Direct Quantification of Protein–Protein Interactions in Living Bacterial Cells
Abstract Quantitative measurement of protein–protein interactions (PPIs) within living cells is vital for understanding their cellular functions at the molecular level and for applications in synthetic biology, protein engineering, and drug discovery. Although several techniques have been developed...
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| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Wiley
2025-05-01
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| Series: | Advanced Science |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/advs.202414777 |
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| Summary: | Abstract Quantitative measurement of protein–protein interactions (PPIs) within living cells is vital for understanding their cellular functions at the molecular level and for applications in synthetic biology, protein engineering, and drug discovery. Although several techniques have been developed to measure PPI strength in vitro, direct measurement of PPI strength within living bacterial cells remains challenging. Here, a method for quantitatively measuring PPIs by determining the dissociation constant (Kd) in living E. coli using fluorescence resonance energy transfer (FRET), a technique termed KD‐FRET, is reported. It is found that the direct excitation of the acceptor fluorophore among spectral crosstalks primarily results in non‐interacting pairs exhibiting an apparent Kd, leading to false‐positive signals. KD‐FRET proves highly effective in quantifying various PPI Kd values, including both heterologous and homologous pairs. Moreover, KD‐FRET enables the quantification of Kd for interaction pairs that are unmeasurable in vitro owing to their instability under standard buffer conditions. KD‐FRET is successfully applied in the development of a novel synthetic biology tool to enhance naringenin production in E. coli and lycopene production in S. cerevisiae by precisely engineering metabolic pathway. These results demonstrate the potential of KD‐FRET as a powerful tool for studying PPIs in their native cellular environments. |
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| ISSN: | 2198-3844 |