Selective deamidation of proteins and peptides is a reaction of great interest, whether it has physiological significance as in protein aging, or occurs as a disturbing event in the preparation of natural or recombinant proteins. Deamidation of bovine pancreatic ribonuclease A, RNase A, a classical model protein, has been reported to occur only after denaturation of the protein, or under harsh conditions. In this paper convenient procedures are described for selective deamidation of Asna7 in native RNase A under mild conditions. Furthermore, for the first time, both products of deamidation were isolated: the aspartyl and the isoaspartyl containing protein derivatives. Replacement of Asns7 with either residue lowers the catalytic activity of the enzyme, on RNA and on model substrates, except when a dinucleotide with a purine on the 5’ side is the substrate. In the latter case an intriguing increase in the specificity constant is observed. The Aspe7 derivative was found to refold, after full denaturation and reduction, at the same rate as the fully amidated protein, whereas the iso-Asp“ derivative refolded at half that rate. It is hypothesized that this effect is due to a delayed formation of disulfide 65-72 for the presence of the abnormal isopeptide bond between residues 67 and 68.

Selective Deamidation of Ribonuclease A / DI DONATO, Alberto; Ciardiello, M. A.; de Nigris, M.; Piccoli, Renata; Mazzarella, Lelio; D'Alessio, Giuseppe. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - STAMPA. - 268:(1993), pp. 4745-4751.

Selective Deamidation of Ribonuclease A

DI DONATO, ALBERTO;PICCOLI, RENATA;MAZZARELLA, LELIO;D'ALESSIO, GIUSEPPE
1993

Abstract

Selective deamidation of proteins and peptides is a reaction of great interest, whether it has physiological significance as in protein aging, or occurs as a disturbing event in the preparation of natural or recombinant proteins. Deamidation of bovine pancreatic ribonuclease A, RNase A, a classical model protein, has been reported to occur only after denaturation of the protein, or under harsh conditions. In this paper convenient procedures are described for selective deamidation of Asna7 in native RNase A under mild conditions. Furthermore, for the first time, both products of deamidation were isolated: the aspartyl and the isoaspartyl containing protein derivatives. Replacement of Asns7 with either residue lowers the catalytic activity of the enzyme, on RNA and on model substrates, except when a dinucleotide with a purine on the 5’ side is the substrate. In the latter case an intriguing increase in the specificity constant is observed. The Aspe7 derivative was found to refold, after full denaturation and reduction, at the same rate as the fully amidated protein, whereas the iso-Asp“ derivative refolded at half that rate. It is hypothesized that this effect is due to a delayed formation of disulfide 65-72 for the presence of the abnormal isopeptide bond between residues 67 and 68.
1993
Selective Deamidation of Ribonuclease A / DI DONATO, Alberto; Ciardiello, M. A.; de Nigris, M.; Piccoli, Renata; Mazzarella, Lelio; D'Alessio, Giuseppe. - In: THE JOURNAL OF BIOLOGICAL CHEMISTRY. - ISSN 0021-9258. - STAMPA. - 268:(1993), pp. 4745-4751.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/456207
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