Meinhold Derrick, Beach Michael, Shao Yongping, Osuna Robert, Colón Wilfredo
Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, 110 8th Street, Troy, New York 12180, USA.
Biochemistry. 2006 Aug 15;45(32):9767-77. doi: 10.1021/bi060672n.
Two crossed-linked variants of the homodimeric DNA binding protein factor for inversion stimulation (FIS) were created via engineering of single intermolecular disulfide bonds. The conservative S30C and the nonconservative V58C FIS independent mutations resulted in FIS crossed-linked at the A helix (C30-C30) and at the middle of the B helix (C58-C58). This study sought to investigate how the location of an intermolecular disulfide bond may determine the effect on stability and its propagation through the structure to preserve or alter the denaturation cooperativity of FIS. The oxidized and reduced S30C and V58C FIS exhibited a far-UV CD spectrum and DNA binding affinities that were similar to WT FIS, indicating no significant changes in secondary and tertiary structure. However, the reduced and oxidized forms of the mutants revealed significant differences in the stability and equilibrium denaturation mechanism between the two mutants. In the reduced state, S30C FIS had very little effect on FIS stability, whereas V58C FIS was 2-3 kcal/mol less stable than WT FIS. Interestingly, while both disulfide bonds significantly increased the resistance to urea- and guanidine hydrochloride (GuHCl)-induced denaturation, oxidized V58C FIS exhibited a three-state GuHCl-induced transition. In contrast, oxidized S30C FIS displayed a highly cooperative WT-like transition with both denaturants. The three-state denaturation mechanism of oxidized V58C FIS induced by the GuHCl salt was reproduced by urea denaturation at pH 4, suggesting that disruption of a C-terminus salt-bridge network is responsible for the loss of denaturation cooperativity of V58C FIS in GuHCl or urea, pH 4. A second mutation on V58C FIS created to place a single tryptophan probe (Y95W) at the C-terminus further implies that the denaturation intermediate observed in disulfide crossed-linked V58C FIS results from a decoupling of the stabilities of the C-terminus and the rest of the protein. These results show that, unlike the C30-C30 intermolecular disulfide bond, the C58-C58 disulfide bond did not evenly stabilize the FIS structure, thereby highlighting the importance of the location of an engineered disulfide bond on the propagation of stability and the denaturation cooperativity of a protein.
通过构建单个分子间二硫键，制备了同二聚体DNA结合蛋白倒位刺激因子（FIS）的两种交联变体。保守的S30C和非保守的V58C FIS独立突变导致FIS在A螺旋（C30-C30）和B螺旋中部（C58-C58）处发生交联。本研究旨在探究分子间二硫键的位置如何决定其对稳定性的影响，以及这种影响如何通过结构传递以维持或改变FIS的变性协同性。氧化型和还原型的S30C和V58C FIS呈现出与野生型FIS相似的远紫外圆二色光谱和DNA结合亲和力，表明其二级和三级结构无显著变化。然而，突变体的还原型和氧化型在稳定性和平衡变性机制上存在显著差异。在还原状态下，S30C FIS对FIS稳定性影响很小，而V58C FIS的稳定性比野生型FIS低2-3千卡/摩尔。有趣的是，虽然两个二硫键均显著增强了对尿素和盐酸胍（GuHCl）诱导变性的抗性，但氧化型V58C FIS在GuHCl诱导下呈现三态转变。相比之下，氧化型S30C FIS在两种变性剂作用下均表现出类似野生型的高度协同转变。pH 4条件下尿素变性重现了GuHCl盐诱导的氧化型V58C FIS的三态变性机制，表明C端盐桥网络的破坏是导致V58C FIS在GuHCl或pH 4尿素中变性协同性丧失的原因。在V58C FIS上引入的第二个突变，即在C端放置单个色氨酸探针（Y95W），进一步表明在二硫键交联的V58C FIS中观察到的变性中间体是由C端与蛋白质其余部分稳定性的解耦导致的。这些结果表明，与C30-C30分子间二硫键不同，C58-C58二硫键并未均匀地稳定FIS结构，从而突出了工程化二硫键位置对蛋白质稳定性传递和变性协同性的重要性。
