Green Fluorescent Calmodulin

 

Green fluorescent protein (GFP) is a spontaneously fluorescent protein originally identified in and cloned from the Pacific jellyfish, Aequoria victoria.  GFP can be expressed in virtually any organism and retain its characteristic fluorescence excitation and emission properties.  It also readily tolerates protein fusions to its N- and C-termini.  Thus, GFP has gained wide use as a reporter gene to monitor the expression patterns encoded by gene promoters and protein distribution when it is fused to a protein of interest.  We are using GFP::CaM fusions to examine CaM diffusion rates and distributions in vivo.  One indication that this fusion protein is fully function and reflects biologically relevant properties of unfused CaM is that it complements a non-revertable CaM knockout in yeast and facilitates yeast growth at near WT rates as illustrated in the Figure, where: represents the growth of yeast harboring the WT yeast CaM gene; represents the growth of yeast expressing unfused Arabidopsis CaM ; represents the growth of yeast expressing CaM::GFP (Arabidopsis CaM fused to the N-terminus of GFP); and represents the growth of yeast expressing GFP::CaM (Arabidopsis CaM fused to the C-terminus of GFP).

 

 

We have also successfully expressed GFP::CaM in microprojectile-bombarded onion cells and are currently asking whether the fusion protein can complement CaM function in T-DNA insertional knockout lines of Arabidopsis CaM that we have identified and are characterizing.

 

We are also using recombinant GFP::CaM in in vitro binding experiments as part of a “split cameleon” to examine the interaction of CaM-binding domains with CaM to determine the Ca2+-dependence of their interaction.  The Figure shows the results of such a binding experiment in which split cameleons consisting of GFP fused with different Arabidopsis CaM isoforms were used to determine the Ca2+-dependence of CaM interaction with CIP111, a plant CaM-binding protein belonging to the AAA family of ATPases.  In this experiment, the upper curve illustrates the interaction of GFP::CaM2 with BFP::CIP111, while the middle curve shows GFP::CaM8 and the lower curve shows GFP::CaM9 interactions with BFP::CIP111.

 

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This page is maintained by Ray Zielinski and was last updated on 31 January 2004.