TRP TRNA LIGASE--X RAY STUDIES OF THE CATALYTIC CYCLE

我们的目标是将发生的蛋白质构象变化联系起来 色氨酰-tRNA合成酶(TrpRS)对tRNA的氨酰化反应 从嗜热性硬脂菌中分离出具有特异性识别和化学成分 底物的变化。我们计划通过解决以下问题来实现这一目标 代表催化的其他阶段的X射线晶体结构 循环，并将它们与现在已知的色氨酸的结构进行比较- 5‘AMP复合体，其结构分析也将扩展到其 衍射极限为1.7A。新的晶体结构包括一个产物。 含色氨基-2‘，3’-三磷酸腺苷的米氏络合物 色氨酸和ATP，一种无配体的酶，以及与 同源tRNA。所有晶型的衍射率都高于3.0A， 通过分子置换来解决。晶体的定量分析 生长条件、小角X射线散射和荧光研究 已经证明这些晶型至少代表两种和 可能有三种不同的构象。 我们将首先关注其配体Trp-的产物络合物晶体。 2‘(3’)三磷酸腺苷，类似于自然产生的酰基转移产物， 色氨酰-tRNA/色氨酸。这种结构应该能提供对tRNA的洞察 TrpRS的结合和羟基专一性。然后我们就会完成 这两个米氏络合物的结构。将这两者进行比较 与腺苷酸盐和产物络合物的结构将 阐明活性部位中所有重要的结合部位，并建议 它们在活化和酰基转移过程中如何变化和相互作用。 突变分析将用于测试由 结构，包括已经提出的关于残基的假设 负责氨基酸的专一性。 对tRNA的特异性识别，特别是对tRNA之间的偶联 反密码子结合和催化酰基转移将通过 制备晶体并解决一种酶的结构：tRNA复合体。 现在可以从携带枯草杆菌的质粒中获得纯化的tRNA/Trp TRNA/Trp基因。之前已经获得了晶体，并显示了 含有化学计量当量的两种物质的电泳法 酶和tRNA/Trp。因此，现在应该可以测试特定的 关于这个复合体的预测是从 腺化复合体，并将其与其他结构进行比较。 催化路径。尤其重要的是要将这个综合体与 之前为GlnRS获得的数据，并解释如何使用 色氨酸与tRNA/Gln的反密码子突变体一起出现。 与已知结构一起，新结构将构成一个 广泛的酶：配体络合物，超过范围 以前可用于其他合成酶的结构。他们应该 因此提供了一个前所未有的机会来研究如何 酶：配体相互作用和蛋白质构象变化 色氨酸活化和酰基转移的催化作用，因此 选择了正确的氨基酸和tRNA。

Our goal is to correlate protein conformational changes that take place during aminoacylation of tRNA by tryptophanyl-tRNA synthetase (TrpRS) from B stearothermophilus with the specific recognition and chemical transformations of the substrates. We plan to accomplish this by solving X-ray crystal structures representing additional stages of the catalytic cycle and comparing them with the now known structure of the trptophanyl- 5'AMP complex, whose structure analysis we will also extend to its diffraction limit of 1.7 A. The new crystal structures include a product complex containing tryptophanyl-2'3'-ATP, the Michaelis complexes with tryptophan and ATP, the ligand-free enzyme, and a complex with the cognate tRNA. All crystal forms diffract to better than 3.0 A and will be solved by molecular replacement. Quantitative analysis of crystal growth conditions, small angle X-ray scattering, and fluorescence studies have demonstrated that these crystal forms represent at least two and probably three different conformations. We will focus first on the product complex crystals whose ligand, trp- 2'(3')ATP, resembles the naturally occurring acyl-transfer product, tryptophanyl-tRNA/trp. That structure should provide insight into tRNA binding and hydroxyl group specificity of TrpRS. Then we will complete the structure of the two Michaelis complexes. Comparing these two structures with that of the adenylate and product complexes will elucidate all the important binding sites in the active site and suggest how they change and interact during activation and acyl-transfer. Mutational analysis will be used to test ideas suggested by the structures, including hypotheses already formulated about residues responsible for amino acid specificity. Specific recognition of tRNA and especially the coupling between anticodon binding and catalysis of acyl transfer will be addressed by preparing crystals and solving the structure of an enzyme:tRNA complex. Purified tRNA/trp is now available from a plasmid bearing the B. subtilis tRNA/trp gene. Crystals have previously been obtained and shown by electrophoresis to contain stoichiometrically equivalent amounts of both enzyme and tRNA/trp. Thus, it should now be possible to test specific predictions about the complex developed from the structure of the adenylate complex, and compare it with other structures along the catalytic path. Especially important will be to compare the complex with that previously obtained for GlnRS, and to explain how suppression with tryptophan occurs with an anticodon mutant of tRNA/gln. Together with the known structure, the new structures will constitute an extensive set of enzyme:ligand complexes, exceeding the range of structures previously available for other synthetases. They should therefore provide an unprecedented opportunity to examine how enzyme:ligand interactions and protein conformations change during catalysis of tryptophan activation and acyl-transfer, and hence how the correct amino acid and tRNA are selected.