Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche (Alicante), Spain.
Biochemistry. 2010 Mar 2;49(8):1577-89. doi: 10.1021/bi901616z.
Neuronal specification is regulated by the activity of transcription factors containing the basic helix-loop-helix motif (bHLH); these regulating proteins include, among others, the neurogenin (Ngn) family, related to the atonal family of genes. Neurogenin 1 (NGN1) is a 237-residue protein that contains a bHLH domain and is involved in neuronal differentiation. In this work, we synthesized the bHLH region of NGN1 (bHLHN) comprising residues 90-150 of the full-length NGN1. The domain is a monomeric natively unfolded protein with a pH-dependent premolten globule conformation, as shown by several spectroscopic techniques (namely, NMR, fluorescence, FTIR, and circular dichroism). The unfolded character of the domain also explains, first, the impossibility of its overexpression in several Escherichia coli strains and, second, its insolubility in aqueous buffers. To the best of our knowledge, this is the first extensive study of the conformational preferences of a bHLH domain under different solution conditions. Upon binding to two DNA E-boxes, the protein forms "fuzzy" complexes (that is, the complexes were not fully folded). The affinities of bHLHN for both DNA boxes were smaller than those of other bHLH domains, which might explain why the protein-DNA complexes were not fully folded.
神经元的特化受含有基本螺旋-环-螺旋基序 (bHLH) 的转录因子的活性调控；这些调节蛋白包括神经基因家族 (Ngn)，与 atonal 基因家族有关。神经基因 1 (NGN1) 是一种 237 个氨基酸残基的蛋白质，含有一个 bHLH 结构域，参与神经元分化。在这项工作中，我们合成了全长 NGN1 第 90-150 位残基的 bHLH 区域 (bHLHN)。该结构域是一个单体的天然无规则卷曲蛋白质，具有 pH 依赖性的预熔球构象，如几种光谱技术（即 NMR、荧光、FTIR 和圆二色性）所示。该结构域的无规则卷曲特性首先解释了它在几种大肠杆菌菌株中无法过表达的原因，其次也解释了它在水性缓冲液中不溶解的原因。据我们所知，这是首次在不同溶液条件下对 bHLH 结构域的构象偏好进行广泛研究。与两个 DNA E-box 结合后，该蛋白形成“模糊”复合物（即复合物没有完全折叠）。bHLHN 与两个 DNA 盒的亲和力均小于其他 bHLH 结构域，这可能解释了为什么蛋白质-DNA 复合物没有完全折叠。
