fragments on the surface of the cell. The basis of cell-mediated immunity MHC-1 On all cell surfaces Displays fragments from internal protein digests Interacts with TC cells to initiate infected cell destruction MHC-2 On surface of phagocytic cells (macrophages, etc.) Displays digested fragments from external protein sources Interacts with TH cells to alert system of infection

18. Important! Internal proteins digested by proteases cannot bind to MHC-2 MHC-1 interaction is a signal to TC cells to destroy the infected cell External proteins that enter the cell by an endosomal pathway cannot bind to MHC-1. MHC-2 interaction is a signal to TH cells that the system is under attack by a virus, bacteria, etc. and to send for help in the form of antibody-producing cells