Protein–Ligand Interactions: Thermodynamic Basis and Mechanistic Consequences


It is difficult to imagine any biological process that is not initiated by the binding of some chemical entity (or ‘ligand’) to a protein. Thus, enzymatic catalysis, signal transduction, ion channel activity, immune responses and the various events involved in genetic expression are all obvious examples of events which, at the molecular level, must begin with the binding of some ligand to a specific binding site on a protein molecule. Furthermore, as each one of these processes proceeds along its reaction pathway, modulation of the affinities of such ligands, positive and negative interactions caused by the binding of other ligands and the release of these ligands either in their original or in altered form constitute obligatory steps in the driving forces and controlling restraints that adapt them to their specific tasks. We extend the basic conceptions set forth in this article to a on more detailed level focused the fundamental nature of ligand–ligand interactions of a protein–ligand complex and the energetic mechanistic consequences to which they lead.

Key Concepts

  • Every biological reaction is initiated by a protein–ligand binding step.
  • Such reactions never involve the binding of only a single ligand or a single step.
  • The binding of two ligands to the same protein always involves a mutual interaction.
  • The product of a ligand‐binding reaction is a new entity in itself; its structure and its properties may differ substantially from the simple sum of those of its initial components.
  • The concept of G Weber's ‘thermodynamic square’ permits the evaluation of a variety of interaction parameters.
  • Patterns of interaction parameters provide a basis for exploring ligand‐binding energy transduction.
  • Measurement of ligand‐binding heat by techniques using higher levels of mathematical integration provides more detailed dissection into parameters such as enthalpy, entropy, heat capacity and their corresponding interaction terms.
  • The ‘fluctuating protein’ concept suggests that protein reactions proceed as multiple traces on multi‐dimensional free‐energy landscapes.
  • Consequences of the thermodynamic complexity of ligand binding suggest new views of such processes as enzymatic catalysis, signalling events and evolutionary aspects.

Keywords: proteins; ligands binding; thermodynamics; co‐operativity; coupling; free energy; enthalpy; entropy; heat capacity; enzymes

Figure 1. The pH dependence of single‐site binding. The pK is assumed to be 7, as indicated by the vertical arrow. The curve is calculated according to eqn (10).
Figure 10. The multiple reaction co‐ordinates of an enzyme reaction plotted on a surface whose vertical axis is ΔG° of the protein component of the reaction with the various conformations available to the protein on one horizontal axis and the successive reaction intermediates on the other. (This portrayal of the energetics of an enzyme reaction is in contrast to the conventional two‐dimensional forms shown in Figure .) The red lines trace the conformational and kinetic interconversions accessible to the enzyme in its various ligand‐bound forms. I indicates a dead‐end inhibitor complex and T a ‘tunnelling’ step. An important feature of the landscape is that the barrier heights between adjacent conformation change with each reaction step as the ligands (substrates and products) undergo chemical changes. Reproduced from Swint‐Kruse L and Fisher HF © Elsevier.
Figure 11. A mechanism of the multiple pathways of the fumarase reaction featuring the details of the ‘iso’ step. The subscripts distinguish the malate binding (m) conformation of the enzyme from that of the fumarate (f) and the malate‐or‐fumarate (mf) binding forms. The superscripts indicate the state of protonation or hydration of the enzyme. This representation of the fumarase mechanism presents a ‘flat’ version of Figure , as the heights of various energy barriers and wells for each intermediate are not yet known. Reproduced with permission from Rose © American Chemical Society.
Figure 12. Thermodynamic parameters for three successive sites of the NADPH and the l‐glutamate‐NADPH complexes of bovine liver glutamate dehydrogenase calculated using the multi‐site interactive programme of Wiseman et al. (). Reproduced with permission from Fisher 2012 © Springer Science+Business Media, LLC.
Figure 2. The logarithmic form of the pH dependence of single‐site binding. The pK is indicated by the intersection of the extrapolations of the two linear portions of the curve.
Figure 3. The pH dependence of binding involving two sites, where the group with the lower pK must be unprotonated and the group with the higher pK must be protonated. f, the fraction of the protein that exists in the specific monoprotonated form required for ligand binding is plotted as a function of pH calculated by the equation: f = 1/(1 + K1/[H+] + H+/K2) using K 1 = 10− 6 and K 2 = 10− 8. The dashed vertical lines indicate the two pK values.
Figure 4. The pH dependence of binding involving two sites, where the group with the lower pK must be protonated and the group with the higher pK must be unprotonated. The dotted lines indicate the behaviour of a system in which each of the two functional groups could bind a ligand independently of the other.
Figure 5. The dependence of the binding constant on the absolute temperature, T, plotted according to the van't Hoff equation.
Figure 6. The temperature dependence of the observed ΔH for a coupled, hidden two‐state system: E Δ H 0 E Δ H 1 E L . It is assumed that [L] is presented in saturating conditions. Reproduced from Fisher, ©John Wiley & Sons.
Figure 7. The temperature dependence of the apparent ΔC p, generated by the system portrayed in Figure . Curves are shown for four different ΔH 0 ° values.
Figure 8. Interaction parameters for A, the enzyme–NADPH–l‐glutamate complex (a positive interaction); and B, for the enzyme–NADPH–ADP complex (a negative interaction of bovine liver glutamate dehydrogenase). Reproduced from Fisher © John Wiley & Sons.
Figure 9. The Lumry ‘free energy complementarity’ concept. (a) An example where no alteration in the activation frets energy of the substrate process occurs. (b) An example where free energy changes in the protein itself are partially complementary to those of the substrate system. The dashed line represents the level of ligand‐binding energy store in the protein molecule itself. Reproduced from Swint‐Kruse L and Fisher HF © Elsevier.


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Fisher, Harvey F(Mar 2015) Protein–Ligand Interactions: Thermodynamic Basis and Mechanistic Consequences. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0001341.pub3]