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조회152

김은지Eunji Kim

서울대학교

Adv. Sci., Mar 24 2025, | https://doi.org/10.1002/advs.202414777 Direct Quantification of Protein-Protein Interactions in Living Bacterial Cells

Soojung Yi ¹, Eunji Kim ², Sora Yang ¹, Gyeongmin Kim ³, Da-Woon Bae ⁴, Se-Young Son ⁴, Bo-Gyeong Jeong ⁴, Jeong Seok Ji ¹, Hyung Ho Lee¹, Ji-Sook Hahn ³, Sun-Shin Cha ⁴, Yeo Joon Yoon ^{2 *}, Nam Ki Lee ^{1 *}

¹Department of Chemistry, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea.
²Natural Products Research Institute, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea.
³Department of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea.
⁴Department of Chemistry and Nanoscience, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul, 03760, Republic of Korea.

^†S.Y. and E.K. contributed equally to this work.
^*Corresponding authors: correspondence to Yeo Joon Yoon or Nam Ki Lee

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 (K_d) 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 K_d, leading to false-positive signals. KD-FRET proves highly effective in quantifying various PPI K_d values, including both heterologous and homologous pairs. Moreover, KD-FRET enables the quantification of K_d 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.

논문정보

형식Research article
게재일2025년 03월 (BRIC 등록일 2025-03-26)
연구진국내 연구진
분야 생명과학 > 구조생물 및 생물물리학

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