Novel fluorescent indicator proteins for monitoring free intracellular Ca2+

Cd/ Calcium (1997) 22(3), 209-216 0 Pearson Professional Ltd 1997

Research

Novel fluorescent indicator proteins for monitoring free intracellular Ca*+ Anthony Persechini, Jennifer A. Lynch, Valerie A. Romoser Department

of Pharmacology

and Physiology,

University

of Rochester

Medical

Center,

Rochester,

New York, USA

Summary We have recently described a fluorescent indicator protein in which red- and blue-shifted variants of green fluorescent protein are joined by the calmodulin-binding sequence from smooth muscle myosin light chain kinase [Romoser V.A., Hinkle P.M., Persechini A. Detection in living ceils of Ca2+-dependent changes in the fluorescence 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 1997; 272: 13270-132741. The fluorescence emission of this protein at 505 nm (380 nm excitation) is reduced by -65% when (Ca’+),-calmodulin is bound, with a proportional increase in fluorescence emission at 440 nm. We have found that fusion of an engineered calmodulin, in which the C- and N-terminal EF hand pairs have been exchanged, to the C-terminus of this protein results in a novel indicator that responds directly to changes in the Ca2+ ion concentration, with an apparent K, value of 100 nM for Ca2+ in the presence of 0.5 mM Mg2+. The affinity of the indicator for Ca2+ can be decreased by altering the amino acid sequence of the calmodulin-binding sequence to weaken its interaction with the intrinsic calmodulin domain. The fluorescence emission of this indicator can be used to monitor physiological changes in the free Ca2+ ion concentration in living cells.

The green fluorescent protein (GFP) from the jellyfish Aequorid Victoria spontaneously forms a fluorophore through an oxygen-dependent cyclization reaction involving Ser-65, Tyr-66 and Gly-67 [l]. The fluorescence characteristics of GFP can be modified by altering its amino acid sequence. This approach has led to the generation of two particularly useful classes of variants: one exhibiting red-shifted excitation spectra, with excitation maxima at -490 nm (compared with -400 nm for the native protein [Z]), and the other exhibiting blueshifted emission spectra, with emission maxima at -450 nm (compared with -505 nm for the native protein 131).

Mitra et al [4] have reported that fusion of red- and blueshifted GFP variants results in a protein with a fluorescence emission peak at 505 nm when excited at 385 nm, which is due to intramolecular fluorescence resonance energy transfer (FRET) from the blue- to the red-shifted fluorophore. This FRET is eliminated when the amino acid sequence linking the two GFP variants is cleaved by a specific protease [4]. We have constructed a similar fusion protein (FIP-CB,,) in which the GFP variants are linked by the Cah&binding sequence from smooth muscle myosin light chain kinase. When FIP-CB,, binds (Ca2+),-CaM, FRET is essentially eliminated and under 380 nm illumination the protein exhibits a 65% reduction in fluorescence emission at 505 nm and a 5-6-fold increase

Received 8 July 1997 Revised 29 July 1997 Accepted 30 July 1997

Abbreviations: BAPTA, 1 ,2-bis(2-aminophenoxy)ethane-N,N,N’,Nr-tetraacetic acid; TRH, thyrotropin releasing hormone; CaM, calmodulin; CaMCN, an engineered CaM in which residues 82-148 and 9-75 have been exchanged:

INTRODUCTION

Correspondence and Physiology,

tot Dr Anthony Persechini, Department University of Rochester Medical Center,

Avenue, Box 711, Rochester, NY 14642, Fax: +l 716 461 3259 E-mail: [email protected]

USA

of Pharmacology 601 Elmwood

FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; BGFP, blue-shifted GFP; RGFP, red-shifted GFP; FIP, fluorescent indicator protein; FIP-CBS,, a fluorescent indicator protein in which BGFP and RGFP domains are joined by the CaM-binding domain from smooth muscle myosin light chain kinase; FIP-CA, a fluorescent indicator protein binds Ca*+ by virtue of intrinsic EF hand Cap+-binding domains.

