PRIM: Proximity Imaging of Green Fluorescent Protein-Tagged Polypeptides

We report a serendipitous discovery that extends the impressive catalog of reporter functions performed by green fluorescent protein (GFP) or its derivatives. When two GFP molecules are brought into proximity, changes in the relative intensities of green fluorescence emitted upon excitation at 395 v...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1998-10, Vol.95 (21), p.12312-12316
Main Authors: De Angelis, Dino A., Miesenbock, Gero, Zemelman, Boris V., Rothman, James E.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Base Sequence
Biological Sciences
Cells
Cellular biology
Chromophores
Dimerization
Dimers
DNA Primers
Energy Transfer
Fluorescence
Green Fluorescent Proteins
HeLa Cells
Humans
Imaging
Luminescent Proteins - chemistry
Molecular Sequence Data
Monomers
Peptides
Peptides - chemistry
Proteins
Spectrometry, Fluorescence
Wavelengths
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Summary:We report a serendipitous discovery that extends the impressive catalog of reporter functions performed by green fluorescent protein (GFP) or its derivatives. When two GFP molecules are brought into proximity, changes in the relative intensities of green fluorescence emitted upon excitation at 395 vs. 475 nm result. These spectral changes provide a sensitive ratiometric index of the extent of self-association that can be exploited to quantitatively image homo-oligomerization or clustering processes of GFP-tagged proteins in vivo. The method, which we term proximity imaging (PRIM), complements fluorescence resonance energy transfer between a blue fluorescent protein donor and a GFP acceptor, a powerful method for imaging proximity relationships between different proteins. However, unlike fluorescence resonance energy transfer (which is a spectral interaction), PRIM depends on direct contact between two GFP modules, which can lead to structural perturbations and concomitant spectral changes within a module. Moreover, the precise spatial arrangement of the GFP molecules within a given dimer determines the magnitude and direction of the spectral change. We have used PRIM to detect FK1012-induced dimerization of GFP fused to FK506-binding protein and clustering of glycosylphosphatidylinositol-anchored GFP at cell surfaces.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.95.21.12312