title: Roles of electrostatics and conformation in protein-crystal interactions.
abstract: In vitro studies have shown that the phosphoprotein osteopontin (OPN) inhibits the nucleation and growth of hydroxyapatite (HA) and other biominerals. In vivo, OPN is believed to prevent the calcification of soft tissues. However, the nature of the interaction between OPN and HA is not understood. In the computational part of the present study, we used molecular dynamics simulations to predict the adsorption of 19 peptides, each 16 amino acids long and collectively covering the entire sequence of OPN, to the {100} face of HA. This analysis showed that there is an inverse relationship between predicted strength of adsorption and peptide isoelectric point (P<0.0001). Analysis of the OPN sequence by PONDR (Predictor of Naturally Disordered Regions) indicated that OPN sequences predicted to adsorb well to HA are highly disordered. In the experimental part of the study, we synthesized phosphorylated and non-phosphorylated peptides corresponding to OPN sequences 65-80 (pSHDHMDDDDDDDDDGD) and 220-235 (pSHEpSTEQSDAIDpSAEK). In agreement with the PONDR analysis, these were shown by circular dichroism spectroscopy to be largely disordered. A constant-composition/seeded growth assay was used to assess the HA-inhibiting potencies of the synthetic peptides. The phosphorylated versions of OPN65-80 (IC(50) = 1.93 microg/ml) and OPN220-235 (IC(50) = 1.48 microg/ml) are potent inhibitors of HA growth, as is the nonphosphorylated version of OPN65-80 (IC(50) = 2.97 microg/ml); the nonphosphorylated version of OPN220-235 has no measurable inhibitory activity. These findings suggest that the adsorption of acidic proteins to Ca2+-rich crystal faces of biominerals is governed by electrostatics and is facilitated by conformational flexibility of the polypeptide chain.
authors.affiliation: School of Dentistry and Department of Biochemistry, University of Western Ontario, London, Ontario, Canada.
authors.forename: Paul V
authors.initials: PV
authors.lastname: Azzopardi
authors.forename: Jason
authors.initials: J
authors.lastname: O'Young
authors.forename: Gilles
authors.initials: G
authors.lastname: Lajoie
authors.forename: Mikko
authors.initials: M
authors.lastname: Karttunen
authors.forename: Harvey A
authors.initials: HA
authors.lastname: Goldberg
authors.forename: Graeme K
authors.initials: GK
authors.lastname: Hunter
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003198:Computer Simulation; D003460:Crystallization; D017886:Durapatite / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D053495:Osteopontin / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D055672:Static Electricity
chemical_list: D010455:Peptides; D053495:Osteopontin; D017886:Durapatite