Interaction of RGG and HTH motifs with nucleic acids: a study with rationally designed synthetic and recombinant polypeptides

The aim of this work is to provide data on the weak interactions between proteins and nucleic acids, using a sequence-specific DNA-binding protein (the recombinant 1-69 N-terminal domain of the phage 434 cl repressor, R69), and a non-specific nucleic acid binding domain (the arginine rich RGG domain of human nucleolin produced by solid phase synthesis) as in vitro model systems. Pyrene fluorescence spectroscopy and disulfide crosslinking via C-terminally attached Cys residues showed that cognate DNA acts as template for R69 dimerization even in the absence of the native C-terminal dimerization domain. Dimeric binding of R69 to DNA results in intensive pyrene excimer fluorescence resulting from the stacking of two C-terminally attached pyrene labels. The'fluorescence of the pyrene monomer (383nm) is quenched in a salt/sensitive manner by both cognate and non-cognate DNA molecules, probably due to electrostatic interactions with the DNA backbone. DNA sequences as short as the half-site are sufficient to promote dimer formation in a detectable manner. On the other hand, studies with artificial hybrid operators prove that two half sites provide complete specificity to the complex assembly. These studies suggest that sequential binding of monomers is a plausible pathway for the assembly of R69 on DNA. The RGG motif is present in many specific and non-specific nucleic acid binding proteins and its arginine residues undergo methylation by protein arginine methyltransferase type I as a part of various, not entirely known regulatory events. We used novel methods of solid phase peptide synthesis to produce methylated and non-methylated peptides spanning sequences from residues 646-706 of the human nucleolin C-terminal domain and we used them to study nucleic acid binding in vitro. Melting curve assays, double filter binding assays and circular dichroism studies showed that the non-sequence specific nucleic acid binding is sensitive to the geometry of the nucleic acid as it binds ssDNA>RNA>dsDNA. While binding strength is not substantially influenced by dimethylation, the ability of the peptides to modify the nuclecic acid structure, detected by CD spectroscopy, is substantially decreased upon methylation