Fluorescent Indicator Proteins (FIPs)

FIPs as used in our lab consist of multiple fusion proteins comprising a CaM-binding peptide derived from a protein of interest flanked by GFP derivatives similar to those originally described by Persechini and colleagues shown in the figure (Romoser et al., 1997). The GFP variants are selected for their ability to act as a donor-acceptor fluorophore pair for Förster resonance energy transfer (FRET). In the figure, blue fluorescent protein (BFP, B in the figure) and GFP (G) were used as the donor and acceptor, respectively, but cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) are more commonly used because they do not bleach as rapidly and their fluorescence properties are more favorable than the BFP-GFP donor-acceptor pair. In the presence of CaM and low levels of free Ca2+, CaM does not interact with the CaM-binding peptide portion of the FIP and FRET is high. When free Ca2+ levels rise, on the other hand, CaM binds to the CaM-binding domain of the FIP and physically separates the donor and acceptor GFP derivatives; when this happens FRET is reduced. As can be seen in the figure, CaM binding to the FIP can be observed (and quantified) by measuring either the change in GFP fluorescence emission or the ratio of GFP-to-BFP fluorescence emission as a function of Ca2+-CaM concentration.


We are using FIPs in which the CaM-binding domain is derived from skeletal muscle myosin light chain kinase (Kd for CaM < 1 nM) or from the cyclic nucleotide gated cation channel CNGC2 (Kd for CaM ~10 nM). The figure on the right demonstrates the interaction between CaM and the CaM-binding domain of CNGC2 measured using FIP-CNGC2. Panel A shows the purity of recombinant FIP-CNGC2, AtCaM4 and a negative control FIP (FIP-NC) that does not interact with CaM and the fluorescence emission responses of mixtures of FIPs and CaM titrated with buffered free Ca2+ ranging from 0 to 1.1 mM. Panel B illustrates the results of binding experiments in which AtCaM4 binding by FIP-CNGC2 was measured at saturating free Ca2+ levels. This figure was taken from Hua et al. (2003), which details the methods for making FIP-based measurements of binding and gives more specific details on the construction and expression of FIP-CNGC2 and FIP-NC.





          More recently, we have used FIP-CNGC2 and FIP-CNGC1 to demonstrate apparent affinity differences among different CaM proteins for the CaM-binding domains of these channel proteins.




Some Basic Background on FRET

        FRET is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon.  FRET is dependent on the inverse sixth power of the intermolecular separation, making it useful over distances comparable with the dimensions of biological macromolecules.  This makes FRET a useful technique for investigating biological processes that cause changes in molecular proximity.  The rate of energy transfer from a donor to an acceptor (kT) is given by


(Eq. 1)          kT (r) = (1/tD)(Ro/r)6


Where:        tD = the decay rate of the donor in the absence of the acceptor

                   r = the donor to acceptor (D-A) distance

                   Ro = the Förster distance


        The Förster distance (Ro) is the D-A distance at which energy transfer efficiency is 50% (i.e., 50% of excited donors are deactivated by FRET). The rate of FRET is strongly dependent upon the D-A distance, being inversely proportional to r6.  Förster distances ranging from 20 to 90 Å are most convenient for studies of biological macromolecules; these are comparable to the diameters of many proteins, the thicknesses of biological membranes, and the distance between sites on multisubunit proteins.  Any process that affects the D-A distance affects the energy transfer rate and allows the process to be quantified.  For this reason FRET has been referred to as a spectroscopic ruler.  The table below shows the Förster distances between donor-acceptor pairs commonly used in cell and molecular biological studies.



Ro (Å)











QSY-7 dye









In practice, kT is not easily measured and not necessarily that informative for biologists.  A more useful parameter is the efficiency (E) with which energy is transferred from the donor to the acceptor.  This efficiency can be expressed as a ratio of the rates of energy donation to an acceptor divided by the total rate of energy dissipation by the donor (the sum of fluorescence and donation).


(Eq. 2)                   E = kT/(tD-1 + kT)


By substituting Eq. 1 for kT in Eq. 2, one can obtain


(Eq. 3)                   E = Ro6/(Ro6 + r6)


This equation describes the exquisite distance dependence of FRET.  If Ro is arbitrarily set to a value of 1, then by inspection, one can see that if r = Ro, E = 0.5 (or 50%) (i.e., 16/(16 + 16)); if r = 0.1 Ro, then E = 16/(16 + 0.16) = 0.999999; and if r = 10 Ro, then E = 16/(16 + 106) = 0.000001 (!)


Primary Conditions for FRET

          Donor and acceptor molecules must be in close proximity (typically 10–100 Å).  The absorption spectrum of the acceptor must overlap fluorescence emission spectrum of the donor.   Quantitatively, the rate of FRET is determined in part by the degree of spectral overlap defined by the overlap integral, J(l) (see Lakowicz, 1999 for a full derivation of the overlap integral).  Donor and acceptor transition dipole orientations must be approximately parallel.  This condition is usually met some portion of the time in freely diffusible materials (e.g., proteins in solution).  On a practical note, although the donor emission and acceptor excitation spectra must overlap, the fluorescence emission maxima of the donor and acceptor must be sufficiently separated so as to be measurable.



·        Clontech brochure on Living ColorsTM autofluorescent proteins.

·        Ali, R., Zielinski, R.E., and Berkowitz, G.A. (2006) Expression of plant cyclic nucleotide-gated cation channels in yeast. J.Exp. Bot. 57, 125-138.

·        Hua, B.-G., Mercier, R. W., Zielinski, R. E., and Berkowitz, G. A. (2003)  Functional interaction of calmodulin with a plant cyclic nucleotide gated cation channel. Plant Physiol. Biochem. 41, 945-954.

·        Lakowicz, J.R.  1999.  Principles of Fluorescence Spectroscopy, 2nd ed., Kluwer Academic/Plenum Publishers, NY, 698 pp. (The “Bible” of fluorescence.  Chapter 13 gives a very thorough presentation of the theory and applications of FRET)

·        Miyawaki, A. and Tsien, R.Y. 2000. Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods Enzymol. 327, 472-500.

·        Molecular Probes, Inc. This site has a tremendous amount of information on applications of fluorescence technology, including FRET.

·        Persechini, A. and Cronk, B. 1999.  The relationship between the free concentrations of Ca2+ and Ca2+- calmodulin in intact cells. J. Biol. Chem. 274, 6827-6830.

·        Romoser, V. A., Hinkle, P M.. and Persechini, A.  1997. Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence: A new class of fluorescent indicators.  J. Biol. Chem. 272, 13270-13274.