Uranium-chelating peptides derived from EF-hand calcium-binding motif useful for uranium biodetection and biodecontamination

Abstract

Uranium-chelating polypeptides comprising at least one helix-loop-helix calcium-binding (EF-hand) motif which comprises a deletion of at least two amino acid in the 12-amino-acid calcium-binding loop sequence, and their use for uranium biodetection and biodecontamination.

Claims

1. A polypeptide comprising at least one helix-loop-helix calcium-binding (EF-hand) motif with deletion of two amino acid residues in the 12-amino-acid calcium-binding loop sequence selected from the group consisting of deletion in positions 1 and 2, and deletion in positions 2 and 3, wherein said polypeptide binds uranyl.

2. The polypeptide of claim 1, wherein said EF-hand motif(s) are derived from signaling EF-hand protein(s) of the calmodulin superfamily selected from the group consisting of calmodulin and troponin C.

3. The polypeptide of claim 1, which comprises two or four EF-hand motifs, wherein at least one EF-hand motif comprises said deletion in the calcium-binding loop sequence, and the other EF-hand motif(s) comprise said deletion or not.

4. The polypeptide of claim 2, which is a calmodulin domain 1 variant comprising two EF-hand motifs, respectively from the EF-hand1 and the EF-hand2 of calmodulin protein(s).

5. The polypeptide of claim 3, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 20 and 60.

6. A fusion protein comprising a polypeptide according to claim 1 fused to another protein moiety.

7. The fusion protein of claim 6, which is a cameleon protein comprising tandem fusions of a fluorescence-donor protein, a polypeptide which comprises two or four EF-hand motifs, wherein at least one EF-hand motif comprises said deletion in the calcium-binding loop sequence and the other EF-hand motif(s) comprise said deletion or not, and a fluorescence-acceptor protein.

8. The fusion protein of claim 7, which comprises a sequence selected from the group consisting of SEQ ID NO: 35, 38, 61, 63 and 67.

9. The polypeptide of claim 1 or a fusion protein comprising a polypeptide according to claim 1 fused to another protein moiety, which is immobilized onto a solid support.

10. A polynucleotide encoding the polypeptide of claim 1.

11. A host cell comprising with the polynucleotide of claim 10, wherein the host cell is not an organism.

12. A non-human transgenic organism comprising the polynucleotide of claim 10.

13. A method of detecting uranium contamination in a sample comprising contacting the sample with a uranyl chelating agent comprising: i. the polypeptide of claim 1, ii. a fusion protein comprising a polypeptide according to claim 1 fused to another protein moiety, iii. a host cell comprising a polynucleotide encoding the polypeptide of claim 1 or a fusion protein comprising a polypeptide according to claim 1 fused to another protein moiety, or iv. A non-human transgenic organism comprising a polynucleotide encoding the polypeptide of claim 1 or a fusion protein comprising a polypeptide according to claim 1 fused to another protein moiety, and detecting the presence of uranyl chelation in the sample.

14. A method of decontaminating or bio-remediating a sample containing uranium comprising contacting the sample with a uranyl chelating agent comprising: i. the polypeptide of claim 1, ii. a fusion protein comprising a polypeptide according to claim 1 fused to another protein moiety, iii. a host cell comprising a polynucleotide encoding the polypeptide of claim 1 or a fusion protein comprising a polypeptide according to claim 1 fused to another protein moiety, or iv. A non-human transgenic organism comprising a polynucleotide encoding the polypeptide of claim 1 or a fusion protein comprising a polypeptide according to claim 1 fused to another protein moiety.

15. The polypeptide of claim 3, which consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 20 and 60.

16. The fusion protein of claim 7, which consists of a sequence selected from the group consisting of SEQ ID NO: 35, 38, 61, 63 and 67.

17. A polynucleotide encoding a fusion protein comprising the polypeptide according to claim 1 fused to another protein moiety.

Description

(1) In addition to the above arrangements, the invention also comprises other arrangements, which will emerge from the description which follows, which refers to exemplary embodiments of the subject of the present invention, with reference to the attached drawings in which:

(2) FIG. 1 is a schematic of calmodulin. A. Schematic of domain 1 of Paramecium tetraurelia calmodulin (PDB code 1EXR) which comprises two calcium-binding sites (site 1 and site 2) which are part of a canonical EF-hand motif (EF-hand1 and EF-hand2, respectively for site 1 and site 2). B. Structural model of the calcium-binding site 1 of Arabidopsis thaliana calmodulin showing the coordinating amino acids. C. Amino acid sequence of calmodulin site 1 from Arabidopsis thaliana (CaM-WT) and from the peptide CaM1 and CaMΔ analysed in the present study. In the CaM1 and CaMΔ peptides, the metal-binding ability of site 2 was impaired by replacing the aspartate residues in positions 57 and 59 of calmodulin (positions 58 and 60 of CaM1) with alanines. In addition, the threonine residue in position 31 and the lysine residue in position 32 of CaM1 (positions 10 and 11 of site 1) have each been substituted with an alanine residue to obtain an efficient CK2 consensus sequence that targets phosphorylation of threonine 9 (T9) of the metal-binding loop (T30 in the peptide) and a tyrosine was introduced at position 28 of peptide CaM1 (position 7 of the metal-binding loop) to allow the monitoring of uranyl and calcium-binding and the determination of their binding affinities, by following tyrosine fluorescence emission at 302 nm.

