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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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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
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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:
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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
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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
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Simple binding Receptor occupancy: Mass action: (Langmuir isotherm) kinetic parameters equilibrium parameter 1/k off = residence time in the bound state
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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
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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
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From G. Hummer CO binds to the porphyrin ring of heme exactly where O 2 binds
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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
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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
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Cooperativity is due to tight intersubunit interactions n – Hill coefficient independent binding cooperative binding
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Protein Kinase A spatially organizes ATP and peptide chain to facilitate the phosphorylation reaction (old book)
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Intracellular signaling adapter domains SH2 and SH3 Proline-rich sequence Segment containing phosphotyrosine Fig 16-11 Fig 16-23
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PDZ domains spatially organize ion channel/receptor complexes in synapses “Postsynaptic density” complex (old book)
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Fatty acid binding protein (FABP) Fig. 10-24
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Common theme: hormones promote dimerization of receptors Fig. 16-7
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The Growth Hormone sequentially binds to two receptors first binding event second receptor is then recruited Fig 15-3
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Binding of the Epidermal Growth Factor (EGF) leads to receptor dimerization not by cross-linking but by exposing ‘sticky’ loops Fig. 16-17
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Antibody (IgG) CDR = complementarity determining region
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The lymph system and lymph nodes See Chapter 24
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Clonal selection of B lymphocytes: prolifereation and differentiation of these cells is induced by an encounter with an antigen recognized by the surface receptor
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The immunoglobulin fold and the hypervariable regions Fig. 24-12
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Variability of sequence in hypervariable loops
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The antigen recognition site Fig. 24-13
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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
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The recognition site exposes flexible loops typically with many polar residues
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