Lecture 6 Protein-protein interactions Affinities (cases of simple and cooperative binding) Examples of Ligand-protein interactions Antibodies and their generation 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 shortrange interactions 0.8 U1( r)
describes both repulsion and attraction E p (r ) E0 (r0 / r )12 2 E0 (r0 / r ) 6 Uo 1 12 2x 6 r = r0 U( x) Uo x 2 steric repulsion 1
r = 0.89r0 U( x) Bond stretching is often considered in the harmonic approximation: U ( x) 12 ( x x0 ) 2 0 r = r0 (attraction=minimum) 1
0.6 0.8 1 1.2 1.4 x 1.6 1.8
Here is a typical form in which energy of interactions between two proteins or protein and small molecule can be written Etot qel Asteric Bdisp Edesolv Ionic pairs + H-bonding Van der Waals removal of water from the contact
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
Simple binding Receptor occupancy: [ LR ] [ LR ] B [totalR ] [ R] [ LR ] K [ L] B b 1 K b [ L] L R LR Mass action:
Kd [ LR ] K b [ L][ R] [ LR ] k Kb on [ L][ R ] k off equilibrium parameter 1/koff = residence time in the bound state kinetic parameters
1 Kb [ L] B K d [ L] (Langmuir isotherm) Receptor occupancy is a hyperbolic function of [L ] (Langmuir adsorption isotherm) Bmax [ L]
B K d [ L] Kd = 1 Kd = 3 Kd = 10 1 0.8 B1( x) B2( x) 0.6
B3( x) Kd has the dimension of concentration and should be measured in the same units as L (M). 0.4 0.2 0 0
10 20 30 40 50 L Note that for a shallow curve it is hard to say where it saturates Oxygen and Hemoglobin
97% of O2 is carried in the form of Oxyhemoglobin (HbO2) 3% - dissolved in plasma physiological range P1/2 = 28 mm Hg When PO2 changes from 100 to 40 mm Hg, the saturation decreases from 98 to 75% CO binds to the porphyrin ring of heme exactly where O 2 binds From G. Hummer
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 [ RLn ] 1 [ L ]n [ R ] K n [ RLn ] 1
[ L]n [ Rtot RLn ] K n 1 n=4 n=2 0.8 B1( x) n=1 0.6
B2( x) rearrange [ RLn ] [ L ]n Bn [ Rtot ] K n [ L]n Hill equation, n is Hill coefficient B3( x) 0.4 0.2
0 0 1 2 3 L 4
5 Hemoglobin vs Myoglobin [ L ]n Bn K n [ L ]n Myoglobin, n = 1 1 0.8 B1( x) 0.6
Hemoglobin, n = 2.8 B3( x) 0.4 0.2 0 0 2 4
6 pO2 (kPa) pO2 in tissues 8 10 Cooperativity is due to tight intersubunit interactions B x kd x independe
nt binding cooperative binding B x n kd x n n Hill coefficient Protein Kinase A spatially organizes ATP and peptide chain to facilitate the phosphorylation reaction (old book)
Intracellular signaling adapter domains SH2 and SH3 Segment containing phosphotyrosine Fig 16-11 Proline-rich sequence Fig 16-23 PDZ domains spatially organize ion channel/receptor complexes in synapses Postsynaptic density complex
(old book) Fatty acid binding protein (FABP) Fig. 10-24 Common theme: hormones promote dimerization of receptors Fig. 16-7 The Growth Hormone sequentially binds to two receptors Fig 15-3 first binding event second receptor is then recruited
Binding of the Epidermal Growth Factor (EGF) leads to receptor dimerization not by cross-linking but by exposing sticky loops Fig. 16-17 Antibody (IgG) CDR = complementarity determining region The lymph system and lymph nodes See Chapter 24 Clonal selection of B lymphocytes: prolifereation and differentiation of these cells is induced by an
encounter with an antigen recognized by the surface receptor The immunoglobulin fold and the hypervariable regions Fig. 24-12 Variability of sequence in hypervariable loops The antigen recognition site Fig. 24-13 Combinatorial diversity of antibodies Light chain coding regions: V variable C constant
VL CL 100 5 variants Heavy chain coding regions: VH D
100 30 CH 4 variants therefore, total number of combinations is ~ 6,000,000 see Lodish (4th edition) The recognition site exposes flexible loops typically with many polar residues
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