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in its 440/505 fluorescence emission ratio. These spectral changes are completely reversible and are of sufficient magnitude to be used for monitoring the intracellular free (Ca2+),-CaM concentration [5]. We have recently generated additional indicator proteins in which native or engineered CaMs are fused to FIP-CB,, or variants with modified &M-binding amino acid sequences. These indicators respond directly to changes in the free Ca2+ion with spectral changes similar to the those exhibited by FIP-CB,, in response to changes in the free (Ca2+)4-CaM concentration. MATERIALS Expression

AND

METHODS

and purification

of proteins

The vectors used for expression of Ca2+-binding fluorescent indicator proteins in bacteria are based on the FIP-CB,, expression vector that we have described previously [5]. The indicators used for this study were generated by fusing the N-terminus of CaMCN, in which the native C- and N-terminal EF hand pairs are exchanged [6], to the C-terminus of the blue-shifted GFP domain of FIP-CB,, or to variants with the CaM-binding linker sequence altered as shown in Figure 1. These manipulations were accomplished using standard techniques in molecular biology as detailed elsewhere [5,7]. The junction between the blue-shifted GFP domain and the N-terminus of CaMCN by the sequence: D-E-L-Y-K-L-E-A-D-Q-L-T, where the amino acids indicated in boldface are at the C-terminus of the GFP domain and the N-terminus of CaMCN, respectively. The ammo acids in italics, L-E, are encoded by the sequence of an XhoI site used to make the construction. Bacterial expression vectors were transformed into Eschetic~id coli strain BL21 @E3), and proteins were expressed and purified as described previously 151.The concentrations of fluorescent indicator protein solutions were estimated as described previously 151. In vitro

measurements

of FIP-CB,,

fluorescence

Steady-state fluorescence measurements were performed using a Photon Technology International (Monmouth Junction, NJ, USA) QuantaMasterTM photon counting spectrofluorometer. Reaction volumes (2-3 ml) were incubated at 37°C in a stirred cuvette. Excitation and emission slit widths were -5 nm. The standard buffer used for in vitro experiments contained 25 mM Tris, 0.1 M KC1 and 0.5 mM MgCI,, pH 74. For experiments in which the free Ca2+ion concentration was varied, buffers included 3 mM 1,2-bis(o-amino-5-5’-dibromophenoxy)ethane-N,N,N’,N’-tetraacetic acid (Br,BAPTA) was added to the standard buffer. Aliquots of CaCI, solutions Cell Calcium (1997) 22(3), 209-2 16

RGFP

R Q R Q

R Q R Q

K K K K

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BGFP

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A A A A

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R R Q Q

A A A A

I I I I

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L: L: L: L:

FIP-CA3 FIP-CA8 FIP-CA9 FIP-CA10

Fig. 1 Schematic for FIP-CA indicators. A cartoon depicting a possible structure for Ca2+-saturated FIP-CA,. CaMCN is depicted as two hemispheres joined by a flexible tether; bound Ca2+ ions are shown as filled circles. The smooth muscle myosin light chain kinase CaM-binding sequence is indicated by the heavy line connecting the red-shifted (RGFP) and blue-shifted (BGFP) GFP domains. The N-terminal lobe of CaMCN, with its exchanged EF hand pairs, is assumed to associate with the N-terminal region of the smooth muscle myosin light chain kinase CaM-binding sequence, which is opposite the orientation seen in the complex between this sequence and native CaM 1151. The unaltered CaMbinding sequence is present in FIP-CA,; R to Q substitutions made to produce the other FIP-CAs are indicated in the figure.

standardized by atomic absorbance spectroscopy were added to achieve various free Ca2+ ion concentrations, which were calculated using the MaxChelator program [8]. Data for titrations of fluorescent indicator proteins with Ca2+ion were fit to an equation of the form:

