Isotope Enriched Active Site Structure of EF-Tu by Endor

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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. ***