Author

Paul Ala

Date of Award

7-1994

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry

Supervisor

Daniel S.C. Yang

Abstract

Single crystals of tryptophanyl-tRNA synthetase from Bacillus subtilis were grown by vapor diffusion at 23℃ from 0.1 M Tris-HCl pH 7.0 solutions containing polyethylene glycol 8000, magnesium ATP and L-tryptophan. The crystals diffracted to 2.9 A (55% complete with 14885 accepted reflections two times above the sigma level and Rsym = 5.4%) at -180℃, and belong to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions: a = 145.09 ± 0.12 A, b = 108.56 ± 0.12 A, c = 49.37 ± 0.06 A and one dimer in the asymmetric unit. The refined model of tryptophanyl-tRNA synthetase from Bacillus stearothermophilus was used as a starting model for molecular replacement. Cross-rotation and translation functions calculated at various resolutions ranges produced a unique solution 4 and 15 times the r.m.s. level, respectively. The crystal structure of tryptophanyl-tRNA synthetase, complexed with the tryptophanyl adenylate intermediate has been refined to a crystallographic R-value of 23.4% for reflections between 8 and 3.1 A. In the final model, the root-mean-square deviation from ideality for bond distances is 0.02 A and for angles is 4.6°. Each monomer contains two domains: an α/β domain consisting of five parallel β-strands (residues 1 to 180) and a helical domain (residues 181 to 326). Residues 181 to 295 which are located in surface loops in the helical domains have high thermal factors and unfavourable dihedral angles. The substrate, Trp-AMP, binds in a deep pocket ~20 A long in the middle of the α/β domain. The indole and adenine rings of Trp and AMP are positioned close to hydrophobic residues located at the bottom of the pocket. The hydroxyl groups of the ribose ring, the carbonyl oxygen and amide nitrogen of Trp are bound to the top of the pocket within hydrogen bonding distance to Asn18 and Gln147, respectively. The results of point mutations in the vicinity of the active site are examined in light of this structure.

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Biochemistry Commons

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