Identification of the amino acid residues of the amino terminus of vimentin responsible for DNA binding by enzymatic and chemical sequencing and analysis by MALDI-TOF

The amino acid residues responsible for stable binding of nucleic acids by the intermediate filament (IF) subunit protein vimentin were identified by a combination of enyzmatic and chemical ladder sequencing of photo-cross-li nked vimentin-oligodeoxyribonucleotide complexes and analysis by MALDI-TOF mass spectrometry. Three tryptic peptides of vimentin (vim(28-35), vim(36-4 9), and vim(50-63)) were found to be cross-linked to oligo(dG.BrdU)(12).dG. 3'-FITC. From a methodological standpoint, it was necessary to remove the b ulk of the bound oligonucleotide by digestion with nuclease P1 to get repro ducible spectra for most of the peptides studied. Additionally, removal of the phosphate group of the residually bound dUMP or modification of the ami no terminus of the peptide-oligonucleotide complexes with dimethylaminoazob enzene isothiocyanate dramatically improved the quality of the MALDI-TOF sp ectra obtained, particularly for the Vim(28-35) peptide. A single Tyr resid ue within each of these peptides (Tyr(29), Tyr(37), and Tyr(52)) was unequi vocally demonstrated to be the unique site of cross-linking in each peptide . These three Tyr residues are contained within the two beta-ladder DNA-bin ding wings proposed for the middle of the vimentin non-alpha-helical head d omain. The experimental approach described should be generally applicable t o the study of protein-nucleic acid interactions and is currently being emp loyed to characterize the DNA-binding sites of several other IF subunit pro teins.