Characterization of the nucleic acid binding region of the intermediate filament protein vimentin by fluorescence polarization

Employing deletion mutant proteins and fluorescein-labeled oligodeoxyribonu cleotides in a fluorescence polarization assay, the nucleic acid binding si te of the intermediate filament (IF) subunit protein vimentin was localized to the middle of the arginine-rich, non-alpha-helical, N-terminal head dom ain. While deletion of the first few N-terminal residues (up to amino acid 17) had almost no effect, deletions of residues 25-64 or 25-68 essentially abolished the binding of nucleic acids by the respective proteins. Proteins with smaller deletions, of residues 25-39 or 43-68, were still able to bin d nucleic acids quite well at low ionic strength, but only the proteins con taining the first DNA-binding wing (residues 27-39) retained the ability to stably bind nucleic acids at physiological ionic strength. These results w ere confirmed by data obtained with two synthetic peptides whose sequences correspond to the smaller deletions. Nitration experiments showed that one or more of the tyrosines in the head domain are responsible for the stable binding by intercalation. interestingly, the residues responsible for bindi ng nucleic acids can be deleted without major influence on the in vivo poly merization properties of the mutant proteins. Only the protein with the lar gest internal deletion, of residues 25-68, failed to form filaments in vivo . Since the N-terminal head domains of IF proteins are largely exposed on t he filament surface, but nevertheless essential for filament assembly, thes e results support the model that the middle of the head domain of vimentin may loop out from the filament surface and thus be available for interactio ns with other cellular structures or molecules.