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PEDS Advance Access originally published online on September 9, 2005
Protein Engineering Design and Selection 2005 18(10):487-495; doi:10.1093/protein/gzi054
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Chemical synthesis of the RGD-protein decorsin: Pro->Ala replacement reduces protein thermostability

Erica Frare1, Patrizia Polverino de Laureto1, Elena Scaramella1, Fiorella Tonello1, Oriano Marin1, Renzo Deana2 and Angelo Fontana1,3

1CRIBI Biotechnology Centre and 2Department of Biological Chemistry, University of Padua, Viale G. Colombo 3, 35121 Padua, Italy

3 To whom correspondence should be addressed. E-mail: angelo.fontana{at}unipd.it

Decorsin is a 39-residue polypeptide chain, crosslinked by three disulfide bridges, that strongly inhibits platelet aggregation. We report the chemical synthesis and characterization of analogs of decorsin with the aim of investigating the role of proline residues in protein structure, stability and biological activity. Decorsin analogs have been synthesized in which one (P23A and P24A decorsin) or two (P23,24A decorsin) proline residues have been substituted by alanine. The crude synthetic polypeptides were purified by reversed-phase HPLC in their reduced form and allowed to refold oxidatively to their disulfide-crosslinked species. The homogeneity of the synthetic mini-proteins, and also the correct pairing of the three disulfide bridges, were established by a number of analytical criteria, including fingerprinting analysis of the refolded synthetic analogs by using thermolysin and proteinase K as proteolytic enzymes. Replacement of proline by alanine results in a significant and cumulative decrease of the high thermal stability (Tm 74°C) of native decorsin. The mono-substituted analogs display a Tm of 66–67°C, while the double-substituted analog a Tm of 50°C. On the other hand, the overall secondary and tertiary structures were not affected by the Pro->Ala exchanges, as judged from circular dichroism measurements. Platelet aggregation assays established that the proline substitutions do not impair significantly the biological activity of decorsin. The results of this study clearly indicate that proline residues contribute significantly to the protein thermal stability. Our results are in line with the ‘proline rule’, previously advanced for explaining the unusual thermal stability of thermophilic enzymes, which usually show an enhanced content of proline residues with respect to their mesophilic counterparts.

Keywords: circular dichroism/decorsin/peptide synthesis/RGD-protein/thermal stability

Received July 27, 2005; accepted July 28, 2005.

Edited by Lynne Regan and Andreas Plückthun


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