Wang Q, Tolstonog G V, Shoeman R, Traub P
Max-Planck-Institut für Zellbiologie, Rosenhof, 68526 Ladenburg, Germany.
Biochemistry. 2001 Aug 28;40(34):10342-9. doi: 10.1021/bi0108305.
A combination of enzymatic and chemical ladder sequencing of photo-cross-linked protein-single-stranded oligodeoxyribonucleotide complexes and analysis by MALDI-TOF mass spectrometry was employed to identify the amino acid residues responsible for the stable binding of nucleic acids in several intermediate filament (IF) subunit proteins. The IF proteins studied included the type I and type II cytokeratins K8, K18, and K19; the type III proteins desmin, glial fibrillary acidic protein (GFAP), peripherin, and vimentin; and the type IV neurofilament triplet protein L (NF-L). The site of nucleic acid binding was localized to the non-alpha-helical, amino-terminal head domain of all of the IF proteins tested. GFAP, which has the shortest head domain of the proteins tested, cross-linked via only two amino acid residues. One of these residues was located within a conserved nonapeptide domain that has been shown to be required for filament formation. One or more cross-linked residues were found in a similar location in the other proteins studied. The major binding site for nucleic acids for most of the proteins appears to be localized within the middle of the head domain. The two exceptions to this generalization are GFAP, which lacks these residues, and NF-L, in which a large number of cross-linked residues were found scattered throughout the first half of the head domain. Control experiments were also done with two bacteriophage ssDNA-binding proteins, as well as actin and tubulin. The single sites of cross-linkage observed with the bacteriophage proteins, Phe(183) for the T4 gene 32 protein and Phe(73) for the M13 gene 5 protein, were in good agreement with literature data. Actin and tubulin could not be cross-linked to the oligonucleotide. Aside from the insight into the biological activity of IF proteins that these data provide, they also demonstrate that this analytical method can be employed to study a variety of protein-nucleic acid interactions.
采用酶促和化学阶梯测序相结合的方法，对光交联的蛋白质 - 单链寡脱氧核糖核苷酸复合物进行测序，并通过基质辅助激光解吸电离飞行时间质谱（MALDI - TOF质谱）分析，以鉴定几种中间丝（IF）亚基蛋白中负责核酸稳定结合的氨基酸残基。所研究的IF蛋白包括I型和II型细胞角蛋白K8、K18和K19；III型蛋白结蛋白、胶质纤维酸性蛋白（GFAP）、外周蛋白和波形蛋白；以及IV型神经丝三联体蛋白L（NF - L）。核酸结合位点定位于所有测试的IF蛋白的非α螺旋氨基末端头部结构域。GFAP是所测试蛋白中头部结构域最短的，仅通过两个氨基酸残基发生交联。其中一个残基位于一个保守的九肽结构域内，该结构域已被证明是丝形成所必需的。在其他所研究的蛋白中，在类似位置发现了一个或多个交联残基。大多数蛋白的核酸主要结合位点似乎位于头部结构域的中部。这个普遍规律的两个例外是缺乏这些残基的GFAP和在头部结构域前半部分发现大量交联残基散布的NF - L。还对两种噬菌体单链DNA结合蛋白以及肌动蛋白和微管蛋白进行了对照实验。在噬菌体蛋白中观察到的单交联位点，T4基因32蛋白的Phe(183)和M13基因5蛋白的Phe(73)，与文献数据高度一致。肌动蛋白和微管蛋白不能与寡核苷酸交联。这些数据不仅为IF蛋白的生物学活性提供了深入了解，还表明这种分析方法可用于研究多种蛋白质 - 核酸相互作用。
