Phase-resolved spectral measurements with several two tryptophan containing proteins

Area of Science:

• Biophysics
• Biochemistry
• Spectroscopy

Background:

• Proteins containing multiple tryptophan (Trp) residues present complex fluorescence emission spectra.
• Resolving individual Trp fluorescence is crucial for understanding protein structure and dynamics.

Purpose of the Study:

• To apply frequency domain fluorescence techniques to resolve component emission spectra of multi-tryptophan proteins.
• To investigate energy transfer mechanisms between tryptophan residues.
• To demonstrate the utility of this approach for studying protein-ligand interactions.

Main Methods:

• Multifrequency phase/modulation fluorescence measurements.
• Phase-sensitive detection with the Gratton and Jameson algorithm.
• Selective solute quenching experiments.
• Multifrequency lifetime measurements.

Main Results:

• Protein fluorescence was consistently described by a double exponential decay.
• Phase-resolved emission spectra were determined for each decay time component.
• Independent emission of Trp residues was supported in most proteins studied.
• Evidence of energy transfer from internal to surface Trp residues was observed in apoazurin.

Conclusions:

• Frequency domain fluorescence spectroscopy effectively resolves individual tryptophan emission spectra in proteins.
• The technique can identify energy transfer processes, as seen in apoazurin.
• This method is valuable for studying fluorescence changes in protein-ligand complexes.