ANALYSIS OF CROSS LINKED COMPLEX OF THIOREDOXIN REDUCTASE & THIOREDOXIN

来自大肠杆菌的硫氧还蛋白还原酶(TrxR)催化 NADPH还原蛋白质硫氧还蛋白(Trx)中的二硫键。 据推测，在催化过程中，TrxR在两个 构象，其中只有一种被x射线实际观察到 结晶学(Waksman等人，J.Mol.比奥尔。236：800,1994)。这个 第二构象与底物Trx反应。模型建造 表明这两种构象有很大的旋转差异(65) NADP结合域相对于FAD结构域的位置。C135S TrxR的突变体和Trx的C32S突变体通过 二硫化物(王等，生物化学35：4812,1996)。王某的证据 等人的研究。暗示这个建筑群被锁在第二层(建议) 构象，所以对这个或相关复合体的分析将允许 确定该构象的结构。两个交联体 制备了C138-C35和C138-C32的配合物，用于结晶 审判。只有TrxR(C138)-Trx(C32)络合物已结晶 以具有足够分辨率的形式可重现。此表单可以 有时会在我们的家用仪器(R轴IV)上建立索引 原始正交晶胞(a=88，b=158，c=458)，但数据收集 由于R轴仪器无法分辨 长长的细胞边缘。国际象棋F-1光束线的分辨能力 大的晶胞边缘对于研究这种晶体形态是至关重要的。 在国际象棋比赛中收集了九个部分数据集。对数据进行测量 到2.7到3.3的分辨率Rsym值为3.9-9.4%，但 由于快速的晶体衰变(即使在超低温下)，没有 单个数据集的所有数据都已完成。可以合并两个数据集 产生一组几乎完整的在太空中被索引的强度 P212121组。总体完成度为3.3是95%，具有 再合并8.2%。交联剂的所有组分的结构 复杂性是已知的，但由于其规模，分析具有挑战性 不对称单位。初始交叉旋转和平移 搜索使用FAD域名的二聚体作为搜索模型，并已 将这些中心区域定位在7-8个蛋白质二聚体中的四个 晶体密度测量预测出现在不对称 单位。使用NADP域和其他组合进行搜索 片段，使用各种分子置换算法，是 正在进行中。

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.