F--F ~ Fmax - Fmin

=

Eq. I

where F is the fluorescence emission measured at 505 nm, Fminis the value at a saturating Ca2+ concentration, Fmaxis the value in the absence of bound Ca2+, n is a cooperativity coefficient, and a is a correction for underestimation of Fmax.Data presented for Ca2+ titrations are representative of at least three determinations. Identical results were obtained in the presence and absence of 5 pM exogenous CaM. Transient-kinetic fluorescence measurements were performed at 37°C using a Kin-Tee (University Park, PA, USA) two-syringe stopped-flow fluorimeter with a dead time of - 1.8 ms. FIP-CA fluorescence emission was excited at 380 nm and was monitored using a 510 nm bandpass filter in the emission light path. Data from 6-8 individual experiments were averaged to obtain the final traces. 0 Pearson Professional Ltd 1997

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Emission A

Free [Ca”] Fig. 2 Titration of FIP-CA, and FIP-CA, with Caz+ ion. The final concentration of FIP-CA, or FIP-CA, used in titration experiments was 50 nM. The relative change in fluorescence emission at 505 nm was calculated as (Fman - F)/(F,,,a - F,J, where F is the measured emission and Fmin is the minimum emission. Curves were generated according to Equation 1 fluorescence emission, Fmax is the maximum with K, and n values of 100 nM and 1 .i3 for FIP-CA,, and 280 nM and 2.1 for FIP-CA,. A typical set of spectra for titration of FIP-CA, with CaZ+ ion is shown in the inset to the figure. Fluorescence emission decreases at 505 nm and increases at 440 nm when Cap+, ion is bound to the indicator. Spectra drawn with dashed lines represent the fluorescence emission of FIP-CB,, in the presence and absence of a saturating (Ca*+),-CaM concentration. In analogy with the Ca*+ indicators, FIP-CB,, fluorescence emission increases at 505 nm and decreases at 440 nm when (Ca’+),-CaM is bound.

Non-linear least-squares curve fits were performed using the Prism software package (GraphPad, Inc.).

measured at 500 ms intervals with a 5 10 nm bandpass filter in the emission light path.

Measurements microinjected

RESULTS

of FIP-CA, cells

fluorescence

in

Human embryonic kidney cells (HEK-293) stably transfected with an epitope-tagged TRH receptor [9] were grown on glass coverslips to 60-80% confluence, rinsed in Hanks balanced salt solution, and placed in a SykesMoore chamber maintained at 37°C. Microinjections were performed on an Eppendorf Transjector 5246 equipped with a Micromanipulator 5 171 using Femtotips from Eppendorf (Madison, WI, USA). Microinjection solutions containing 100 PM FIP-CA3 were centrifuged and injected at pressures of 50-100 hectopascals for 0.1 s. Successful injections were visualized in brightfield and by observing at 530 nm the fluorescence emission of RGFP excited directly at 495 nm. Cells were allowed to recover for at least 30 min after microinjections. Dynamic measurements were then performed using a Dage CCD72 camera and Geniisys image intensifier system (Michigan City, IN, USA) and IMAGE-l/AT analysis software from Universal Imaging (Media, PA, USA). The fluorescence emission of FIP-CA, excited at 380 nm was

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DISCUSSION

We initially investigated the feasibility of constructing Caz+ indicators based on FIP-CB,, by fusing it either to native calmodulin (CaM) or to an engineered CaM in which the N- and C-terminal EF hand pairs have been exchanged (CaMCN). The latter approach was taken based on molecular modeling studies, which indicate that with native CaM bound to the linker in FIP-CB,, fusion of the N-terminus of CaM to the C-terminus of the BGFP creates a sterically unfavorable structure. With CaMCN bound to FIP-CB,,, the positions of the two EF hand pairs should be swapped relative to native CaM 161,which places the Nterminus of the protein in a more sterically favorable position in relation to the C-terminus of the BGFP domain. Consistent with this, we have found that fusion of native CaM to FIP-CB,, results in indicators whose fluorescence emission spectra exhibit complex dependencies on the free Ca2+concentration (data not shown). In contrast, the fluorescence emission spectra of indicators based on fusions with CaMCN exhibit monophasic dependencies on the free Ca2+concentration.

