Development of a method for structural characterization of DNA-binding proteins with multiple structural domains

为了建立具有多个结构域的dna结合蛋白的结构表征方法，对转录因子HSF3的一个dna结合域及其靶蛋白的复合体进行了化学交联和质谱分析。在对非共价蛋白-蛋白复合物的分子离子进行观察后，用BS^3（双（磺基琥珀酰亚基）亚硫酸酯）对Lys侧链的两个氨基进行交联。通过SDS-PAGE分离交联蛋白复合物，还原烷基化，并在凝胶中用胰蛋白酶消化。利用MALDI-TOFMS对该酶解物进行了分析，并对序列进行了分子质量分配，从而确定了连接的Lys残基。随着进一步的改进，该方法有望用于具有多结构域的蛋白质的表征，其三级结构难以用x射线晶体学或核磁共振来解决。对具有多个结构域的转录因子HSF3和TFIIE进行了更多的全长表达和纯化。由于HSF3在初始系统中的表达水平较低，因此对几种质粒和宿主大肠杆菌进行了检测。TFIIE是两种TFIIEα和β的异四聚体，在表达水平（TFIIEα）和纯化过程中的稳定性（TFIIEβ）方面存在困难。在这些蛋白的表达和纯化系统建立后，将上述方法应用于这些蛋白。用氢-氘交换和傅里叶变换离子回旋共振质谱法研究了肽与磷脂相互作用后的结构变化。蜂毒蛋白是一种从蜜蜂中分离出来的溶血肽，在不含脂质的情况下，蜂毒蛋白的结构是随机的，而在有磷脂的情况下，蜂毒蛋白的结构是稳定的。根据磷脂种类的不同，可以识别出不同的行为。该方法可用于膜结合蛋白的结构表征。少

In order to develop a method for structural characterization of DNA-binding proteins with multiple structural domains, chemical cross-linking and mass spectrometry analyses were carried out on a complex of a DNA-binding domain of transcription factor HSF3 and its target protein. After the observation of the molecular ion of the non-covalent protein-protein complex, cross-linking of two amino groups of Lys side chains were performed with BS^3 (bis(sulfosuccinimidyl) suberate). The cross-linked protein complex was isolated by SDS-PAGE, reduced and alkylated, and digested with trypsin in the gel. The digest was analyzed by MALDI-TOFMS and assignment of molecular masses to the sequence was carried out, resulting in the identification of linked Lys residues. With further improvement, this method is promising for the characterization of a protein with multiple structural domains, whose tertiary structure is difficult to be solved with X-ray crystallography or NMR.Expression and purification … More of the full-length HSF3 and TFIIE, transcription factors with multiple structural domains, was carried out. Since the expression level of HSF3 was low with the initial system, several kinds of plasmid and host E. coli were examined. In the case of TFIIE, heterotetramer of two TFIIEα and β, there were difficulties in the expression level (TFIIEα) and in the stability during the purification process (TFIIEβ). The method mentioned above will be applied to these proteins after the establishment of their expression and purification systems.Structural changes on a peptide by the interaction with phospholipids were investigated with hydrogen-deuterium exchange and Fourier-transform ion cyclotron resonance mass spectrometry. Melittin, a hemolytic peptide isolated from honeybee, was in quite random structure in the absence of lipids while it was in some stable structure in the presence of phospholipids. Different behavior was recognized according to the kinds of phospholipids. This method might be applicable to characterize the structure of membrane-bound proteins. Less