Publication: Role of electrostatics and conformation in protein-crystal interactions

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Title Role of electrostatics and conformation in protein-crystal interactions
Authors/Editors* P.V. Azzopardi, J. O'Young, G. Lajoie, M. Karttunen, H.A. Goldberg and G.K. Hunter.
Where published* PLoS One
How published* Journal
Year* 2010
Volume 5
Number 2
Pages e9330
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 constantcomposition/seeded growth assay was used to assess the HA-inhibiting potencies of the synthetic peptides. The phosphorylated versions of OPN65-80 (IC50 = 1.93 mg/ml) and OPN220-235 (IC50 = 1.48 mg/ml) are potent inhibitors of HA growth, as is the nonphosphorylated version of OPN65-80 (IC50 = 2.97 mg/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.
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