Characterization of aminoacyl-adenylates in B. subtilis tryptophanyl-tRNA synthetase, by the fluorescence of tryptophan analogs 5-hydroxytryptophan and 7-azatryptophan.
The tryptophan analogs 5-hydroxytryptophan (5HW) and 7-azatryptophan (7AW) are capable of being biosynthetically incorporated into bacterial proteins and can be used as intrinsic fluorescence probes of protein structure, function and dynamics. A prerequisite for analog incorporation is their recognition by tryptophanyl-tRNA synthetase (TrpRS) and the formation of the analog aminoacyladenylate in the enzyme's active site. The binding of 5HW and 7AW to B. subtilis TrpRS and the stability of the corresponding aminoacyladenylates of 5HW and 7AW were examined using their unique spectroscopic properties. The adenylate of 7AW in the active site of TrpRS exhibited intense fluorescence with a 10.5 ns fluorescence decay time. Enzyme-bound 7AW-adenylate was a long-lived intermediate with a half-life of over 9 hours. Enzyme-bound 5HW-adenylate fluorescence was quenched compared to that of 5HW in solution. The 5HW-adenylate/TrpRS complex was much less stable than that of 7AW, with a half-life of 33 minutes. Rapid hydrolysis of the 5HW-adenylate may explain the apparent proofreading observed which prohibits 5HW incorporation into proteins in the presence of tryptophan. Hydrolysis of the adenylates of both analogs restored the fluorescence parameters towards those of the analogs in solution. Neither 1-methyltryptophan nor 5-methoxytryptophan were capable of forming long-lived aminoacyladenylate intermediates in TrpRS. This study provides perspectives on the usefulness of 5HW and 7AW as intrinsic fluorescence probes of protein structure. The enhanced fluorescence of 7AW suggests its location in a buried hydrophobic environment in the protein. Exposure to water results in significant fluorescence quenching. These studies clearly demonstrate the utility of Trp analogs for the elucidation of molecular details of protein structure and dynamics.
Hogue
C W
CW
Division of Biological Science, National Research Council of Canada, Ottawa, Ont.
Szabo
A G
AG
D006916:5-Hydroxytryptophan / Q000737:chemistry*; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry*; D005240:Feasibility Studies; D005456:Fluorescent Dyes; D007700:Kinetics; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry; D014369:Tryptophan-tRNA Ligase / Q000737:chemistry*
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; C006657:7-azatryptophan; D014364:Tryptophan; D006916:5-Hydroxytryptophan; D014369:Tryptophan-tRNA Ligase