Efficient synthesis and comparative studies of the arginine and Nω, Nω-dimethylarginine forms of the human nucleolin glycine/arginine rich domain

Sotir Zahariev, Corrado Guarnaccia, Francesco Zanuttin, Alessandro Pintar, Gennaro Esposito, Gordana Maravić, Bernard Krust, Ara G. Hovanessian, Sándor Pongor

Research output: Contribution to journalArticle

Abstract

The Gly- and Arg-rich C-terminal region of human nucleolin is a 61-residue long domain involved in a number of protein-protein and protein-nucleic acid interactions. This domain contains 10 aDma residues in the form of aDma-GG repeats interspersed with Phe residues. The exact role of Arg dimethylation is not known, partly because of the lack of efficient synthetic methods. This work describes an effective synthetic strategy, generally applicable to long RGG peptides, based on side-chain protected aDma and backbone protected dipeptide Fmoc-Gly-(Dmob)Gly-OH. This strategy allowed us to synthesize both the unmodified (N61Arg) and the dimethylated (N61aDma) peptides with high yield (∼26%) and purity. As detected by NMR spectroscopy, N61Arg does not possess any stable secondary or tertiary structure in solution and Nω,Nω-dimethylation of the guanidino group does not alter the overall conformational propensity of this peptide. While both peptides bind single-stranded nucleic acids with similar affinities (Kd = 1.5 × 10-7 M), they exhibit a different behaviour in ssDNA affinity chromatography consistent with the difference in pKa values. It has been previously shown that N61Arg inhibits HIV infection at the stage of HIV attachment to cells. This study demonstrates that Arg-dimethylated C-terminal domain lacks any inhibition activity, raising the question of whether nucleolin expressed on the cell-surface is indeed dimethylated.

Original languageEnglish (US)
Pages (from-to)17-28
Number of pages12
JournalJournal of Peptide Science
Volume11
Issue number1
DOIs
StatePublished - Jan 1 2005

Fingerprint

Nimodipine
Glycine
Arginine
Peptides
Nucleic Acids
Affinity chromatography
Proteins
Dipeptides
Affinity Chromatography
Nuclear magnetic resonance spectroscopy
HIV Infections
Magnetic Resonance Spectroscopy
HIV
nucleolin
N,N-dimethylarginine

Keywords

  • Backbone protection
  • Dimethylarginine
  • HIV
  • Nucleolin
  • RGG box

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry

Cite this

Efficient synthesis and comparative studies of the arginine and Nω, Nω-dimethylarginine forms of the human nucleolin glycine/arginine rich domain. / Zahariev, Sotir; Guarnaccia, Corrado; Zanuttin, Francesco; Pintar, Alessandro; Esposito, Gennaro; Maravić, Gordana; Krust, Bernard; Hovanessian, Ara G.; Pongor, Sándor.

In: Journal of Peptide Science, Vol. 11, No. 1, 01.01.2005, p. 17-28.

Research output: Contribution to journalArticle

Zahariev, Sotir ; Guarnaccia, Corrado ; Zanuttin, Francesco ; Pintar, Alessandro ; Esposito, Gennaro ; Maravić, Gordana ; Krust, Bernard ; Hovanessian, Ara G. ; Pongor, Sándor. / Efficient synthesis and comparative studies of the arginine and Nω, Nω-dimethylarginine forms of the human nucleolin glycine/arginine rich domain. In: Journal of Peptide Science. 2005 ; Vol. 11, No. 1. pp. 17-28.
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AU - Guarnaccia, Corrado

AU - Zanuttin, Francesco

AU - Pintar, Alessandro

AU - Esposito, Gennaro

AU - Maravić, Gordana

AU - Krust, Bernard

AU - Hovanessian, Ara G.

AU - Pongor, Sándor

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AB - The Gly- and Arg-rich C-terminal region of human nucleolin is a 61-residue long domain involved in a number of protein-protein and protein-nucleic acid interactions. This domain contains 10 aDma residues in the form of aDma-GG repeats interspersed with Phe residues. The exact role of Arg dimethylation is not known, partly because of the lack of efficient synthetic methods. This work describes an effective synthetic strategy, generally applicable to long RGG peptides, based on side-chain protected aDma and backbone protected dipeptide Fmoc-Gly-(Dmob)Gly-OH. This strategy allowed us to synthesize both the unmodified (N61Arg) and the dimethylated (N61aDma) peptides with high yield (∼26%) and purity. As detected by NMR spectroscopy, N61Arg does not possess any stable secondary or tertiary structure in solution and Nω,Nω-dimethylation of the guanidino group does not alter the overall conformational propensity of this peptide. While both peptides bind single-stranded nucleic acids with similar affinities (Kd = 1.5 × 10-7 M), they exhibit a different behaviour in ssDNA affinity chromatography consistent with the difference in pKa values. It has been previously shown that N61Arg inhibits HIV infection at the stage of HIV attachment to cells. This study demonstrates that Arg-dimethylated C-terminal domain lacks any inhibition activity, raising the question of whether nucleolin expressed on the cell-surface is indeed dimethylated.

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KW - Dimethylarginine

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KW - RGG box

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