Da. Deangelis et al., PRIM - PROXIMITY IMAGING OF GREEN FLUORESCENT PROTEIN-TAGGED POLYPEPTIDES, Proceedings of the National Academy of Sciences of the United Statesof America, 95(21), 1998, pp. 12312-12316
We report a serendipitous discovery that extends the impressive catalo
g of reporter functions performed by green fluorescent protein (GFP) o
r 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 ca
n be exploited to quantitatively image homo-oligomerization or cluster
ing processes of GFP-tagged proteins in vivo. The method, which we ter
m proximity imaging (PRIM), complements fluorescence resonance energy
transfer between a blue fluorescent protein donor and a GEP acceptor,
a powerful method for imaging proximity relationships between differen
t proteins. However, unlike fluorescence resonance energy transfer (wh
ich is a spectral interaction), PRIM depends on direct contact between
two GFP modules, which can lead to structural perturbations and conco
mitant 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 d
etect FK1012-induced dimerization of GFP fused to FK506-binding protei
n and clustering of glycosylphosphatidglinositol-anchored GFP at cell
surfaces.