Protein‑protein interaction

1. FRET (Fluorescence Resonance Energy Transfer)

1.1 Introduction

Fluorescence Resonance Energy Transfer (FRET) is a physical process based on non‑radiative dipole‑dipole coupling between a donor fluorophore and an acceptor fluorophore. When the donor and acceptor are separated by 1‑10 nm with appropriate dipole orientation, energy absorbed by the donor upon excitation is transferred non‑radiatively to the acceptor, resulting in donor fluorescence quenching and enhanced acceptor emission. FRET efficiency is inversely proportional to the sixth power of the donor‑acceptor distance, rendering it exquisitely sensitive to intermolecular separation. This makes FRET an indispensable technique for investigating protein‑protein interactions, conformational dynamics, and real‑time molecular processes in living cells.

1.2 Products

1.3 Applications

1. Protein‑Protein Interaction Detection
   Two target proteins are fused with donor (e.g., CFP) and acceptor (e.g., YFP) fluorescent proteins, respectively. Upon protein interaction, the donor and acceptor approach each other, enhancing the FRET signal; conversely, the signal diminishes or disappears. This technique enables real‑time tracking of interaction spatiotemporal dynamics in living cells and has been widely applied in receptor oligomerization, signal pathway complex assembly, and transcription factor functional studies.

2. Real‑Time Enzyme Activity Monitoring
   FRET probes are designed with enzyme‑cleavable linkers connecting the donor and acceptor; enzyme activity cleaves the peptide, separating the donor and acceptor and reducing FRET efficiency. This technique has been successfully applied to dynamic monitoring of protein kinase (PKA, Src, EGFR), caspase, and matrix metalloproteinase (MMP) activities in living cells, providing a high‑throughput platform for drug screening.

3. Biosensor Development
   FRET pairs are fused with sensing elements (e.g., ion‑binding domains, metabolite‑binding proteins, or conformation‑sensitive domains). Target molecule binding or physiological signal changes induce conformational alterations, altering FRET efficiency. Representative applications include calcium ion (Ca²⁺) detection, cAMP level monitoring, pH sensing, and real‑time imaging of GTPase activity (Ras, Rho family).

4. Protein Conformational Change Analysis
   Donor and acceptor fluorophores are labeled at different domains of the same protein. Conformational changes (e.g., folding, allosteric transitions, phosphorylation‑induced rearrangements) alter the distance between them, changing FRET efficiency. This technique resolves structural transitions between active and inactive states, applicable to G protein‑coupled receptors (GPCRs), ion channels, and kinase dynamic conformation studies.

5. Protein‑Nucleic Acid Interaction Studies
   A DNA/RNA‑binding protein is fused with a donor fluorophore, while the nucleic acid probe is labeled with an acceptor fluorophore. Protein‑nucleic acid binding brings the donor and acceptor into proximity, generating a FRET signal. This technique is used to study dynamic binding of transcription factors to promoters/enhancers, RNA‑binding protein target recognition, and CRISPR‑Cas system DNA targeting mechanisms.

1.4 References:

1. Wang S, Chen D, Jiang C, et al. Design and Application of GFP‑based FRET Biosensor. Chinese Journal of Cell Biology, 2012, 34(12): 1171‑1180.
2. Day RN, Davidson MW. The fluorescent protein palette: tools for cellular imaging. Chemical Society Reviews, 2009, 38(10): 2887‑2921.
3. Zhang J, Campbell RE, Ting AY, Tsien RY. Creating new fluorescent probes for cell biology. Nature Reviews Molecular Cell Biology, 2002, 3(12): 906‑918.
4. Kraynov VS, Chamberlain C, Bokoch GM, et al. Localized Rac activation dynamics visualized in living cells. Science, 2000, 290(5490): 333‑337.