Detecting "silent" allosteric coupling.

Using isothermal calorimetry (ITC), we have found one case where a well-characterized allosteric activator showed no sign of allostery in its ΔG° of binding to successive sites on multiple subunits and another case where successive binding showed no ΔG° binding allostery but did show large entropy-compensated flip-flopping enthalpy changes. This behavior, which we have termed "isoergonic cooperativity" and others have referred to as "silent coupling" is quite simply explained by basic linkage theory when reactions are considered beyond the ΔG° level. Thus, direct calorimetric determination of all thermodynamic parameters including ΔH°, ΔS°, ΔG°, ΔC (p)°, and d(ΔC (p)°/dt) provides a more informative depiction of a ligand binding event and its consequences than does the mere measurement of ΔG° alone. We further discuss the benefits and limitations of methods that have previously been used to study silent coupling. In particular, ITC is free of the numerous pitfalls inherent in the application of van't Hoff and Arrhenius plots to allosteric phenomena. Aside from having a 30-fold advantage in precision, ITC is capable of measuring changes in enthalpy directly at five more levels of mathematical differentiation than are available to van't Hoff type approaches.