We shall complete X-ray crystal structures for the B. stearothermophilus tryptophanyl-tRNA synthetase complexed to a substrate, tryptophan (type II*), and to an acyl-transfer product, tryptophanyl-ATP, in order to provide high resolution descriptions of the enzyme at two stages of its catalytic cycle, Michaelis complex and product complex. Major efforts during the forthcoming grant period are expected to be interpretation of electron density maps for these two structures in terms of atomic positions for the non-hydrogen atoms, preliminary structure refinement, and analysis of models for answers to questions about how the ligand binding sites are constructed in the two complexes, tertiary structural homology between this enzyme and other tRNA ligases whose structures are known, and the ability of the enzyme to catalyze various non-physiological reactions. Side chains in the catalytic and/or specificity sites of the enzyme will be evaluated for potential site-directed mutagenesis experiments to further probe their role in binding and catalysis. Enzyme conformations in the two crystal forms will be compared to identify differences related to catalysis. Structural studies will begin on crystals of the cognate tRNA, and efforts to prepare and characterize crystals of the enzyme complexed to a non-hydrolyzable adenylate intermediate and of an enzyme:tRNA complex will continue. Having cloned the gene for the enzyme and acquired an area detector for rapid, precise data collection we are in a very strong position to solve several of these new crystal structures during the forthcoming grant period. This progress will bring us considerably closer to achieving the long-term goal of this research program, which is to describe the entire process of activation and aminoacylation in this system in structural terms, including factors responsible for specific recognition of the correct tRNA substrate.
| Carter Jr, C W; Doublie, S; Coleman, D E (1994) Quantitative analysis of crystal growth. Tryptophanyl-tRNA synthetase crystal polymorphism and its relationship to catalysis. J Mol Biol 238:346-65 |
| Smith, F R; Lattman, E E; Carter Jr, C W (1991) The mutation beta 99 Asp-Tyr stabilizes Y--a new, composite quaternary state of human hemoglobin. Proteins 10:81-91 |
| Carter Jr, C W; Green, D C; Toomim, C S et al. (1985) Two-step purification of tryptophan-accepting tRNA from Bacillus stearothermophilus. Anal Biochem 151:515-9 |
