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-linked vimentin-oligodeoxyribonucleotide complexes and analysis by MALDI-TOF mass spectrometry. Three tryptic peptides of vimentin (vim(28)(-)(35), vim(36)(-)(49), 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 bulk of the bound oligonucleotide by digestion with nuclease P1 to get reproducible spectra for most of the peptides studied. Additionally, removal of the phosphate group of the residually bound dUMP or modification of the amino terminus of the peptide-oligonucleotide complexes with dimethylaminoazobenzene isothiocyanate dramatically improved the quality of the MALDI-TOF spectra obtained, particularly for the vim(28)(-)(35) peptide. A single Tyr residue within each of these peptides (Tyr(29), Tyr(37), and Tyr(52)) was unequivocally demonstrated to be the unique site of cross-linking in each peptide. These three Tyr residues are contained within the two beta-ladder DNA-binding wings proposed for the middle of the vimentin non-alpha-helical head domain. The experimental approach described should be generally applicable to the study of protein-nucleic acid interactions and is currently being employed to characterize the DNA-binding sites of several other IF subunit proteins.
Wang
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Q
Max-Planck-Institut für Zellbiologie, Rosenhof, 68526 Ladenburg, Germany.
Shoeman
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R
Traub
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P
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D007381:Intermediate Filament Proteins / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014746:Vimentin / Q000737:chemistry*
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D007381:Intermediate Filament Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D014746:Vimentin; D004247:DNA