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


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 degrees C) of native decorsin. The mono-substituted analogs display a Tm of 66-67 degrees C, while the double-substituted analog a Tm of 50 degrees 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. Study holds ProTherm entries: 19715, 19716, 19717, 19718 Extra Details: circular dichroism; decorsin; peptide synthesis; RGD-protein; thermal stability

Submission Details

ID: 5pqTEmTj3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:51 p.m.

Version: 1

Publication Details
Frare E;de Laureto PP;Scaramella E;Tonello F;Marin O;Deana R;Fontana A,Protein Eng. Des. Sel. (2005) Chemical synthesis of the RGD-protein decorsin: Pro-->Ala replacement reduces protein thermostability. PMID:16155118
Additional Information

Study Summary

Number of data points 7
Proteins Decorsin ; Decorsin
Unique complexes 4
Assays/Quantities/Protocols Experimental Assay: Tm ; Derived Quantity: dTm

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Decorsin P17350 DECO_MACDE