STOCHASTIC ENDOR FOR ENZYME ACTIVE SITE STRUCTURE

将开发一种创新的技术和方法方法 确定True的三维活动站点结构 一种新方法在酶催化反应中的应用 电子核双共振(Endor)光谱的一种称为 随机-Endor。细菌伸长因子Tu(EF-Tu)将为 分离出一种天然丰度为1H-或19F-的过氚蛋白- 取代氨基酸选择性地结合在活性中心位置 通过营养缺陷型大肠杆菌在过氢培养基上的生长。 将获得EF-Tu.V02+(鸟苷5‘-(5’-(β， γ-亚胺)三磷酸配合物和EF-TuV02+GTP配合物(生成 通过光解切割结合在蛋白质上的“笼状”-GTP)，其中 VO2+离子是Hg2+的顺磁性替代物。随机-Endor 将被用来确定这一新的方法是否具有很大的 增强的信噪比能力超过传统、连续 波(CW)END0R可以用来检测1H和19F共振 其残基位点被特异性地结合到去氢化蛋白中。这个 随机-E&D0R应用的局限性将被评估为 确定准确信号所需的同位素浓缩水平 检测并确定这种新Endor的分辨率 残基距离(6-Il)的部分重叠共振方法 A)从顺磁位置。主体超细的测定 特定活性中心残基的偶联成分将允许直接 它们与金属离子的距离和它们的取向 相对于VO2+离子的磁轴。成功地应用了 随机-ENDOR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中心结构 解决方案。

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.