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For this study we produced four different FIP-CAs with integral CaMCN domains (Fig. 1). In FIP-CA,, CaMCN is fused to the FIP-CB,, sequence as described under Materials and methods. Based on the thermodynamics of the system, weakening the interaction between the integral CaM-binding sequence and the CaMCN domain should reduce the affinity of the indicator for Ca2+ ion [ 10,111. To determine whether this approach could be used to alter the indicator Ca2+-binding properties, FIPCA,, FIP-CA,, and FIP-CA,, were generated by replacing Arg residues in the Can/l-binding linker sequence with Gln residues, as indicated in Figure 1. Since the positivelycharged residues in CaM-binding sequences are thought to play a critical role in establishing the CaM-binding affinities of these regions, these replacements would be expected to weaken the interaction between the CaMCN domain and the linker [ 12,131. The fluorescence emission spectra of all four FIP-CAs examined exhibit essentially identical changes in response to bound Ca2+ion. FIP-CA, and FIP-CA, also have the same apparent affinities for Ca2+ion, while the apparent affinities of both FIP-CA,, and FIP-CA, are -3-fold weaker. These observations suggest that only the two Arg residues in the C-terminal half of the &M-binding sequence contribute significantly to the apparent Ca2+-binding affinities of the indicators. Due to the redundancies in properties of these indicators, only FIP-CA, and FIP-CA, were characterized in detail. Data for spectral titrations of FIP-CA, and FIP-CA, with Ca2+ ion are shown in Figure 2. Data for both indicators were fit according to Equation 1 with co-operativity coefficients of -2, indicating that changes in FRET are coupled to binding of two Ca2+ ions. This suggests that binding of Ca2+to one of the EF hand pairs in CaMCN is spectrally silent. The spectral Kd values derived for binding of Ca2+to FIP-CA, and FIP-CA, are 100 and 280 nM, respectively. A representative set of emission spectra determined for FIP-CA, at free Ca2+ ion concentrations ranging from I0 uM are shown in the inset to Figure 2; essentially identical Ca2+-dependent spectral changes are seen with FIP-CA,. The fluorescence emission of FIP-CA, or FIP-CA9 at 505 nm decreases by -30% when Caz+ is bound, with a corresponding increase in emission at 440 nm. The 4401505 emission ratios for the two indicators range from - 1.5 in the absence of bound Caz+ to -2.5 at a saturating Ca2+ concentration (Fig. 3). The maximal responses of two indicators are about half that of FIP-CB,, (Fig. 2). We am currently attempting to improve the magnitude of the indicator response by changing the length and composition of the amino acid sequence connecting the GFP and calmodulin domains and by utilizing brighter GFP variants. of FIP-CA, and FIP-CA, The pH-dependencies responses were determined at pH 6.5, ZO and 24 (Fig. 3). Cell Calcium (1997) 22(3),

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Fig. 3 The effect of pH changes on the 440/505 emission ratios for FIP-CA, and FIP-CA, measured at different free Ca*+ concentrations. Data for FIP-CA, (upper panel) and FIP-CA, (lower panel) wore measured at pH 6.5 (open triangles), 7.0 (filled triahgles) and 7.4 (filled squares). The buffer conditions were as described under Materials and methods except that, at pH 6.5 and 7.0, 25 mM Tris-HCI was replaced by 50 mM MOPS. A 50 nM final concentration of indicator was used for these experiments.

