Lanthanide-binding tags as luminescent probes for studying protein interactions

Area of Science:

• Biochemistry
• Biophysics
• Molecular Biology

Background:

• Protein-peptide interactions are crucial in cellular processes.
• Existing methods for studying these interactions have limitations.
• Lanthanide-binding tags (LBTs) offer a luminescent alternative to fluorescent proteins.

Purpose of the Study:

• To develop and validate a new method for studying protein-peptide interactions using Tb(III) luminescence.
• To demonstrate the specificity and sensitivity of the LRET method for detecting these interactions.
• To enable the measurement of binding affinities and distances in protein-peptide complexes.

Main Methods:

• Utilized luminescence resonance energy transfer (LRET) with terbium (Tb(III)) as the luminescent donor.
• Engineered genetically encodable lanthanide-binding tags (LBTs) for protein labeling.
• Synthesized phosphopeptides with organic fluorophores for interaction studies.
• Employed time-resolved detection to leverage the long luminescence lifetime of Tb(III).

Main Results:

• Demonstrated specific LRET signals between SH2 domains and phosphopeptides, indicating recognition.
• Showed the method's capability to differentiate binding affinities and measure dissociation constants.
• Successfully calculated distances between peptide and protein sites using decay experiments and Förster theory.
• Eliminated background fluorescence using time-resolved detection, enhancing signal clarity.

Conclusions:

• The developed LRET method provides a sensitive and specific approach for studying protein-peptide interactions.
• LBTs offer advantages over traditional fluorescent proteins due to their small size and long luminescence lifetime.
• This technique facilitates the quantitative analysis of binding kinetics and structural information.