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

我们的目标是将蛋白质的构象变化 在tRNA被氨酰-tRNA合成酶（TrpRS）氨酰化期间 从嗜热脂肪B菌中分离得到的具有特异性识别和化学 基质的转化。 我们计划通过解决 X射线晶体结构表示催化剂的附加阶段， 循环，并将它们与现在已知的色氨酸结构进行比较， 5 'AMP复合物的结构分析，我们也将其扩展到其 衍射极限为1.7A。 新的晶体结构包括一种产物 含有异丙基-2 '3 '-ATP的米氏络合物， 色氨酸和ATP，无配体的酶，以及与 同源tRNA。 所有的晶体形式都表现得优于3.0 A， 可以通过分子置换来解决。 晶体定量分析 生长条件、小角X射线散射和荧光研究 已经证明这些晶体形式代表至少两种， 可能有三种不同的构象 我们将首先集中于产物络合物晶体，其配体，色氨酸- 2 '（3'）ATP，类似于天然存在的酰基转移产物， 异氰酰-tRNA/trp. 这种结构应该可以让我们深入了解tRNA TrpRS的结合和羟基特异性。 然后我们将完成 两个米氏络合物的结构。 比较这两 腺苷酸和产物复合物的结构将 阐明了活性部位中所有重要的结合位点，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。 突变分析将被用来测试的想法建议， 结构，包括已经制定的关于残留物的假设 负责氨基酸特异性。 tRNA的特异性识别，尤其是tRNA与 反密码子结合和酰基转移的催化作用将通过 制备晶体并解析酶：tRNA复合物的结构。 现在可以从携带B的质粒中获得纯化的tRNA/trp。枯草 tRNA/trp基因。 晶体先前已经通过以下方法获得和显示： 电泳含有化学计量当量的 酶和tRNA/trp。 因此，现在应该可以测试特定的 关于复杂的预测从结构的发展 腺苷酸复合物，并将其与沿着 催化途径。特别重要的是将络合物与 先前获得的GlnRS，并解释如何抑制与 色氨酸存在于tRNA/Gln的反密码子突变体中。 与已知结构一起，新结构将构成一个 广泛的酶：配体复合物，超过了 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， 酶：配体相互作用和蛋白质构象的变化， 色氨酸活化和酰基转移的催化作用，因此， 选择正确的氨基酸和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.