Protein allostery: · Allosteric: control mediated by a ligand binding at a site that is different from the active site. Conformational dynamics. · Homotropic effects: interactions between identical ligands e.g. O2 binding to haemoglobin. · Heterotropic effects: interactions between different ligands e.g. H+ affecting O2 binding to haemoglobin. · Cooperativity: refers to interactions between identical ligands and these interactions may be positive or negative. Positive: binding starts weak and becomes stronger- negative is vice versa. · Bohr effect: binding of oxygen to haemoglobin. · Often very hard to understand how state A goes to state B. hyperbolic (e.g. O2 binding to myoglobin) and sigmoidal (e.g. O2 binding to hemoglobin) binding curves 100 myoglobin (b) 80 8 Saturation with 02 (%) (a) 40- /hemoglobin 201 0 20 40 1 60 80 100 Venous pressure Arterial pressure po2 (mmHg) . Hyperbolic: starts steep and flattens out as saturation is reached. · Sigmoidal: starts with low affinity but increases as concentration increases, eventually saturation is reached. · Calcium binding to calmodulin induces conformational changes/dynamic changes that allow it to bind to a n.o. different ligands which leads to certain responses within the cell. · Need some degree of flexibility for allostery to be possible. Must be right amount of dynamics: The Goldilocks principle. · Need the right energy differences between states and just the right energy barriers ttactivation energy) separating these states. . Free energy of unbound state favours inactive conformation. unbound protein A: "inactive" conformation B: "active" conformation free energy B A TAG AG+ conformation
· Allosteric modulator changes the relative energies/barriers. Now active state is favoured by the positive modulator binding. · There are many states in which the protein may exist in. · Energy landscape perspective: 3D representations of energy wells. Deeper well = more energetically favoured conformation. · Induced fit: something binds and forces it into state B vs. conformational selection: selectively binds to state B which was there already- stabilises it and overtime selects this state/conformation. There is some non-binary character to these mechanisms. Q: does the conformational change occur before or after the initial encounter? A+ B (AB) AB* Induced fit A+B k3 k2 kg k3' AB# Conformational selection Allosteric vs. orthosteric inhibition: · Orthosteric: competes for binding by binding to the same site to prevent association with the real ligand. Results in changes in KD values. · Allosteric: bind to another site which is distal to the ligand binding site to cause a conformational change. Ligand can no longer bind. Results in changes in Bmax values.
100 no inhibitor 100 no inhibitor 88 8 9 8 80. 60 40 20 bound (CPM) 8 60 apparent change bound (CPM) in Kp 40 apparent change in B. +orthosteric inhibitor 20 +allosteric inhibitor 10 100 1000 [ligand] (nM) 10000 10 100 1000 [igand] (nM) 10000 Orthosteric inhibition: results in changes in apparent Kg values Allosteric inhibition: results in changes in apparent Bmax values . In allosteric inhibition, n.o. binding sites is decreased and Bmax is decreased. Allostery inhibition versus