Related Experiment Videos
Fluorescence quenching studies of Trp repressor using single-tryptophan mutants
1Department of Physical Biochemistry, Jagellonian University, Kraków, Poland.
Journal of Protein Chemistry
|November 1, 1995
Summary
The Trp repressor protein exists in two distinct conformational states, revealed by analyzing the fluorescence of its tryptophan residues. This finding helps understand protein dynamics and function.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- The Trp repressor (TrpR) regulates gene expression in bacteria.
- Understanding TrpR's conformational flexibility is crucial for its function.
- Tryptophan residues are sensitive probes of protein microenvironments.
Purpose of the Study:
- To resolve the heterogeneous fluorescence emission of single-tryptophan mutants of Trp repressor (W19F and W99F).
- To investigate the distinct microenvironments of tryptophan residues in TrpR.
- To determine if TrpR exists in multiple conformational states.
Main Methods:
- Time-resolved and steady-state fluorescence spectroscopy.
- Fluorescence-quenching-resolved spectra (FQRS) using iodide as a quencher.
- Analysis of fluorescence lifetime as a function of quencher concentration.
Main Results:
- Trp99 emission resolved into two components (338 nm and 328 nm), with one exposed to solvent.
- Trp19 emission resolved into two iodide-quenchable components (322-324 nm).
- Fluorescence lifetime data support the existence of two classes for both Trp99 and Trp19.
Conclusions:
- The Trp aporepressor can exist in at least two distinct ground-state conformational states.
- These conformational states differ in the microenvironment surrounding the tryptophan residues.
- The study provides insights into the dynamic nature of the Trp repressor protein.