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.
Frare
Erica
E
CRIBI Biotechnology Centre and Department of Biological Chemistry, University of Padua, Viale G. Colombo 3, 35121 Padua, Italy.
de Laureto
Patrizia Polverino
PP
Scaramella
Elena
E
Tonello
Fiorella
F
Marin
Oriano
O
Deana
Renzo
R
Fontana
Angelo
A
D000409:Alanine / Q000737:chemistry*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001792:Blood Platelets / Q000187:drug effects; D015815:Cell Adhesion Molecules; D002942:Circular Dichroism; D004355:Drug Stability; D006358:Hot Temperature; D006801:Humans; D020128:Inhibitory Concentration 50; D010449:Peptide Mapping; D010975:Platelet Aggregation Inhibitors / Q000138:chemical synthesis*; D011392:Proline / Q000737:chemistry*; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D011506:Proteins / Q000138:chemical synthesis*; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D015815:Cell Adhesion Molecules; D010975:Platelet Aggregation Inhibitors; D011506:Proteins; C064478:decorsin; D011392:Proline; D000409:Alanine