title: Efficient synthesis and comparative studies of the arginine and Nomega,Nomega-dimethylarginine forms of the human nucleolin glycine/arginine rich domain.
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 ( approximately 26%) and purity. As detected by NMR spectroscopy, N61Arg does not possess any stable secondary or tertiary structure in solution and N(omega),N(omega)-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 (K(d) = 1.5 x 10(-7) M), they exhibit a different behaviour in ssDNA affinity chromatography consistent with the difference in pK(a) 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.
authors.affiliation: Protein Structure and Bioinformatics Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Area Science Park, Padriciano 99, 34012 Trieste, Italy. sotir@icgeb.org
authors.forename: Sotir
authors.initials: S
authors.lastname: Zahariev
authors.forename: Corrado
authors.initials: C
authors.lastname: Guarnaccia
authors.forename: Francesco
authors.initials: F
authors.lastname: Zanuttin
authors.forename: Alessandro
authors.initials: A
authors.lastname: Pintar
authors.forename: Gennaro
authors.initials: G
authors.lastname: Esposito
authors.forename: Gordana
authors.initials: G
authors.lastname: Maravić
authors.forename: Bernard
authors.initials: B
authors.lastname: Krust
authors.forename: Ara G
authors.initials: AG
authors.lastname: Hovanessian
authors.forename: Sándor
authors.initials: S
authors.lastname: Pongor
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D044966:Anti-Retroviral Agents / Q000494:pharmacology; D001120:Arginine / Q000031:analogs & derivatives* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004265:DNA Helicases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D006678:HIV / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D010750:Phosphoproteins / Q000138:chemical synthesis / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D000097796:Nucleolin
chemical_list: D000251:Adenosine Triphosphatases; D044966:Anti-Retroviral Agents; D001120:Arginine; D004265:DNA Helicases; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D000097796:Nucleolin; C487735:dimethylarginine; C059716:helD protein, E coli