Hosur R V, Radha P K, Madan A, Padhy L C
Tata Institute of Fundamental Research, Mumbai, India.
Biophys Chem. 1997 Oct;68(1-3):147-59. doi: 10.1016/s0301-4622(97)00026-4.
The oncogene product c-myb is a transcriptional modulator and is known to play important roles in cell growth and differentiation. It binds to DNA in a sequence specific manner and its cognate sequence motifs have been detected in the genes of proteins implying its role in a variety of regulatory functions. The protein has a DNA binding domain consisting of three imperfect repeats with highly conserved tryptophans at regular spacings in each of the repeats. We have carried out a variety of investigations on the structure and interactions of the DNA binding domain of Drosophila c-myb and its cognate DNA target sequences. The domain has been bacterially over-expressed by subcloning a segment of the gene coding for the domain in a pET 11d vector and transforming it into E. coli BL21 (DE3). Circular dichroism of the protein has revealed that the domain is largely helical in nature. Fluorescence investigations indicated that three out of the nine tryptophans are solvent exposed and the others are buried in the interior. The recombinant protein is able to distinguish between specific and non-specific DNA targets in its binding and the interaction is largely electrostatic in nature in both cases. Dynamic fluorescence quenching experiments suggested that the DNA binding sites on the protein for specific and non-specific DNA targets are physically different. Most of the conserved tryptophans are associated with the specific DNA binding site. Simulated annealing and molecular dynamic simulations in a water matrix have been used to predict an energetically favoured conformation for the protein. Calculation of surface accessibilities of the individual residues shows that nearly 60% of the residues are less than 50% accessible to the solvent. Two and three dimensional NMR experiments with isotopically labelled protein have enabled spin system identification for many residue type and the types of residues involved in hydrophobic core formation in the protein. In an attempt to see the DNA surface possibly involved in specific interaction with the protein, a three-dimensional structure of a 12 mer cognate DNA has been determined by NMR in conjunction with restrained energy minimization. The recognition sequence shows interesting structural characteristics that may have important roles in specific interaction.
癌基因产物c-myb是一种转录调节因子，已知在细胞生长和分化中发挥重要作用。它以序列特异性方式与DNA结合，并且在蛋白质基因中检测到其同源序列基序，这暗示了它在多种调节功能中的作用。该蛋白质具有一个DNA结合结构域，由三个不完美的重复序列组成，每个重复序列中都有规则间隔的高度保守的色氨酸。我们对果蝇c-myb的DNA结合结构域及其同源DNA靶序列的结构和相互作用进行了各种研究。通过将编码该结构域的基因片段亚克隆到pET 11d载体中并将其转化到大肠杆菌BL21(DE3)中，该结构域已在细菌中过量表达。蛋白质的圆二色性表明该结构域在本质上主要是螺旋状的。荧光研究表明，九个色氨酸中有三个暴露于溶剂中，其他则埋藏在内部。重组蛋白在其结合过程中能够区分特异性和非特异性DNA靶标，并且在这两种情况下相互作用在很大程度上都是静电性质的。动态荧光猝灭实验表明，蛋白质上特异性和非特异性DNA靶标的DNA结合位点在物理上是不同的。大多数保守的色氨酸与特异性DNA结合位点相关。在水基质中的模拟退火和分子动力学模拟已被用于预测该蛋白质能量上有利的构象。单个残基的表面可及性计算表明，近60%的残基对溶剂的可及性小于50%。用同位素标记的蛋白质进行的二维和三维NMR实验能够识别许多残基类型的自旋系统以及蛋白质中参与疏水核心形成的残基类型。为了观察可能与蛋白质发生特异性相互作用的DNA表面，通过NMR结合受限能量最小化确定了一个12聚体同源DNA的三维结构。识别序列显示出有趣的结构特征，可能在特异性相互作用中起重要作用。
