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Luminescence studies with trp repressor and its single-tryptophan mutants
M R Eftink1, G D Ramsay, L Burns
1Department of Chemistry, University of Mississippi, University 38677.
Biochemistry
|September 7, 1993
Summary
This study characterizes the two intrinsic tryptophan residues in E. coli trp aporepressor using luminescence spectroscopy. Trp19 and Trp99 exhibit distinct spectral properties and environments, with evidence of energy transfer from Trp19 to Trp99.
Area of Science:
- Biochemistry
- Molecular Biology
- Spectroscopy
Background:
- The trp aporepressor from Escherichia coli is a key regulatory protein.
- Understanding the environment and dynamics of intrinsic tryptophan residues is crucial for elucidating protein function.
- Distinguishing the contributions of individual tryptophan residues can be challenging due to spectral overlap.
Purpose of the Study:
- To resolve and characterize the luminescence properties of the two intrinsic tryptophan residues (Trp19 and Trp99) in E. coli trp aporepressor.
- To assign spectral features to specific tryptophan residues using site-directed mutagenesis.
- To investigate the environmental exposure, dynamics, and potential energy transfer between these residues.
Main Methods:
- Time-resolved and steady-state fluorescence spectroscopy.
- Low-temperature phosphorescence spectroscopy.
- Optically detected magnetic resonance (ODMR) measurements.
- Site-directed mutagenesis (W19F and W99F mutants).
- Solute fluorescence quenching studies.
Main Results:
- Trp19 and Trp99 exhibit distinct fluorescence emission wavelengths, decay times, quantum yields, and rotational freedom.
- Trp19 is generally less exposed and more rigidly associated with the protein, while Trp99 is more exposed and rotationally mobile.
- Phosphorescence spectra clearly resolve Trp19 (407 nm) and Trp99 (415 nm) emission maxima.
- ODMR measurements reveal characteristic zero-field splitting parameters for each tryptophan.
- Evidence for resonance energy transfer from Trp19 to Trp99 was observed in the wild-type protein.
Conclusions:
- The study successfully differentiated the spectral and dynamic properties of Trp19 and Trp99 in E. coli trp aporepressor.
- The findings provide insights into the local protein environment and conformational dynamics around these tryptophan residues.
- Resonance energy transfer from Trp19 to Trp99 suggests a specific spatial arrangement and electronic coupling between these residues within the protein structure.