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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Wiley
2025-05-01
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| Series: | Advanced Science |
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| Online Access: | https://doi.org/10.1002/advs.202414777 |
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| author | Soojung Yi Eunji Kim Sora Yang Gyeongmin Kim Da‐Woon Bae Se‐Young Son Bo‐Gyeong Jeong Jeong Seok Ji Hyung Ho Lee Ji‐Sook Hahn Sun‐Shin Cha Yeo Joon Yoon Nam Ki Lee |
| author_facet | Soojung Yi Eunji Kim Sora Yang Gyeongmin Kim Da‐Woon Bae Se‐Young Son Bo‐Gyeong Jeong Jeong Seok Ji Hyung Ho Lee Ji‐Sook Hahn Sun‐Shin Cha Yeo Joon Yoon Nam Ki Lee |
| author_sort | Soojung Yi |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-c91790ba56c84665a7b09a7d77d9465f |
| institution | Kabale University |
| issn | 2198-3844 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Wiley |
| record_format | Article |
| series | Advanced Science |
| spelling | doaj-art-c91790ba56c84665a7b09a7d77d9465f2025-08-20T03:47:33ZengWileyAdvanced Science2198-38442025-05-011219n/an/a10.1002/advs.202414777Direct Quantification of Protein–Protein Interactions in Living Bacterial CellsSoojung Yi0Eunji Kim1Sora Yang2Gyeongmin Kim3Da‐Woon Bae4Se‐Young Son5Bo‐Gyeong Jeong6Jeong Seok Ji7Hyung Ho Lee8Ji‐Sook Hahn9Sun‐Shin Cha10Yeo Joon Yoon11Nam Ki Lee12Department of Chemistry Seoul National University 1 Gwanak‐ro, Gwanak‐gu Seoul 08826 Republic of KoreaNatural Products Research Institute College of Pharmacy Seoul National University 1 Gwanak‐ro, Gwanak‐gu Seoul 08826 Republic of KoreaDepartment of Chemistry Seoul National University 1 Gwanak‐ro, Gwanak‐gu Seoul 08826 Republic of KoreaDepartment of Chemical and Biological Engineering Institute of Chemical Processes Seoul National University 1 Gwanak‐ro, Gwanak‐gu Seoul 08826 Republic of KoreaDepartment of Chemistry and Nanoscience Ewha Womans University 52 Ewhayeodae‐gil, Seodaemun‐gu Seoul 03760 Republic of KoreaDepartment of Chemistry and Nanoscience Ewha Womans University 52 Ewhayeodae‐gil, Seodaemun‐gu Seoul 03760 Republic of KoreaDepartment of Chemistry and Nanoscience Ewha Womans University 52 Ewhayeodae‐gil, Seodaemun‐gu Seoul 03760 Republic of KoreaDepartment of Chemistry Seoul National University 1 Gwanak‐ro, Gwanak‐gu Seoul 08826 Republic of KoreaDepartment of Chemistry Seoul National University 1 Gwanak‐ro, Gwanak‐gu Seoul 08826 Republic of KoreaDepartment of Chemical and Biological Engineering Institute of Chemical Processes Seoul National University 1 Gwanak‐ro, Gwanak‐gu Seoul 08826 Republic of KoreaDepartment of Chemistry and Nanoscience Ewha Womans University 52 Ewhayeodae‐gil, Seodaemun‐gu Seoul 03760 Republic of KoreaNatural Products Research Institute College of Pharmacy Seoul National University 1 Gwanak‐ro, Gwanak‐gu Seoul 08826 Republic of KoreaDepartment of Chemistry Seoul National University 1 Gwanak‐ro, Gwanak‐gu Seoul 08826 Republic of KoreaAbstract 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.https://doi.org/10.1002/advs.202414777biosensorFRETliving cellsprotein–protein interactionssynthetic biology |
| spellingShingle | Soojung Yi Eunji Kim Sora Yang Gyeongmin Kim Da‐Woon Bae Se‐Young Son Bo‐Gyeong Jeong Jeong Seok Ji Hyung Ho Lee Ji‐Sook Hahn Sun‐Shin Cha Yeo Joon Yoon Nam Ki Lee Direct Quantification of Protein–Protein Interactions in Living Bacterial Cells Advanced Science biosensor FRET living cells protein–protein interactions synthetic biology |
| title | Direct Quantification of Protein–Protein Interactions in Living Bacterial Cells |
| title_full | Direct Quantification of Protein–Protein Interactions in Living Bacterial Cells |
| title_fullStr | Direct Quantification of Protein–Protein Interactions in Living Bacterial Cells |
| title_full_unstemmed | Direct Quantification of Protein–Protein Interactions in Living Bacterial Cells |
| title_short | Direct Quantification of Protein–Protein Interactions in Living Bacterial Cells |
| title_sort | direct quantification of protein protein interactions in living bacterial cells |
| topic | biosensor FRET living cells protein–protein interactions synthetic biology |
| url | https://doi.org/10.1002/advs.202414777 |
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