Isotope Enriched Active Site Structure of EF-Tu by Endor

9513538 Makinen一种将角度选择电子核双共振(Endor)光谱与分子生物学技术相结合，选择性地以同位素生物合成富集蛋白质的新方法，将用于确定细菌延伸因子Tu(EF-Tu)在GTP水解中具有催化活性的活性中心结构。EF-Tu将从克隆了嗜热嗜热菌tufB基因的大肠杆菌JM109中分离出来，以过量生产耐热蛋白。将从以氚藻水解液为培养基生长的细菌中分离出氚蛋白。营养缺陷型菌株也被适当地设计为过量生产耐热的EF-Tu，将用于在氚培养基上生长，以结合特定部位的同位素浓缩氨基酸。由于在蛋白质中金属离子位置半径为8埃的范围内只有一个酪氨酸、一个赖氨酸、一个半胱氨酸、一个valine和一个Pro，因此将选择这些氨基酸所特有的营养缺陷型菌株。虽然蛋白质中的其他位置也将根据其初级序列对每种氨基酸进行同位素浓缩，但它们离金属离子太远，无法具有显著的光谱贡献。在初步的研究中，我们已经确定了钒(VO2+)离子特异性地替代了镁离子，并支持EF-Tu催化的GTP的水解。这种阳离子将被用作角度选择的Endor的顺磁探针，以确定Endor的金属到核的距离，并指定Endor活性核相对于由VO2+的g张量定义的磁轴的位置。Ef-Tu：VO2+：GTP络合物在溶液中的催化活性结构将通过溶液的快速冷冻猝灭产生，用于Endor光谱的结构表征。还测定了EF-Tu：VO2+：GDP，EF-Tu：VO2+：GDPCP和EF-Tu：VO2+GDPCP三元配合物的活性中心结构，其中后两个核苷酸分别代表GTP的非水解性(，(-亚胺)和(，(-亚甲基)类似物)。这些方法还将扩展到测定EF-Tu：VO2+：GTP与氨基酰基-tRNA的络合物的活性中心结构。细菌伸长因子Tu(EF-Tu)是蛋白质生物合成的重要组成部分，与高等生物细胞中的多种G-蛋白具有氨基酸序列同源性。为了实现其功能，EF-Tu与GTP结合并水解物。活性部位含有GTP的EF-Tu的催化活性形式的结构尚不清楚。蛋白质-GTP复合体的结构将通过一系列电子核双共振(Endor)研究来完成，该研究旨在测量催化活性的金属离子与附近氨基酸残基侧链上的磁性核之间的原子间距离。这些结构信息将适用于理解各种其他同源G-蛋白的结构-功能关系。***

9513538 Makinen A new approach combining angle selected electron nuclear double resonance (ENDOR) spectroscopy with molecular biological techniques to selectively enrich proteins biosynthetically with isotopes will be applied to determine the catalytically competent active site structure of the bacterial elongation factor Tu (EF-Tu) in GTP hydrolysis. EF-Tu will be isolated from E. coli JM109 into which the tufB gene of Thermus thermophilus has been cloned to overproduce the thermostable protein. The perdeuterated protein will be isolated from bacteria grown on deuterated algal hydrolyzate as the culture medium. Auxotrophic strains also suitably engineered to overproduce thermostable EF-Tu will be used for growth on deuterated medium to incorporate site specifically isotopically enriched amino acids. Since there is only one tyrosine, one lysine, one cysteine, one valine, and one proline within an 8 angstrom radius of the metal ion site in the protein, auxotropic strains specific for these amino acids will be chosen. Although other sites in the protein will be also isotopically enriched for each amino acid according to its primary sequence, they are too distant from the metal ion to have prominent spectral contributions. In preliminary studies we have already determined that the vanadyl (VO2+) ion specifically substitutes for Mg2+ and supports hydrolysis of GTP catalyzed by EF-Tu. This cation will be employed as the paramagnetic probe for angle selected ENDOR to determine metal-to-nucleus distances by ENDOR and to assign positions of ENDOR active nuclei with respect to magnetic axes defined by the g tensor of VO2+). The catalytically competent structure of the EF-Tu:VO2+:GTP complex in solution will be generated by rapid freeze-quenching of the solution for structural characteriztion by ENDOR spectroscopy. The active site structure of other ternary complexes, namely the EF-Tu:VO2+:GDP, EF-Tu:VO2+:GDPCP and the EF-Tu:VO2+GDPCP complexes, where the latter two nucleotides represent the nonhydrolyzable (,( -imido and (,(-methylene analogs of GTP, respectively, will be also determined. The methods will be also extended to determine active site structure of EF-Tu:VO2+:GTP complexed to amino acyl-tRNA. %%% The bacterial elongation factor Tu(EF-Tu) is an essential component in the biosynthesis of proteins and shows amino acid sequence homology with a variety of G-proteins in cells of higher organisms. In order to carry out its function, EF-Tu binds and hydrolyzes GTP. The structure of the catalytically active form of EF-Tu with GTP in the active site is not known. Determination of the structure of the protein-GTP complex will be accomplished by a series of electron nuclear double resonance (ENDOR) studies designed to measure interatomic distances between a catalytically active metal ion and magnetic nuclei on side-chains of nearby amino acid residues. The structural information will be applicable to understanding structure-function relationships of a wide variety of other homologous G-proteins. ***