Winston R L, Millar D P, Gottesfeld J M, Kent S B
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Biochemistry. 1999 Apr 20;38(16):5138-46. doi: 10.1021/bi982856a.
The basic helix-loop-helix domain of the Drosophila transcription factor Deadpan (Dpn) was prepared by total chemical protein synthesis in order to characterize its DNA binding properties. Circular dichroism spectroscopy was used to correlate structural changes in Dpn with physiologically relevant monovalent (KCl) and divalent (MgCl2) cation concentrations. In addition, we have used electrophoretic mobility shift assay (EMSA) and fluorescence anisotropy methods to determine equilibrium dissociation constants for the interaction of Dpn with two biologically relevant promoters involved in neural development and sex determination pathways. In this study, we have optimized DNA binding conditions for Dpn, and we have found a markedly higher DNA binding affinity for Dpn than reported for other bHLH domain transcription factors. Dpn binds as a homodimer (Kd = 2.6 nM) to double-stranded oligonucleotides containing the binding site CACGCG. In addition, we found that Dpn bound with the same affinity to a single or tandem binding site, indicating no cooperativity between adjacent DNA-bound Dpn dimers. DNA binding was also monitored as a function of physiologically relevant KCl and MgCl2 concentrations, and we found that this activity was significantly different in the presence and absence of the nonspecific competitor poly(dI-dC). Moreover, Dpn displayed moderate sequence selectivity, exhibiting a 100-fold higher binding affinity for specific DNA than for poly(dI-dC). This study constitutes the first detailed biophysical characterization of the DNA binding properties of a bHLH protein.
为了表征果蝇转录因子Deadpan（Dpn）的DNA结合特性，通过全化学蛋白质合成制备了其基本螺旋-环-螺旋结构域。利用圆二色光谱法将Dpn的结构变化与生理相关的单价（KCl）和二价（MgCl2）阳离子浓度相关联。此外，我们使用电泳迁移率变动分析（EMSA）和荧光各向异性方法来确定Dpn与参与神经发育和性别决定途径的两个生物学相关启动子相互作用的平衡解离常数。在本研究中，我们优化了Dpn的DNA结合条件，并且发现Dpn的DNA结合亲和力明显高于其他bHLH结构域转录因子的报道值。Dpn以同二聚体形式（Kd = 2.6 nM）结合到含有结合位点CACGCG的双链寡核苷酸上。此外，我们发现Dpn对单个或串联结合位点的结合亲和力相同，这表明相邻的DNA结合Dpn二聚体之间没有协同作用。还监测了DNA结合作为生理相关KCl和MgCl2浓度的函数，并且我们发现在存在和不存在非特异性竞争者聚（dI-dC）的情况下这种活性存在显著差异。此外，Dpn表现出适度的序列选择性，对特定DNA的结合亲和力比对聚（dI-dC）高100倍。这项研究构成了对bHLH蛋白DNA结合特性的首次详细生物物理表征。
