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Lecture 6

Lecture 6. Protein-protein interactions Affinities (cases of simple and cooperative binding) Examples of Ligand-protein interactions Antibodies and their generation.

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Lecture 6

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  1. Lecture 6 Protein-protein interactions Affinities (cases of simple and cooperative binding) Examples of Ligand-protein interactions Antibodies and their generation

  2. Even without NET CHARGES on the molecules, attractive interactions always exist. In the presence of random thermal forces all charge-dipole or dipole-dipole interactions decay steeply (as 1/r4 or 1/r6) Long-range and short-range interactions 1/r 1/r2 1/r6 1/r4

  3. Interatomic interaction: Lennard-Jones potential describes both repulsion and attraction r = r0 steric repulsion r = 0.89r0 Bond stretching is often considered in the harmonic approximation: r = r0 (attraction=minimum)

  4. Here is a typical form in which energy of interactions between two proteins or protein and small molecule can be written Ionic pairs + H-bonding removal of water from the contact Van der Waals

  5. What determines affinity and specificity? Tight stereochemical fit and Van der Waals forces Electrostatic interactions Hydrogen bonding Hydrophobic effect All forces add up giving the total energy of binding: Gbound– Gfree= RT lnKd

  6. Simple binding Receptor occupancy: Mass action: 1/koff= residence time in the bound state (Langmuir isotherm) kinetic parameters equilibrium parameter

  7. Bmax Kd = 1 1 Kd = 3 Kd = 10 0.8 B1 ( x ) 0.6 B2 ( x ) B3 ( x ) 0.4 0.2 0 0 10 20 30 40 50 L Receptor occupancy is a hyperbolic function of [L] (Langmuir adsorption isotherm) Kd has the dimension of concentration and should be measured in the same units as L (M). Note that for a shallow curve it is hard to say where it saturates

  8. Oxygen and Hemoglobin 97% of O2 is carried in the form of Oxyhemoglobin (HbO2) 3% - dissolved in plasma When PO2 changes from 100 to 40 mm Hg, the saturation decreases from 98 to 75% physiological range P1/2 =28 mm Hg

  9. CO binds to the porphyrin ring of heme exactly where O2 binds From G. Hummer

  10. 1 0.8 0.6 0.4 0.2 0 0 1 2 3 4 5 What if the binding to multiple sites on the same receptor is strictly interdependent (i.e. cooperative)? Probability of binding to one site ~[L] Probability of binding simultaneously to n sites ~[L]n n=4 n=2 n=1 B1 ( x ) B2 ( x ) rearrange B3 ( x ) Hill equation, n is Hill coefficient L

  11. 1 0.8 B1 ( x ) 0.6 B3 ( x ) 0.4 0.2 0 0 2 4 6 8 10 pO2 (kPa) Hemoglobin vs Myoglobin Myoglobin, n = 1 Hemoglobin, n = 2.8 pO2 in tissues

  12. Cooperativity is due to tight intersubunit interactions independent binding cooperative binding n – Hill coefficient

  13. Protein Kinase A spatially organizes ATP and peptide chain to facilitate the phosphorylation reaction (old book)

  14. Intracellular signaling adapter domains SH2 and SH3 Segment containing phosphotyrosine Proline-rich sequence Fig 16-11 Fig 16-23

  15. PDZ domains spatially organize ion channel/receptor complexes in synapses “Postsynaptic density” complex (old book)

  16. Fatty acid binding protein (FABP) Fig. 10-24

  17. Common theme: hormones promote dimerization of receptors Fig. 16-7

  18. The Growth Hormone sequentially binds to two receptors Fig 15-3 second receptor is then recruited first binding event

  19. Binding of the Epidermal Growth Factor (EGF) leads to receptor dimerization not by cross-linking but by exposing ‘sticky’ loops Fig. 16-17

  20. Antibody (IgG) CDR = complementarity determining region

  21. The lymph system and lymph nodes See Chapter 24

  22. Clonal selection of B lymphocytes: prolifereation and differentiation of these cells is induced by an encounter with an antigen recognized by the surface receptor

  23. The immunoglobulin fold and the hypervariable regions Fig. 24-12

  24. Variability of sequence in hypervariable loops

  25. The antigen recognition site Fig. 24-13

  26. Light chain coding regions: CL VL 100 5 variants Heavy chain coding regions: D CH VH 4 variants 100 30 Combinatorial diversity of antibodies V – variable C – constant therefore, total number of combinations is ~ 6,000,000 see Lodish (4th edition)

  27. The recognition site exposes flexible loops typically with many polar residues

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