PRIM - PROXIMITY IMAGING OF GREEN FLUORESCENT PROTEIN-TAGGED POLYPEPTIDES

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.