(3) FIG. 2 represents Structural models obtained by molecular dynamics of the CaM1P-U (A), CaMΔP-U (B) CaM1-U (C) and CaMΔ-U (D) complexes. E=glutamic acid, D=aspartic acid, Y=tyrosine, T=threonine, Tp=Phosphothreonine.

(4) FIG. 3 shows the binding thermograms of the CaMΔ and CaMΔP peptides with uranyl at pH 6 (A) and pH 7 (B). Conditions: 10 μM CaMΔ or CaMΔP, 100 μM IDA, 20 mM MES and 100 mM KCl. Full square: CaMΔP. Full circle: CaMΔ.

(5) FIG. 4 shows the binding thermograms of the CaMΔ with uranyl in the absence (empty triangle) and in presence (empty circle) of 10 mM CaCl.sub.2 Conditions: 10 μM CaMΔ, 100 μM IDA, 20 mM MES and 100 mM KCl.

(6) FIG. 5 shows the growth curve of three E. coli strains transformed with respectively, the expression vector without insert (as a control) or vectors expressing recombinant CaM1 or CaMΔ peptide, in the presence or absence of uranyl acetate (U). Control (empty circle). Control+U (full circle). CaM1 (empty triangle). CaM1+U (full triangle). CaMΔ(empty square). CaMΔ+U (full square). IPTG was added at t=105 min; 50 μM uranyl acetate (U) or 100 μM sodium acetate were added at t=135 min.

(7) FIG. 6 shows the emission spectra of cameleon biosensor WT protein (excited at 440 nM) at varying CaCl.sub.2 concentrations between 0 μM to 10 μM. 0 μM Ca (full diamond). 2 μM Ca (empty circle). 4 μM Ca (empty triangle). 6 μM Ca (full circle). 8 μM Ca (cross). 10 μM Ca (empty square). Vertical arrow indicates an increase in FRET detection with increasing Ca concentrations.

(8) FIG. 7 shows the emission spectra of cameleon biosensor WT protein (excited at 440 nM) at varying uranyl nitrate concentrations between 0 μM to 10 μM. 0 μM UO.sub.2 (full black circle). 2 μM UO.sub.2 (empty square). 4 μM UO.sub.2 (full triangle). 6 μM UO.sub.2 (empty circle). 8 μM UO.sub.2 (full grey circle). 10 μM UO.sub.2 (empty diamond). Vertical arrow indicates an increase in FRET detection with increasing uranyl concentrations.

(9) FIG. 8 shows the transmission ATR-FTIR spectra of the gold strip covered with a 5 nm layer of polyacrylic acid and the CaMΔ(upper spectrum), with the polyacrylic acid layer only (middle spectrum) and with a 50 nm layer of polyacrylic acid and the CaMΔ(lower spectrum).

(10) FIG. 9 shows XPS spectra recorded on the grafted gold strip. The upper spectrum is recorded in a spot exposed to the protein and the lower spectrum is recorded on a spot that was not contacted with the protein solution.

(11) FIG. 10 shows structural models of the Ef-hand2 obtained by molecular dynamics simulations. This figure shows that uranyl coordination may be efficiently achieved in shorter binding loops, i.e. using deletion of at least two residues.

EXAMPLE 1

Construction and Characterization of Calmodulin Peptides CaMΔ, CaMΔ3 and CaMΔ-WT

(12) 1. Methods

(13) The recombinant peptides were produced in E. coli. A histidine-tag followed by the Tobacco Etch Virus protease (TEV) recognition sequence was introduced at the N-terminus, allowing the purification of the pepti using two subsequent chromatography steps on Ni-columns.

(14) 1.1 Engineering and Purification of Calmodulin Derived Peptides

(15) The CaM1 construct containing the Arabidopsis thaliana sequence of calmodulin domain 1 was obtained as previously described (Pardoux et al., PLoS One, 2012, 7, e41922) and used as a template for new constructs. The CaM1 construct (nucleotide sequence SEQ ID NO: 39/amino acid sequence SEQ ID NO: 40) comprises the following mutations, by reference to CaM1 amino acid sequence: (1) C28Y mutation to allow the monitoring of uranyl- and calcium-binding and the determination of their binding affinities, by following tyrosine fluorescence emission at 302 nm, (2) T31A and K32A mutations to enable efficient phosphorylation of T30 by CK2, and (3) D58A and D60A mutations to inactivate the metal-binding site 2 of domain 1.

