Direct Quantification of Protein-Protein Interactions in Living Bacterial Cells.

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Quantitative measurement of protein-protein interactions (PPIs) is crucial for understanding cellular functions.
• Existing in vitro methods struggle to accurately measure PPI strength within living cells.
• Challenges include the instability of some interaction pairs under standard buffer conditions.

Purpose of the Study:

• To develop and validate a method for quantitatively measuring PPIs within living bacterial cells.
• To determine the dissociation constant (Kd) of PPIs using fluorescence resonance energy transfer (FRET).
• To address limitations of existing techniques, including false-positive signals from spectral crosstalk.

Main Methods:

• Development of KD-FRET (Kd measurement using FRET) for in vivo PPI quantification.
• Utilizing living E. coli as a model system.
• Characterizing and correcting for spectral crosstalk to ensure accurate Kd measurements.

Main Results:

• KD-FRET accurately quantifies dissociation constants (Kd) for various heterologous and homologous PPIs in living E. coli.
• The method successfully measures Kd for interaction pairs unstable under in vitro conditions.
• KD-FRET enabled the engineering of metabolic pathways for enhanced naringenin and lycopene production.

Conclusions:

• KD-FRET is a robust tool for quantitative in vivo measurement of PPIs.
• The technique overcomes limitations of in vitro assays and spectral crosstalk.
• KD-FRET has significant potential for synthetic biology, drug discovery, and understanding cellular processes.