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

2. 1/r 2 1/r 6 1/r Long-range and short- range interactions 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/r 4 or 1/r 6 ) 1/r 4

3. Interatomic interaction: Lennard-Jones potential describes both repulsion and attraction r = r 0 ( attraction=minimum ) r = 0.89r 0 r = r 0 steric repulsion Bond stretching is often considered in the harmonic approximation:

4. Van der Waals 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

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: G bound – G free = RT ln K d

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

7. Receptor occupancy is a hyperbolic function of [ L ] (Langmuir adsorption isotherm) B1x( B2x( B3x( L 01020304050 0 0.2 0.4 0.6 0.8 1 ) ) ) K d = 1 K d = 3 K d = 10 B max K d 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. 97% of O 2 is carried in the form of Oxyhemoglobin (HbO 2 ) 3% - dissolved in plasma P 1/2 = 28 mm Hg When P O2 changes from 100 to 40 mm Hg, the saturation decreases from 98 to 75% physiological range Oxygen and Hemoglobin

9. From G. Hummer CO binds to the porphyrin ring of heme exactly where O 2 binds

10. What if the binding to multiple sites on the same receptor is strictly interdependent (i.e. cooperative)? Hill equation, n is Hill coefficient 012345 0 0.2 0.4 0.6 0.8 1 B1x() B2x() B3x() L n=2 n=4 n=1 rearrange Probability of binding to one site ~[L] Probability of binding simultaneously to n sites ~ [L] n

11. Myoglobin, n = 1 Hemoglobin, n = 2.8 pO 2 (kPa) 0246810 0 0.2 0.4 0.6 0.8 1 B1x() B3x() pO 2 in tissues Hemoglobin vs Myoglobin

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

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 Proline-rich sequence Segment containing phosphotyrosine 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 first binding event second receptor is then recruited Fig 15-3

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: VLVL CLCL 1005 variants Heavy chain coding regions: VHVH DCHCH 10030 4 variants therefore, total number of combinations is ~ 6,000,000 Combinatorial diversity of antibodies see Lodish (4th edition) V – variable C – constant

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