As the pH is decreased from 24 to 6.5, the fluorescence emission of the indicators at a nominal free Ca2+ concentration below 10 nM increases at 440 nm and decreases at 505 nm, consistent with a decrease in FRET between the RGFP and BGFP chromophores. However, these spectral changes are superimposed on an overall decrease in the fluorescence emission at both 440 and 505 nm, so it was felt that these spectral data are best presented as 440/505 emission ratios. The apparent Kd values for Ca2+ binding to FIP-CA, and FIP-CA, are not significantly affected by changes in pH over the range examined (Fig. 3). This is consistent with the observation that between values of 6.5 and Z4, changes in pH have no apparent effect on either Ca2+-binding to CaM or binding of (Ca2+),-CaM to FIP-CB,, ([14]; A. Persechini, unpublished observations). Changes in the indicator are 0 Pearson

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Time (s) Fig. 4 Time courses for the increase in FIP-CA fluorescence emission at 510 nm associated with release of Ca*+ from the indicators. For measurements of the increase in fluorescence after chelation of Ca2+ ion, syringe A contained 400 nM FIP-CA in the standard buffer plus 20 f.rM CaCI, and syringe B contained the standard buffer plus 10 mM EGTA. Data for FIP-CA, (open symbols) and FIP-CA, (filled symbols) are shown. Data for both fluorescence time courses were fit to double exponential equations. For FIP-CA,, the rate constants are 7.6 and 0.63 s-l, and for FIP-CA, the rate constants are 34.4 and 1.5 s-‘. For both indicators, the two rate processes each correspond to half the total amplitude of the fluorescence change.

most significant at low Ca2+ ion concentrations (Fig. 3). Between pH 70 and 6.5, more extensive changes in the emission ratios occur, although for FIP-CA, these are still substantially greater at low Ca2+ concentrations (Fig. 3). Thus, these indicators appear suitable for use at a controlled pH near the normal physiological value. Stopped-flow experiments were performed at 37°C to determine the maximum response times for these indicators. FIP-CB,, FIP-CA, and FIP-CA, all exhibit a significant mixing artefact consisting of a rapid increase in fluorescence emission at 5 10 mn, followed by a decrease in fluorescence that occurs with an apparent rate constant of 150-200 s-l. This process obscures all other fluorescence transients with rates greater than -50 s-l. For purposes of assessing the response times of the indicators this limitation is not serious, since the rate-limiting fluorescence transitions for the indicators appear to be much slower than 50 s-l. There is biphasic increase in fluorescence emission at 5 10 nm when Caz+ is released from the indicators, with each phase accounting for half the total magnitude of the fluorescence increase (Fig. 4). The rate constants for the two phases are 0.63 and 76 s-l for FIP-CA,, and 1.4 and 34.4 s-l for FIP-C%. As seen in Figure 5, when the Ca2+-depleted indicators are rapidly mixed with a high CaZ+ concentration Q 25 I.&I), both exhibit a rapid initial decrease in fluorescence, followed by a relatively small amplitude decrease with a rate constant of 1.8 s-’ for FIP-C& and 1.3 s-l for FIP-CA,. The slower process is independent of the Ca2+ concentration. Thus, 0 Pearson

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either indicator responds rapidly enough to monitor increases in the Ca2+concentration occurring with a halftime of 0.5 s or greater, FIP-CA, is also suitable for measuring decreases in the Ca2+ concentration occuring with similar rates, while FIP-CA, can be used to monitor decreases occurring with half-times greater than 1 s. Both of these indicators would appear to respond rapidly enough to monitor changes in [Caz+],in non-excitable cells. We have investigated the potential utility of these indicators for monitoring [Ca2+li in HEK-293 cells stably transfected with the Gq,-coupled Ca2+-mobilizing receptor for thyrotropin releasing hormone (TRH). The 380 mn excited fluorescence emission at 510 mn was monitored at 500 ms intervals in cells microinjected with FIP-CA, while [Ca2+li was manipulated (Fig. 6). The maximum fractional reduction in fluorescence emission measured in cells is - 15*/o, which is half the maximum Ca2+-dependent reduction measured in vitro. We have previously observed that the maximum fractional reduction in the FIP-CB,, fluorescence emission at 510 nm measured in cells co-injected with CaM is also about half the maximum fluorescence decrease measured in vitro [5]. As noted previously for FIP-CB,,, microinjected FIP-CA, exhibits a diffuse pattern of fluorescence and appears to he excluded from the nucleus [5]. Measurements performed using Fura- indicate that [Ca2+li in these cells exceeds 1 FM after addition of Ca2+ and ionomycin [5]. Given that Fm, is the fluorescence emission at 5 10 nm after addition of Ca2+and ionomycin, Cell Calcium

