J. Mol. Biol. (1996) 260, 446–466 A Concerted Tryptophanyl-adenylate-dependent Conformational Change in Bacillus subtilis Tryptophanyl-tRNA Synthetase Revealed by the Fluorescence of Trp92 Christopher W. V. Hogue 1 , Sylvie Doublie ´ 2 , Hong Xue 3 Jeffery T. Wong 3 , Charles W. Carter Jr 2 and Arthur G. Szabo 4 * 1 Department of Biochemistry A semi-conserved tryptophan residue of Bacillus subtilis tryptophanyl- tRNA synthetase (TrpRS) was previously asserted to be an essential University of Ottawa, 451 residue and directly involved in tRNA Trp binding and recognition. The Smyth Road, Ottawa crystal structure of the Bacillus stearothermophilus TrpRS tryptophanyl-5'- Ontario, Canada K1H 8M5 adenylate complex (Trp-AMP) shows that the corresponding Trp91 is 2 Department of Biochemistry buried and in the dimer interface, contrary to the expectations of the earlier and Biophysics CB 7260 assertation. Here we examine the role of this semi-conserved tryptophan University of North Carolina residue using fluorescence spectroscopy. B. subtilis TrpRS has a single at Chapel Hill, Chapel Hill tryptophan residue, Trp92. 4-Fluorotryptophan (4FW) is used as a NC 27599-7260, USA non-fluorescent substrate analog, allowing characterization of Trp92 fluorescence in the 4-fluorotryptophanyl-5'-adenylate (4FW-AMP) TrpRS 3 Department of Biochemistry complex. Complexation causes the Trp92 fluorescence to become quenched University of Toronto by 70%. Titrations, forming this complex under irreversible conditions, Toronto, Ontario, Canada show that this quenching is essentially complete after half of the sites are M5S 1A8 filled. This indicates that a substrate-dependent mechanism exists for the 4 The Department of inter-subunit communication of conformational changes. Trp92 fluor- Chemistry and Biochemistry escence is not efficiently quenched by small solutes in either the apo- or University of Windsor complexed form. From this we conclude that this tryptophan residue is not Windsor, Ontario, Canada solvent exposed and that binding of the Trp92 to tRNA Trp is unlikely. N9B 3P4 Time-resolved fluorescence indicates conformational heterogeneity of B. subtilis Trp92 with the fluorescence decay being best described by three discrete exponential decay times. The decay-associated spectra (DAS) of the apo- and complexed- TrpRS show large variations of the concentration of individual fluorescence decay components. Based on recent correlations of these data with changes in the local secondary structure of the backbone containing the fluorescent tryptophan residue, we conclude that changes observed in Trp92 time-resolved fluorescence originate primarily from large perturbations of its local secondary structure. The quenching of Trp92 in the 4FW-AMP complex is best explained by the crystal structure conformation, in which the tryptophan residue is found in an -helix. The amino acid residue cysteine is observed clearly within the quenching radius (3.6 Å) of the conserved tryptophan residue. These tryptophan and cysteine residues are neighbors, one helical turn *Corresponding author Present addresses: C. W. V. Hogue, National Center for Biotechnology Information, Building 38A, NIH, 8600 Rockville Pike, Bethesda MD 20894, USA; S. Doublie ´, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston MA 02115, USA; H. Xue and J. T. Wong, Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. Abbreviations used: TrpRS, tryptophanyl-tRNA synthetase; Trp-AMP, tryptophanyl-adenylate; 4FW, 4-fluorotryptophan; DAS, decay-associated spectra; 4FW-AMP, L-4-fluorotryptophanyl-adenylate; PPi, Na2 pyrophosphate; PPiase, inorganic pyrophosphatase; DTNB, dithionitrobenzoic acid; IPTG, isopropyl -D-thiogalactopyranoside; ESI electrospray ionization; EDTA, ethylenediaminetetraacetic acid; NATA, N-acetyltryptophanamide; PMSF, phenylmethylsulfonyl fluoride; GdHCl, guanidine hydrochloride; CP1, connective polypeptide 1. The notation xxxRS where xxx is the IUPAC-IUPAB three-letter code of an amino acid refers to the aminoacyl-tRNA synthetase (aaRS) for that amino acid. 0022–2836/96/280446–21 $18.00/0  1996 Academic Press Limited