The role of secondary structure in the entropically driven amelogenin self-assembly

Amelogenin, the major extracellular enamel matrix protein, plays critical roles in controlling enamel mineralization. This generally hydrophobic protein self-assembles to form nanosphere structures under certain solution conditions. To gain clearer insight into the mechanisms of amelogenin self-assembly, we first investigated the occurrences of secondary structures within its sequence. By applying isothermal titration calorimetry (ITC), we determined the thermodynamic parameters associated with protein-protein interactions and with conformational changes during self-assembly. The recombinant porcine full length (rP172) and a truncated amelogenin lacking the hydrophilic C-terminal (rP148) were used. Circular dichroism (CD) measurements performed at low concentrations (< 5 mu M) revealed the presence of the polyproline-type II (PPII) conformation in both amelogenins in addition to a-helix and unordered conformations. Structural transition from PPII/unordered to beta-sheet was observed for both proteins at higher concentrations (> 62.5 mu M) and upon self-assembly. ITC measurements indicated that the self-assembly of rP172 and rP148 is entropically driven (+Delta S-A) and energetically favorable (-Delta G(A)). The magnitude of enthalpy (Delta H-A) and entropy changes of assembly (Delta S-A) were smaller for rP148 than rP172, whereas the Gibbs free energy change of assembly (Delta G(A)) was not significantly different. It was found that rP172 had higher PPII content than rP148, and the monomer-multimer equilibrium for rP172 was observed in a narrower protein concentration range when compared to rP148. The large positive enthalpy and entropy changes in both cases are attributed to the release of ordered water molecules and the associated entropy gain (due to the hydrophobic effect). These findings suggest that PPII conformation plays an important role in amelogenin self-assembly and that rP172 assembly is more favorable than rP148. The data are direct evidence for the notion that hydrophobic interactions are the main driving force for amelogenin self-assembly.