ANALYSIS OF CROSS LINKED COMPLEX OF THIOREDOXIN REDUCTASE & THIOREDOXIN

硫氧还蛋白还原酶交联复合物的分析

• 批准号: 6120511
• 负责人: MARTHA L LUDWIG
• 金额: $ 0.02万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-15 至 1999-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6120511
• 关键词: bioimaging /biomedical imaging; biological products; biomedical resource; proteins; radiography; structural biology

Thioredoxin reductase (TrxR) from Escherichia coli catalyzes the reduction of a disulfide in the protein thioredoxin (Trx) by NADPH. It is presumed that during catalysis TrxR alternates between two conformations, only one of which has been actually observed by x-ray crystallography (Waksman et al., J. Mol. Biol. 236:800, 1994). The second conformation reacts with the substrate Trx. Model building suggests that the two conformations differ by a large rotation (65) of the NADP binding domain relative to the FAD domain. The C135S mutant of TrxR and the C32S mutant of Trx have been crosslinked via a disulfide (Wang etal., Biochemistry 35:4812, 1996). Evidence of Wang et al. suggests that this complex is locked in the second (proposed) conformation, so analysis of this or a related complex would allow the determination of the structure of that conformation. Two crosslinked complexes, C138-C35 and C138-C32,were prepared for crystallization trials. Only the TrxR(C138)-Trx(C32) complex has crystallized reproducibly in a form with adequate resolution. This form could occasionally be indexed on our home instrument (R-AXIS IV) in a primitive orthorhombic cell (a=88, b=158, c=458
) but data collection were precluded by the inability of the R-AXIS instrument to resolve the long cell edge. The ability of the CHESS F-1 beamline to resolve large unit cell edges was critical for studies of this crystal form. Nine partial datasets were collected at CHESS. The data were measured to resolutions of 2.7 to 3.3
with Rsym values of 3.9-9.4%, but because of rapid crystal decay (even at ultralow temperatures), none of the individual datasets was complete. Two datasets could be merged to produce one almost complete set of intensities indexed in space group P212121. The overall completeness to 3.3
is 95% with an Rmerge of 8.2%. Structures of all the constituents of the crosslinked complex are known, but the analysis is challenging because of the size of the asymmetric unit. Initial cross rotation and tranlation searches used the dimer of FAD domains as a search model and have located these central domains in four of the 7-8 protein dimers which crystal density measurements predict are present in the asymmetric unit. Searches with the NADP domains and with other combinations of fragments, using a variety of molecular replacement alogorithms, are in progress.

来自大肠杆菌的硫氧还蛋白还原酶（TrxR）催化 通过NADPH还原蛋白质硫氧还蛋白（Trx）中的二硫化物。 据推测，在催化过程中，TrxR在两个 构象，其中只有一个已被实际观察到的x射线 结晶学（Waksman等人，J. Mol. 236：800，1994）。 的 第二构象与底物Trx反应。模型构建 表明这两种构象相差很大（65) 相对于FAD结构域的NADP结合结构域。 C135S TrxR的C32 S突变体和Trx的C32 S突变体已经通过一种交联剂交联。 二硫化物（Wang埃塔尔，Biochemistry 35：4812，1996）。 王的证据 等人认为，这种复杂的是锁定在第二（建议） 构象，因此对该复合物或相关复合物的分析将允许 确定该构象的结构。 二交联 制备了C138-C35和C138-C32配合物用于结晶 审判 只有TrxR（C138）-Trx（C32）复合物结晶 以具有足够分辨率的形式可再现。 这种形式可以 偶尔在我们的家用仪器（R-AXIS IV）上索引， 原始正交晶胞（a=88，B=158，c=458
）但数据收集 由于R-AXIS仪器无法分辨 长细胞边缘。 CHESS F-1光束线分辨 大的晶胞边缘对于研究这种晶形是关键的。 在CHESS收集了9个部分数据集。 测量数据 决议2.7至3.3
Rsym值为3.9- 9.4%，但 由于快速的晶体衰变（即使在超低温下）， 个人数据集是完整的。 可以合并两个数据集 产生一个几乎完整的空间强度索引 P212121组。 整体完整性达到3.3
是95%， Rmerge为8.2%。 交联的所有组分的结构 复杂的是已知的，但分析是具有挑战性的，因为大小 不对称的单位。 初始交叉旋转和平移 搜索使用FAD结构域的二聚体作为搜索模型， 在7-8个蛋白质二聚体中的4个中定位了这些中心结构域， 晶体密度测量预测存在于不对称的 单位 与NADP结构域以及与NADP结构域的其他组合的结合 片段，使用各种分子置换算法， 进行中。