We studied the interaction between a synthetic peptide (sequence Ac-GXGGFGGXGGFXGGXGG-NH(2), where X = arginine, N(omega),N(omega)-dimethylarginine, DMA, or lysine) corresponding to residues 676-692 of human nucleolin and several DNA and RNA substrates using double filter binding, melting curve analysis and circular dichroism spectroscopy. We found that despite the reduced capability of DMA in forming hydrogen bonds, N(omega),N(omega)-dimethylation does not affect the strength of the binding to nucleic acids nor does it have any effect on stabilization of a double-stranded DNA substrate. However, circular dichroism studies show that unmethylated peptide can perturb the helical structure, especially in RNA, to a much larger extent than the DMA peptide.
Circular dichroism and electrophoretic mobility shift studies were performed to confirm that dimerized N‐terminal domains of bacterial repressors containing helix‐turn‐helix motifs are capable of high‐affinity and specific DNA recognition as opposed to the monomeric N‐terminal domains. Specific, high‐affinity DNA binding proteins were designed and produced in which two copies of the N‐terminal 1‐62 domain of the bacteriophage 434 repressor are connected either in a dyad‐symmetric fashion, with a synthetic linker attached to the C‐termini, or as direct sequence repeats. Both molecules bound to their presumptive cognate nearly as tightly as does the natural (full‐length and non‐covalently dimerized) 434 repressor, showing that covalent dimerization can be used to greatly enhance the binding activity of individual protein segments. Circular dichroism spectroscopy showed a pronounced increase in the alpha‐helix content when these new proteins interacted with their cognate DNA and a similar, although 30% lower, increase was also seen upon their interaction with non‐cognate DNA. These results imply that a gradual conformational change may occur when helix‐turn‐helix motifs bind to DNA, and that a scanning mechanism is just as plausible for this motif class as that which is proposed for the more flexible basic‐leucine zipper and basic‐helix‐loop‐helix motifs.
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