Isotope Enriched Active Site Structure of EF-Tu by Endor

本文将角选择电子核双共振（ENDOR）技术与分子生物学技术相结合，利用同位素选择性富集生物合成蛋白质，以确定细菌延伸因子Tu （EF-Tu）在GTP水解中的催化活性位点结构。EF-Tu将从大肠杆菌JM109中分离得到，该菌株克隆了嗜热热菌的tufB基因以过量产生热稳定蛋白。将从以氘化海藻水解物为培养基的细菌中分离出过氘化蛋白质。营养不良菌株也经过适当的工程改造，以过量产生耐热的EF-Tu，将用于在氘化培养基上生长，以加入特定位置的同位素富集氨基酸。由于在蛋白质中金属离子位点的8埃半径内只有一个酪氨酸、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具有特异性的营养不足菌株。虽然蛋白质中的其他位点也会根据其初级序列对每个氨基酸进行同位素富集，但它们离金属离子太远，没有显著的光谱贡献。在初步研究中，我们已经确定钒基（VO2+）离子特异性替代Mg2+并支持EF-Tu催化的GTP水解。该阳离子将作为顺磁探针，用于选定角度的ENDOR，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。通过对溶液进行快速冷冻淬火，生成EF-Tu:VO2+:GTP配合物的催化活性结构，并用ENDOR光谱对其结构进行表征。其他三元配合物，即EF-Tu:VO2+:GDP， EF-Tu:VO2+:GDPCP和EF-Tu:VO2+GDPCP配合物的活性位点结构也将被确定，其中后两个核苷酸分别代表GTP的不可水解的(,（-亚胺）和(,（-亚甲基）类似物。该方法还将扩展到确定EF-Tu:VO2+:GTP与氨基酰基trna络合的活性位点结构。细菌伸长因子Tu（EF-Tu）是蛋白质生物合成的重要成分，其氨基酸序列与高等生物细胞中多种g蛋白具有同源性。EF-Tu结合并水解GTP以发挥其功能。在活性位点有GTP的EF-Tu的催化活性形式的结构尚不清楚。确定蛋白质- gtp复合物的结构将通过一系列电子核双共振（ENDOR）研究来完成，这些研究旨在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。该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. ***