Künne A G, Allemann R K
Department of Chemistry, ETH-Zürich, Switzerland.
Biochemistry. 1997 Feb 4;36(5):1085-91. doi: 10.1021/bi962185l.
MASH-1, a member of the basic-helix-loop-helix (BHLH) family of transcription factors, promotes the differentiation of committed neuronal precursor cells. In vitro, MASH-1 displays only marginal DNA sequence specificity. We have produced a MASH-1 variant, MASH-GGC, by introducing the tripeptide Gly-Gly-Cys at the C-terminal end of the BHLH domain. Under reducing conditions the properties of MASH-GGC and of the BHLH domain of MASH-1 were very similar. Like MASH-1, reduced MASH-GGC showed little specificity of DNA binding. CD spectroscopy revealed that both proteins underwent a conformational change from a largely unfolded to a mainly alpha-helical conformation upon binding to DNA. When the subunits of MASH-GGC were linked through a disulfide bond, the folded conformation was stable over a wide concentration range (2.5 nM to 2 microM) even in the absence of DNA. Oxidized MASH-GGC bound to E-box-containing sequences half-maximally at 148 nM, compared to 458 nM for the reduced form. Therefore, even when the change from a monomeric to a dimeric species was taken into account, the affinity for E-box-containing DNA sequences was increased. Surprisingly, the apparent dissociation constant for the complex with DNA not containing E-box sequences was increased upon oxidation. Therefore, despite the large distance between the disulfide bridge and the protein-DNA interface, covalently linking the subunits of MASH-1 increased the specificity of DNA binding significantly. In vivo, such an increase of the intrinsic DNA binding specificity might be achieved through interactions with other proteins of the transcriptional machinery.
MASH-1是转录因子基本螺旋-环-螺旋（BHLH）家族的成员，可促进定向神经元前体细胞的分化。在体外，MASH-1仅表现出有限的DNA序列特异性。我们通过在BHLH结构域的C末端引入三肽Gly-Gly-Cys，产生了一种MASH-1变体MASH-GGC。在还原条件下，MASH-GGC和MASH-1的BHLH结构域的特性非常相似。与MASH-1一样，还原后的MASH-GGC显示出几乎没有DNA结合特异性。圆二色光谱显示，两种蛋白质在与DNA结合时都经历了从基本上未折叠到主要为α-螺旋构象的构象变化。当MASH-GGC的亚基通过二硫键连接时，即使在没有DNA的情况下，折叠构象在很宽的浓度范围（2.5 nM至2 μM）内也是稳定的。氧化后的MASH-GGC与含E-box序列的结合半最大浓度为148 nM，而还原形式为458 nM。因此，即使考虑到从单体到二聚体形式的变化，对含E-box DNA序列的亲和力也增加了。令人惊讶的是，与不含E-box序列的DNA形成的复合物的表观解离常数在氧化后增加。因此，尽管二硫桥与蛋白质-DNA界面之间的距离很大，但共价连接MASH-1的亚基显著增加了DNA结合的特异性。在体内，这种内在DNA结合特异性的增加可能通过与转录机制的其他蛋白质相互作用来实现。
