Iakoucheva L M, Kimzey A L, Masselon C D, Smith R D, Dunker A K, Ackerman E J
Pacific Northwest National Laboratory (PNNL), Molecular Biosciences Department, Richland, Washington 99352, USA.
Protein Sci. 2001 Jul;10(7):1353-62. doi: 10.1110/ps.ps.40101.
The DNA repair protein XPA recognizes a wide variety of bulky lesions and interacts with several other proteins during nucleotide excision repair. We recently identified regions of intrinsic order and disorder in full length Xenopus XPA (xXPA) protein using an experimental approach that combined time-resolved trypsin proteolysis and electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry (MS). MS data were consistent with the interpretation that xXPA contains no post-translational modifications. Here we characterize the discrepancy between the calculated molecular weight (31 kDa) for xXPA and its apparent molecular weight on SDS-PAGE (multiple bands from approximately 40-45 kDa) and gel filtration chromatography ( approximately 92 kDa), as well as the consequences of DNA binding on its anomalous mobility. Iodoacetamide treatment of xXPA prior to SDS-PAGE yielded a single 42-kDa band, showing that covalent modification of Cys did not correct aberrant mobility. Determination of sulfhydryl content in xXPA with Ellman's reagent revealed that all nine Cys in active protein are reduced. Unexpectedly, structural constraints induced by intramolecular glutaraldehyde crosslinks in xXPA produced a approximately 32-kDa monomer in closer agreement with its calculated molecular weight. To investigate whether binding to DNA alters xXPA's anomalous migration, we used gel filtration chromatography. For the first time, we purified stable complexes of xXPA and DNA +/- cisplatin +/- mismatches. xXPA showed at least 10-fold higher affinity for cisplatin DNA +/- mismatches compared to undamaged DNA +/- mismatches. In all cases, DNA binding did not correct xXPA's anomalous migration. To test predictions that a Glu-rich region (EEEEAEE) and/or disordered N- and C-terminal domains were responsible for xXPA's aberrant mobility, the molecular weights of partial proteolytic fragments from approximately 5 to 25 kDa separated by reverse-phase HPLC and precisely determined by ESI-FTICR MS were correlated with their migration on SDS-PAGE. Every partial tryptic fragment analyzed within this size range exhibited 10%-50% larger molecular weights than expected. Thus, both the disordered domains and the Glu-rich region in xXPA are primarily responsible for the aberrant mobility phenomena.
DNA修复蛋白XPA可识别多种大体积损伤，并在核苷酸切除修复过程中与其他几种蛋白相互作用。我们最近采用了一种结合时间分辨胰蛋白酶蛋白水解和电喷雾电离接口与傅里叶变换离子回旋共振（ESI-FTICR）质谱（MS）的实验方法，确定了全长非洲爪蟾XPA（xXPA）蛋白中的内在有序和无序区域。MS数据与xXPA不包含翻译后修饰的解释一致。在此，我们描述了xXPA的计算分子量（31 kDa）与其在SDS-PAGE上的表观分子量（约40-45 kDa的多条带）和凝胶过滤色谱法（约92 kDa）之间的差异，以及DNA结合对其异常迁移率的影响。在SDS-PAGE之前用碘乙酰胺处理xXPA产生了一条单一的42-kDa条带，表明半胱氨酸的共价修饰并未纠正异常迁移率。用埃尔曼试剂测定xXPA中的巯基含量表明，活性蛋白中的所有九个半胱氨酸都是还原态的。出乎意料的是，xXPA中分子内戊二醛交联诱导的结构限制产生了一个约32-kDa的单体，与其计算分子量更接近。为了研究与DNA的结合是否会改变xXPA的异常迁移，我们使用了凝胶过滤色谱法。我们首次纯化了xXPA与DNA+/-顺铂+/-错配的稳定复合物。与未损伤的DNA+/-错配相比，xXPA对顺铂DNA+/-错配的亲和力至少高10倍。在所有情况下，DNA结合都没有纠正xXPA的异常迁移。为了检验富含谷氨酸的区域（EEEEAEE）和/或无序的N端和C端结构域导致xXPA异常迁移率的预测，通过反相HPLC分离并由ESI-FTICR MS精确测定的约5至25 kDa的部分蛋白水解片段的分子量与其在SDS-PAGE上的迁移率相关。在这个大小范围内分析的每个部分胰蛋白酶片段的分子量都比预期大10%-50%。因此，xXPA中的无序结构域和富含谷氨酸的区域主要是异常迁移现象的原因。
