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PRIM: proximity imaging of green fluorescent protein-tagged polypeptides
D A De Angelis1, G Miesenböck, B V Zemelman
1Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 251, New York, NY 10021, USA. d-deangelis@ski.mskcc.org
Summary
Green fluorescent protein (GFP) proximity imaging (PRIM) detects protein self-association. Changes in green fluorescence intensity ratios reveal protein clustering and dimerization in vivo.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Green fluorescent protein (GFP) and its derivatives are widely used as reporters in biological research.
- Existing methods like Förster resonance energy transfer (FRET) image proximity between different proteins.
Purpose of the Study:
- To introduce a novel method, proximity imaging (PRIM), for detecting and quantifying the self-association of GFP-tagged proteins.
- To demonstrate PRIM's utility in visualizing protein homo-oligomerization and clustering in vivo.
Main Methods:
- Exploiting spectral changes in GFP fluorescence intensity ratios upon excitation at 395 nm versus 475 nm when two GFP molecules are in close proximity.
- Developing PRIM as a ratiometric index to measure the extent of protein self-association.
Main Results:
- Demonstrated that proximity-induced spectral shifts in GFP can quantitatively assess protein self-association.
- Successfully applied PRIM to detect FK1012-induced dimerization of GFP-fused FK506-binding protein.
- Visualized clustering of glycosylphosphatidylinositol-anchored GFP at cell surfaces using PRIM.
Conclusions:
- PRIM offers a sensitive, direct method to image protein homo-oligomerization and clustering in vivo.
- PRIM complements FRET by detecting direct contact between identical protein modules, providing insights into protein complex formation.