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Time (s) Fig. 5 Time courses for the decrease in FIP-CA fluorescence emission at 510 nm associated with Ca2+ binding to the indicators. For measurements of the fluorescence decrease associated with Cap+ binding to the indicators, syringe A contained 400 nM FIP-CA in the standard buffer plus 100 PM EGTA and syringe B contained the standard buffer plus 150 f.rM CaCI,. Data for FIP-CA, (open symbols) and FIP-CA, (filled symbols) are shown. Data for both fluorescence time courses were fit to double exponential equations. For FIP-CA, the rate constants are 151 and 1.3 s-l, and for FIP-CA, the rate constants are 189 and 1.8 s-j. The faster rate process is dominated by a mixing artifact (see Results and Discussion).

and assuming that Fmaxis the fluorescence emission at 510 nm after addition of BAPTA, we can estimate [Caz+], according to the relation:

Eq. 2

[Ca”], = I$ (S)“”

where F is the fluorescence emission measured at 510 nm and values for Kd (100 nM) and n (1.8) are those determined from in vitro titration data (Fig. 2). Using this approach, we estimate that [Ca2+li was initially 75 nM in the group of cells examined and that it was reduced to below 50 nM after addition of 3 mM BAPTA. The indicator response appears to be close to saturation at the [CaZ+], evoked by 4 )LM TRH (Fig. 6). Based on a Kd value of 100 nM for CaZCbinding to the indicator, the indicator response should be 80% saturated at a [Caz+li of 400 nM. Thus, the maximal [Caz+li produced in response to TRH certainly exceeds 230 nM, and is probably 2 400 r&I. These values for [Ca2+11are consistent with those estimated under similar conditions using Fura- [5]. The indicators we have described represent a new tool for monitoring [Ca2+li in living cells. The effective range of these indicators is about 5-fold in [Ca2+li,and their cooperative dependencies on the free Caz+ concentration Cell Calcium

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make them well suited to monitoring small changes in [Ca2+li. It is clear that indicators with different Kd values for CaZ+can be easily engineered. These indicators bind CaZ+ion through the action of EF hand structures, just as do most physiologically important Ca2+-binding proteins. Thus, the [Ca2+li reported by FIP-CAs should accurately reflect the effective free CaZ+concentration experienced by the EF hand Ca’+-binding proteins in the cell. What sets these indicators apart from others that are currently available is that they combine the following characteristics: 1. Ca2+-dependent changes in their emission spectra can be monitored as the ratio of the emission at 440 and 505 mn, which provides the advantages of ratiometric quantitation, notably its relative insensitivity to variations in path-length and concentration. 2. Their Ca2+-dependent fluorescence responses are fully reversible and require no specialized cofactors. 3. They can be easily manufactured using simple procedures.

and engineered

4. They can be expressed transiently or stably in cells 0 Pearson

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Fig. 6 Changes in FIP-CA, fluorescence emission measured in HEK-293 cells stably expressing the TRH receptor. Data are presented as the relative change in fluorescence emission at 510 nm (F/FO), where FL) is the average of the first 10 measurements. The trace shown represents the average of data from 11 cells. Additions of 3 mM BAPTA, 4 uM TRH, 5 mM CaCI, and 500 nM ionomycin were made at the indicated times. Cells were microinjected with purified FIP-CA, at a concentration of 100 pM; we estimate the final intracellular concentration to be from I-10 PM. This is consistent with the 6-fold range seen in the initial measurements of fluorescence emission in the 11 cells examined. Estimated values for [Ca*+li were calculated as described in the text.

