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Histidine-tryptophan interactions in T4 lysozyme: 'anomalous' pH dependence of fluorescence
1Department of Chemistry, University of Oregon, Eugene 97403, USA.
Abstract:
A variant of T4 lysozyme which contains only a single tryptophan residue (at position 138) has been prepared (W126Y/W158Y designated 'YWY'). Two additional mutations to YWY have been prepared involving replacement of glutamine 105, which hydrogen bonds to the indole N-H of trp 138 in wild type, with either a histidine (YWY/Q105H) or an alanine (YWY/Q105A). The fluorescence properties of these two species are investigated as a function of pH. YWY/Q105A exhibits essentially a single exponential fluorescence decay (5% tau = 0.35 ns 95% tau = 5 ns) and almost no pH dependence in steady state or time resolved fluorescence behavior. In contrast, YWY/Q105H exhibits complex fluorescence decay over the entire pH range used in these experiments. As the pH is lowered from 8 to 4, there is an increase in the quantum yield and a change in the average lifetime (from 2.0 to 3.1 ns). Using this data, the pKa of histidine 105 has been determined to be 5.9. These results are contrasted to those from other proteins which show a pH dependent tryptophan fluorescence associated with a neighboring histidine or other residue. Quenching behavior in terms of the stereochemistry of the tryptophan-histidine interaction and implications of these results for current models of complex fluorescence behavior of single tryptophan proteins are also discussed.
Insights
Researchers studied T4 lysozyme variants with single tryptophan residues. Mutating glutamine 105 to histidine revealed pH-dependent fluorescence, allowing pKa determination for histidine 105 in this protein.
Area of Science:
- Biochemistry
- Protein Engineering
- Spectroscopy
Background:
- T4 lysozyme is a model system for protein studies.
- Single tryptophan variants are useful for fluorescence investigations.
- Understanding residue interactions is key to protein function.
Purpose of the Study:
- To investigate the fluorescence properties of T4 lysozyme variants with a single tryptophan.
- To determine the pKa of a histidine residue (His105) through fluorescence measurements.
- To explore the impact of mutations on tryptophan fluorescence and pH dependence.
Main Methods:
- Site-directed mutagenesis to create T4 lysozyme variants (YWY, YWY/Q105H, YWY/Q105A).
- Steady-state and time-resolved fluorescence spectroscopy.
- pH-dependent fluorescence measurements.
Main Results:
- YWY/Q105A showed minimal pH dependence in fluorescence.
- YWY/Q105H exhibited complex, pH-dependent fluorescence decay.
- Quantum yield and average fluorescence lifetime of YWY/Q105H changed with pH, yielding a pKa of 5.9 for His105.
Conclusions:
- Histidine 105 in close proximity to Tryptophan 138 influences fluorescence properties in a pH-dependent manner.
- The determined pKa of 5.9 provides insights into the protonation state of His105.
- Results contribute to understanding complex fluorescence behavior in single-tryptophan proteins.