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Synthesis and characterization of the Nω ; , Nω ; -dimethylarginine rich C-terminal region of human nucleolin

Both versions of C-terminal region of human nucleolin ChNu (646-706), containing Arg (I), respectively Nω ; , Nω ; -dimethylarginine (II), from the family of RGG-rich proteins, were successfully synthesized by an automated Fmoc methodology for SPPS using dimethoxybenzyl as a reversible amide bond protecting group. The cleavage/deprotection conditions were optimized and ChNu's were purified to homogeneity >95% by a combination of IE- and RP-HPLC. The purity/identity of peptides were confirmed by ESI-MS, AAA, SDS-PAGE, RP HPLC in two different systems and by IE HPLC. Arg-dimethylation increases hydrophobicity, as determined by RP-HPLC, and decreases basicity (IE-HPLC). In double filter binding assays (I) and (II) show very similar binding potency to single-stranded DNA (ssDNA) with values comparable to that of recombinant fusion ChNu (Kd ~ 2X10-7 M). The nucleic acid binding affinity is substantially increased in comparison to our published result on short peptides and correlates with the higher number of RGG, respectively aDma-GG-repeats. In a ssDNA-agarose affinity chromatography experiment Arg-dimethylation decreased the electrostatic interaction with ssDNA. Both (I) and (II) show very similar binding potency to G4 DNA and do not exhibit helicase activity in a M13 phage assay. (I) is efficiently, but not exhaustively methylated in vitro by recombinant protein arginine N-methyltransferase type I. 1H NMR spectra of (I) and (II) are characterized by the lack of dispersion for NH and Hα ; ; chemical shifts suggesting the absence of globular structure.

peptide synthesis; nucleolin; dimethylation

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