Sulfhydryl chemistry detects three conformations of the lipid binding region of Escherichia coli pyruvate oxidase.
Sulfhydryl chemistry detects three conformations of the lipid binding region of Escherichia coli pyruvate oxidase.
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巯基化学可检测大肠杆菌丙酮酸氧化酶脂质结合区域的三种构象。
DOI：
10.1021/bi9709102
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
CronanJr,JE
中科院分区：
文献类型：
--
作者：
Chang,YY;CronanJr,JE
位点特异性二硫交联实验检测到大肠杆菌酸酯氧化酶（PoxB）的c端段在底物结合时发生构象变化，PoxB是一种脂质激活的同型四聚体酶。， J. E., Jr. (1995)J.生物化学。270、7896−7901]。C端脂质结合区仅在底物、丙酮酸和硫胺素焦磷酸辅因子存在时才交联，这表明一对C端非常接近。我们现在系统地在c端区域的18个氨基酸位置替换了半胱氨酸，得到了21个蛋白质的面板，每个蛋白质都有一个残基变成了半胱氨酸。这些蛋白质已经通过二硫交联和半胱氨酸侧链对各种巯基试剂的可及性进行了研究。在没有丙酮酸的情况下，修饰的PoxB蛋白的半胱氨酸残基不能形成二硫键，通常不能与大的、刚性的亲水巯基试剂4-乙酰氨基-4 ' -[（碘乙酰基）氨基]二苯乙烯-2,2 ' -二磺酸（IASD）反应，在某些情况下与较小的、更疏水的试剂n -乙基马来酰亚胺反应弱。因此，在这种构象中，C末端似乎固定在刚性环境中，与溶剂的接触有限。在丙酮酸存在的情况下，所有C端半胱氨酸残基（除了离C端最远的两个残基）都与巯基试剂反应，并容易形成二硫交联物质，表明转化为具有高度构象自由度的结构。在脂质激活剂Triton X-100或双棕榈酰基磷脂酰甘油的存在下，半胱氨酸取代蛋白的一部分不再与膜不渗透的IASD试剂反应，表明这些蛋白片段渗透到脂质胶束中。对于大多数蛋白质，在存在或不存在脂质激活剂的情况下，在添加氧化剂时可以看到类似程度的二硫形成。一个例外是PoxB D560C，它在脂质存在下更容易交联。此外，在脂质存在的情况下，发现PoxB蛋白的一个亚群交联程度较低。这些蛋白质的行为为两个C末端结合形成功能性脂质结合结构域的模型提供了强有力的支持。这些数据在三个不同的PoxB构象和高度相关的蛋白质的已知晶体结构方面进行了讨论。
Site-specific disulfide cross-linking experiments detected a conformational change within the C-terminal segment ofEscherichia colipyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, upon substrate binding [Chang, Y.-Y., & Cronan, J. E., Jr. (1995)J.Biol.Chem.270, 7896−7901]. The C-terminal lipid binding regions were cross-linked only in the presence of the substrate, pyruvate, and the thiamine pyrophosphate cofactor, indicating close proximity of a pair of C termini. We have now systematically substituted cysteine at 18 additional amino acid positions within the C-terminal region to obtain a panel of 21 proteins each having a single residue changed to cysteine. These proteins have been studied by disulfide cross-linking and by accessibility of the cysteine side chain to a variety of sulfhydryl agents. In the absence of pyruvate, the cysteine residues of the modified PoxB proteins failed to form disulfide bonds, generally failed to react with a large and rigid hydrophilic sulfhydryl reagent, 4-acetamido-4‘-[(iodoacetyl)amino]stilbene-2,2‘-disulfonic acid (IASD), and in some cases reacted weakly with a smaller more hydrophobic reagent,N-ethylmaleimide. Therefore, in this conformation, the C termini appear fixed in a rigid environment having limited exposure to solvent. In the presence of pyruvate, all of the C-terminal cysteine residues (except the two most distal from the C terminus) reacted with both sulfhydryl reagents and readily formed disulfide cross-linked species, indicating conversion to a structure having a high degree of conformational freedom. In the presence of lipid activators, Triton X-100 or dipalmitoylphosphatidylglycerol, a subset of the cysteine-substituted proteins no longer reacted with the membrane-impermeable IASD reagent, indicating penetration of these protein segments into the lipid micelles. For most of the proteins, similar extents of disulfide formation were seen upon addition of an oxidizing agent in the presence or absence of lipid activators. An exception was PoxB D560C which was much more readily cross-linked in the presence of lipid. Moreover, a subset of PoxB proteins that cross-linked to lower extents in the presence of lipids was found. The behavior of these proteins provides strong support for the model in which two C termini associate to form the functional lipid binding domain. These data are discussed in terms of three distinct PoxB conformers and the known crystal structure of a highly related protein.