Module 3: Lesson 1 Protein Binding

Globular Protein Functions Functions/Examples Storage of oxygen/iron (myoglobin, ferritin) Transport and messengers (hemoglobin, serotonin transporter) Defense against pathogens (antibodies, cytokines) Catalysis (enzymes) Structural (actin, myosin) Biological motors (myosin, kinesin) Gene regulation (transcription factors) Keys to Function Reversible binding of ligands Specific ligand-binding sites Induced fit - ligand binding (conformational/dynamic changes) Cooperativity - multiple subunits showing interdependent conformational changes in response to ligand binding Regulated allosteric interactions Protein-Ligand Binding Interaction Basics Reversible, transient processes of equilibrium Ligand + Protein <-> Protein-Ligand complex Ligand: Molecule that binds to proteins Typically small Binding site: Region in protein that binds ligands Ligand binds to protein via non-covalent bonds (weak interactions) Examples of Binding Strength Biotin-avidin: 10^-15 Antibody-antigen: 10^-10 Enzyme-substrate: 10^-8 Typical receptor-ligand interactions: 10^-6 Hormone-receptor: 10^-9 Specificity of Binding and Induced Fit Complementary in size, shape, charge, hydrophilic/hydrophobic interactions Lock and key model (Emil Fischer, 1894) - complementary surfaces are preformed Conformational changes upon ligand binding (Daniel Koshland, 1958) Induced fit, tighter binding/variable affinity of different ligands Ligand and protein change conformations Binding: Quantitative Description Ligand (L) binds reversibly to a site in protein (P) Described quantitatively by the association rate constant (ka) and dissociation rate constant (kd) P + L ⇌ PL Process will reach equilibrium, association/dissociation rates are equal Ka = [PL]/[P][L] = 1/Kd Equilibrium constant = equilibrium dissociation constant Fraction of occupied binding sites (θ) θ = [PL] / ([PL] + [P]) Substituting [PL] with Ka[P][L], eliminating [PL] Rearranging causes equilibrium association constant = equilibrium dissociation constant θ = [PL] / ([PL] + [P]) θ = Ka[L][P] / (Ka[L][P] + [P]) Experimentally: θ = [L] / ([L] + Kd) Ligand concentration Known Kd determined graphically/least squares regression θ = [L] / ([L] + Kd) Y = X / (X + Kd) Ka = 1/[Kd] 1.0 0.5 0