Isotope Enriched Active Site Structure of EF-Tu by Endor

9513538 Makinen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以选择性地用同位素生物合成富集蛋白质的新方法将被应用于确定GTP水解中细菌延伸因子Tu（EF-Tu）的催化活性位点结构。 EF-Tu将从E.将嗜热栖热菌（Thermus thermophilus）的tufB基因克隆到大肠杆菌JM 109中，使其高产耐热蛋白。 将从在作为培养基的氘代藻类水解物上生长的细菌中分离全氘代蛋白质。 也被适当工程化以过量生产热稳定EF-Tu的营养型菌株将用于在氘代培养基上生长以掺入位点特异性同位素富集的氨基酸。由于在蛋白质中金属离子位点的8埃半径内仅存在一个酪氨酸、一个赖氨酸、一个半胱氨酸、一个缬氨酸和一个脯氨酸，因此将选择对这些氨基酸特异性的营养缺陷型菌株。 虽然蛋白质中的其他位点也会根据其一级序列对每种氨基酸进行同位素富集，但它们距离金属离子太远而不能具有突出的光谱贡献。 在初步的研究中，我们已经确定，钒（VO2+）离子专门取代Mg2+和支持水解GTP催化EF-Tu。 该阳离子将用作角度选择ENDOR的顺磁探针，以通过ENDOR确定金属到核的距离，并分配ENDOR活性核相对于由VO2+的g张量定义的磁轴的位置。 溶液中EF-Tu：VO2+：GTP复合物的催化活性结构将通过溶液的快速冷冻淬火产生，用于ENDOR光谱的结构表征。 还将确定其它三元复合物的活性位点结构，即EF-Tu：VO 2+：GDP、EF-Tu：VO 2+：GDPCP和EF-Tu：VO 2 + GDPCP复合物，其中后两个核苷酸分别代表GTP的不可水解的（，）-亚氨基和（，）-亚甲基类似物。 该方法还将扩展到确定EF-Tu：VO2+：GTP复合到氨酰-tRNA的活性位点结构。 细菌延伸因子Tu（EF-Tu）是蛋白质生物合成中的必需组分，并且与高等生物细胞中的多种G蛋白显示氨基酸序列同源性。 为了发挥其功能，EF-Tu结合并水解GTP。 在活性位点具有GTP的EF-Tu的催化活性形式的结构尚不清楚。蛋白质-GTP复合物的结构的测定将通过一系列电子核双共振（ENDOR）研究来完成，所述研究设计用于测量催化活性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。 结构信息将适用于理解各种各样的其他同源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. ***