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Biophys. J. BioFAST: First Published August 17, 2007. doi:10.1529/biophysj.107.113936
© 2007 by the Biophysical Society.

A more recent version of this article appeared on November 15, 2007.
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Rajamani Lakshminarayanan
Daming Fan
Chang Du
Janet Moradian-Oldak
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SUPRAMOLECULAR ASSEMBLIES

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

Rajamani Lakshminarayanan 1, Daming Fan 1, Chang Du 1 and Janet Moradian-Oldak 1*

1 University of Southern California

* To whom correspondence should be addressed. E-mail: joldak{at}usc.edu.

Submitted on May 31, 2007
Revised on July 12, 2007
Accepted on 27 July 2007


  Abstract
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. In order to gain a clearer insight into the mechanisms of amelogenin self-assembly we first investigated the occurrences of secondary structures within its sequence. By applying isothermal titration calorimety (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 (< 5µM) revealed the presence of the polyproline type II (PPII) conformation in mboth amelogenins, in addition to {alpha}-helix and unordered conformations. Structural transition from PPII/unordered to {beta}-sheet was observed for both proteins at higher concentrations (> 62.5 µM)and upon self-assembly. ITC measurements indicated that the self- assembly of rP172 and rP148 is entropically driven (+{Delta}SA) and energetically favorable (- {Delta}GA)). The magnitude of changes in enthalpy ({Delta}HA) and entropy of assembly ({Delta}SA) were smaller for rP148 than rP172, whereas the Gibbs free energy change of assembly ({Delta}GA) 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 slef-assembly and that rP172 assembly is more favorable than rP148. The present data are direct evidence for the notion that hydrophobic interactions are the main driving force for amelogenin self-assembly.

Key Words: PPII structure, amelogenin self-assembly, circular dichroism, enamel matrix, isothermal titration calorimetry, thermodynamics






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Copyright © 2007 by the Biophysical Society.