STOCHASTIC ENDOR FOR ENZYME ACTIVE SITE STRUCTURE

将制定创新的技术和方法 确定真正的三维活性位点结构， 一种新方法在酶催化反应中间体中的应用 电子核双共振（ENDOR）光谱，称为 随机ENDOR 细菌延伸因子Tu（EF-Tu）将是 分离为天然丰度为1H-或19 F-的全氘代蛋白质， 选择性掺入活性位点位置的取代氨基酸 通过营养缺陷型菌株E. 大肠杆菌在全氘代培养基上培养。 将获得EF-Tu.V02+（鸟苷5 ′-（5 ′-（β， γ-亚氨基）三磷酸复合物和EF-TuV 02 +GTP复合物（产生的 通过光解切割与蛋白质结合的“笼状”-GTP），其中 VO ~（2+）是Hg ~（2+）的顺磁性替代物。 随机ENDOR 将被应用于确定这种新的方法， 增强的信噪比能力，优于传统的连续 波（cw）END 0 R可用于检测1H和19 F共振 残基位点特异性地掺入到氘代蛋白质中。 的 将评估随机E & DR应用的局限性， 确定准确信号所需的同位素富集水平 并确定这种新ENDOR的分辨能力 远（6-Il）残基部分重叠共振的方法 A）从顺磁性位点。 主超精细的确定 特定活性位点残基的偶联组分将允许直接 分配它们与金属离子的距离和它们的取向， 相对于VO 2+离子的磁轴。 成功应用 随机END 0 R将有助于开发一种新的通用方法来确定 酶反应中间体的三维活性部位结构 溶液

An innovative technological and methodological approach will be developed to determine the three-dimensional active site structure of true intermediates of enzyme catalyzed reactions by application of a new method of electron nuclear double resonance (ENDOR) spectroscopy known as stochastic-ENDOR. The bacterial elongation factor Tu (EF-Tu) will be isolated as a perdeuterated protein with natural abundance 1H- or 19F- substituted amino acids selectively incorporated in active site positions through growth of auxotrophic strains of E. coli on perdeuterated medium. ENDOR spectra will be obtained for the EF-Tu.V02+(guanosine 5'-(5'-(beta, gamma-imido) triphosphate complex and the EF-TuV02+GTP complex (generated by photolytic cleavage of "caged"-GTP bound to the protein), in which the VO2+ ion serves as a paramagnetic substitute for Hg2+. Stochastic-ENDOR will be applied to determine whether this new methodology with greatly enhanced signal-to-noise capability over that of conventional, continuous wave (cw) END0R can be employed to detect the 1H and 19F resonances of residues site specifically incorporated into the deuterated protein. The limitations of application of stochastic-E&D0R will be assessed to determine the level of isotope enrichment necessary for accurate signal detection and to determine the resolving power of this new ENDOR methodology for partially overlapping resonances of residues distant (6-Il A) from the paramagnetic site. Determination of the principal hyperfine coupling components of specific active site residues will permit direct assignment of their distance from the metal ion and their orientation with respect to magnetic axes of the vo2+ ion. Successful application of stochastic-END0R will help to develop a new general method to determine the three-dimensional active site structure of enzyme reaction intermediates in solution.