(A. Persechini, unpublished observations) and can presumably be targeted to different regions in the cell by fusion to protein segments responsible for specific localization and/or transport. A particularly exciting possibility is to express FIPCAs in transgenic animals, which would greatly facilitate in situ detection of fluctuations in [Caz+li Okabe et al [ 161 have recently demonstrated ubiquitous expression of a GFP variant in transgenic mice, indicating that it should be possible to similarly express FIP-CAs. ACKNOWLEDGEMENTS

This work was supported by PHS Grant No. DK44322 to AP, and National Science Foundation Predoctoral Fellowship No. DGE9253919 to VAR. We would like to thank P. M. Hinkle for use of facilities (supported by PHS Grant No. DK19042 to P. M. Hinkle) for cell microinjections and for measurements of FIP-CA, fluorescence in cells.

REFERENCES 1. Cubitt A.B., Heim R., Adams S.R.,Boyd A.E., Gross L.A., Tsien R.Y. Understanding, improving and using green fluorescent proteins. Trends Biochem Sci 1995; 20: 448-455. 0 Pearson

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2. Delegrave S., Hawtin R.E., SiIva C.M., Yang M.M., Youvan D.C. Red-shifted excitation mutants of the green fluorescent protein. Bio/Technology 1995; 13: 151-154. 3. Heim R., Tsien R.Y. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curv Bioll996; 6: 178-182. 4. Mitra R.D., Silva C.M., Youvan DC. Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein. Gene 1996; 173: 13-12 5. Romoser V.A., HinkIe P.M., Persecbini A. Detection in living cells of Ca*+-dependent changes in the fluorescence of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators. JBiol Chem 1997; 272: 13270-13274. 6. Persechini A., Gansz KJ., Paresi RJ. Activation of myosin light chain kinase and nitric oxide synthase activities by engineered calmodulins with duplicated or exchanged EF hand pairs. Biochemistry 1996; 35: 224-228. 7 Persechini A., Stemmer P.M., Ohashi I. Localization of unique functional determinants in the calmodulin lobes to individual EF hands. JBiol Chem 1996; 271: 32217-32225. 8. Bers D., Patton C., Nuccitelli R. A practical guide to the preparation of CaZ+buffers. In: Nuccitelli R. (Ed.) A Practical Guide to the Study of Calcium in Living Cells, Vol. 40. New York: Academic Press, 1994; 3-29. 9. Shupnik M.A., Week J., Hi&e P.M. Thyrotropin (TSII-releasing hormone stimulates TSH beta promoter activity by two distinct mechanisms involving calcium influx through L type Ca2+ channels and protein kinase C. Mel EndoM-inoZ 1996; 10: 90-99. Cell Calcium

(7997)

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10. Olwin B.B., Edehnan A.M., Krebs E.G., Strom D.R. Quantitation of energy coupling between Caz+,calmodulin, skeletal muscle myosin light chain kinase, and kinase substrates. JBiol Chem 1984; 259: 10949-10955. 11. Olwin B.B., Storm D.R. Calcium binding to complexes of calmodulin and calmodulin binding proteins. Biochemistry 1985; 24: 8081-8096. 12. Wintrode P.L., Privalov P.L. Energetics of target peptide recognition by calmodulin - a calorimetric study. J Mel Biol 1997; 266: 1050-1062.

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13. Crivici A., Ikura M. Molecular and structural basis of target recognition by calmoduhn. Annu Rev Biophys Biomol Struck 1995; 24: 85-116. 14. Linse S.,Helmersson A., For&n S. Calcium binding to calmodulin and its globular domains.JBiol Chem 1991; 266: 8050-8054. 15. Meador W.E., Means A.R., Quiocho F.A. Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulinpeptide complex. Science 1992; 257: 1251-1255. 16. Okabe M., Ikawa M., Kominami K., Nakanishi T., Nishimune Y. Green mice as a source of ubiquitous green cells. FEBS Lett 1997; 407: 3 13-3 19.

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