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首页> 外文期刊>Biochemistry >Characterization of the nucleic acid binding region of the intermediate filament protein vimentin by fluorescence polarization.
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Characterization of the nucleic acid binding region of the intermediate filament protein vimentin by fluorescence polarization.

机译：通过荧光偏振表征中间丝蛋白波形蛋白的核酸结合区。

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摘要

Employing deletion mutant proteins and fluorescein-labeled oligodeoxyribonucleotides in a fluorescence polarization assay, the nucleic acid binding site of the intermediate filament (IF) subunit protein vimentin was localized to the middle of the arginine-rich, non-alpha-helical, N-terminal head domain. 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 bind nucleic acids quite well at low ionic strength, but only the proteins containing the first DNA-binding wing (residues 27-39) retained the ability to stably bind nucleic acids at physiological ionic strength. These results were 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 binding nucleic acids can be deleted without major influence on the in vivo polymerization properties of the mutant proteins. Only the protein with the largest 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 the filament surface, but nevertheless essential for filament assembly, these 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 interactions with other cellular structures or molecules.
机译：在荧光偏振测定中采用缺失突变蛋白和荧光素标记的寡脱氧核糖核苷酸，中间丝（IF）亚基蛋白波形蛋白的核酸结合位点位于富含精氨酸的非α螺旋N末端的中间头域。尽管最初的几个N-末端残基的缺失（直至氨基酸17）几乎没有作用，但是残基25-64或25-68的缺失基本上消除了各个蛋白质对核酸的结合。残基25-39或43-68缺失较小的蛋白质在低离子强度下仍能很好地结合核酸，但只有含有第一个DNA结合翼的蛋白质（残基27-39）保留了结合能力。以生理离子强度稳定结合核酸。这些结果由两个序列对应于较小缺失的合成肽获得的数据证实。硝化实验表明，头部结构域中的一种或多种酪氨酸可通过嵌入稳定结合。有趣的是，可以删除负责结合核酸的残基，而对突变蛋白的体内聚合特性没有重大影响。只有具有最大内部缺失的蛋白（第25-68位残基）无法在体内形成细丝。由于IF蛋白的N末端头部结构域在细丝表面上大量暴露，但是对于细丝组装而言仍然是必不可少的，这些结果支持了波形蛋白头部区域的中部可能从细丝表面环出的模型，因此可以使用与其他细胞结构或分子的相互作用。

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