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Unfolding of trp repressor studied using fluorescence spectroscopic techniques
1School of Pharmacy, University of Wisconsin, Madison 53706.
Biochemistry
|July 28, 1992
Summary
The trp repressor from Escherichia coli unfolds as a two-state dimer to monomer transition. Tryptophan binding stabilizes the repressor, increasing its free energy of unfolding and revealing tetramer dissociation.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Dynamics
Background:
- The trp repressor regulates gene expression in Escherichia coli.
- Understanding protein unfolding is crucial for comprehending protein function and stability.
Purpose of the Study:
- To investigate the unfolding properties of the trp repressor using fluorescence spectroscopy.
- To determine the effect of tryptophan binding on repressor stability and oligomerization.
Main Methods:
- Utilized time-resolved and steady-state fluorescence spectroscopy.
- Monitored urea-induced denaturation via fluorescence emission energy and lifetime.
- Employed a fluorescent probe (DNS) to assess rotational correlation time and detect oligomerization.
Main Results:
- Confirmed a two-state unfolding transition (dimer to monomer) with a free energy of 19.2 kcal/mol.
- Observed urea-induced changes in tryptophan fluorescence decay parameters.
- Identified a tryptophan-promoted, protein concentration-dependent transition attributed to tetramer dissociation.
- Demonstrated increased repressor stability (18.3 to 24.1 kcal/mol) upon tryptophan binding.
Conclusions:
- Trp repressor exhibits a stable dimer structure that dissociates into tetramers upon urea denaturation.
- Tryptophan binding significantly enhances the stability of the trp repressor dimer.
- Fluorescence techniques effectively probe both unfolding and oligomerization states of the trp repressor.