Molecular Signatures of Enzyme−Solid Interactions: Thermodynamics of Protein Binding to α-Zr(IV) Phosphate Nanoplates

Abstract

Isothermal titration calorimetry (ITC) was used to determine the thermodynamics of protein binding to the nanoplates of alpha-Zr(HPO4)2.H2O (alpha-ZrP). The binding constants (K(b)) and DeltaG, DeltaH, and DeltaS have been evaluated for a small set of proteins, and K(b) values are in the range of 2-760 x 10(5) M(-1). The binding of positively charged proteins to the negatively charged alpha-ZrP was endothermic, while the binding of negatively charged proteins was exothermic, and these are contrary to expectations based on a simple electrostatic model. The binding enthalpies of the proteins varied over a range of -24 to +25 kcal/mol, and these correlated roughly with the net charge on the protein (R2 = 0.964) but not with other properties such as the number of basic residues, polar residues, isoelectric point, surface area, or molecular mass. Linear fits to the enthalpy plots indicated that each charge on the protein contributes 1.18 kcal/mol toward the binding enthalpy. Binding entropies of positively charged proteins were favorable (>0) while the binding entropies of negatively charged proteins were unfavorable (<0). The DeltaS values varied over a range of -51 to +98 cal/mol x K, and these correlated very well with the net charge on the protein (R2 = 0.999), but DeltaS is in the opposite direction of DeltaH. The binding or release of cations to/from the protein-solid interface can account for these observations. There was no correlation between the binding free energy (DeltaG(obs)) and any specific molecular properties, but it is likely to be a sum of several opposing interactions of large magnitudes. For the first time, the binding enthalpies and entropies are connected to specific molecular properties. The model suggests that the thermodynamic parameters can be controlled by choosing appropriate cations or by adjusting the net charge on the protein. We hope that physical insights such as these will be useful in understanding the complex behavior of proteins at biological interfaces.