(16) To obtain CaMΔ construct (nucleotide sequence SEQ ID NO: 16/amino acid sequence SEQ ID NO: 17), deletions of K23 and D24 were produced with the QuickChange site-directed mutagenesis kit (STRATAGENE) and specific primer pairs DGD S (SEQ ID NO: 41) and DGD AS (SEQ ID NO: 42), according to the manufacturer's instructions. The engineering plasmid was called pQE-CaMΔ.

(17) To obtain CaMΔ3 construct (nucleotide sequence SEQ ID NO: 55/amino acid sequence SEQ ID NO: 56), deletion of D24 was produced with the QuickChange site-directed mutagenesis kit (STRATAGENE) and specific primer pairs S-Δ3Y (SEQ ID NO: 57) and AS-Δ3Y (SEQ ID NO: 58), according to the manufacturer's instructions. The engineering plasmid was called pQE-CaMΔ3.

(18) We also produced CaMΔ-WT (nucleotide sequence SEQ ID NO: 59/amino acid sequence SEQ ID NO: 60). The protein sequence is the same than those of CaMΔ except that A31 and A32 were replaced by T31 and K32.

(19) Recombinant fusion proteins expressed in E. coli strain M15Rep4 (QIAGEN) were grown at 37° C. in LB medium containing ampicillin (50 μg/mL) and kanamycin (50 μg/mL). Expression was induced by addition of 0.1 mM isopropyl-D-thiogalactoside once OD.sub.600 reached 0.5, and the cultures were further incubated for 5 h at 37° C. Cellular extracts were obtained by French press lysis and a centrifugation step of 30 min at 15000 rpm, and were applied at a 1 mL/min flow rate on a 5 mL HiTrap Chelating Column (GE HEALTHCARE) in buffer A (50 mM Tris-HCl, 0.5 M NaCl, 25 mM imidazole buffer pH 7.5) containing 1 mM 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). The proteins were eluted from the nickel resin at a 4 mL/min flow rate using buffer A supplemented with 150 mM imidazole. The proteins were dialyzed against buffer A and the His-Tags were removed by incubation overnight at 4° C. with TEV protease, followed by separation using a HiTrap Chelating Column. Recombinant proteins were dialyzed against 50 mM Tris-HCl, 150 mM NaCl, pH 7.5. The protein concentrations were measured according to the BC Assay (UPTIMA) with bovine serum albumin as standard. The proteins were concentrated using the Microcon filtration system (Amicon Millipore®), with a cut-off point of 3 kDa.

(20) Phosphorylation of the peptide CaMΔ was performed as previously described in Pardoux et al., PLoS One, 2012, 7, e41922.

(21) 1.2 Tyrosine Fluorescence Titrations

(22) The metal-binding affinity of the various peptides for calcium and uranyl was examined by monitoring the fluorescence intensity of the single tyrosine residue (Tyr28).

(23) The uranyl solutions were prepared extemporaneously by diluting a 0.1 M stock solution of uranyl nitrate (pH 3.5, stored frozen at −20° C.) in the final buffer. Fluorescence titrations in the presence of uranyl were performed using a 10 μM peptide solution in MES (20 mM, pH 6) or Tris (20 mM, pH 7) buffer with 100 mM KCl and 100 μM iminodiacetate (IDA). Fluorescence titrations in the presence of calcium were performed in a 10 μM peptide solution in MES (20 mM, pH 6) or Tris (20 mM, pH 7) buffer with 100 mM KCl. To remove any trace of calcium from the samples, each sample solution was incubated 1 h with Chelex®-100 before uranyl or calcium addition.

(24) Spectra were collected on a Cary Eclipse spectrofluorimeter at 25° C., with 270 nm excitation. Emission was observed from 290 to 350 nm. The excitation and emission slits were 10 nm. A 15 min equilibration time was respected before each measurement. The reported stability constants are averages of three experimental values.

(25) Competition experiments between calmodulin-derived peptides CaM peptides and IDA were performed to determine the conditional dissociation constants of the peptide-uranyl complexes at pH 6 and pH 7. IDA has a moderate affinity for uranyl and forms three major complexes: UO.sub.2IDA, [UO.sub.2(IDA).sub.2].sup.2−, and [(UO.sub.2).sub.2(IDA).sub.2(OH).sub.2].sup.2−. The conditional stability constants of these three species were calculated from the pK.sub.as and the stability constants at 25° C. and 0.1 M ionic strength given by Jiang et al. (Inorg. Chem., 2003, 42, 1233-1240). These three conditional stability constants were fixed in the spectral data analysis, which was performed using the program SPECFIT (Binstead et al., Specfit Global Analysis System Version 3.0.34, 2003). Identical values were obtained for the conditional stability constants of the UO.sub.2-P complexes (where P stands for peptide), either considering that the UO.sub.2-P complex emits or not. In the former case, the spectrum of the UO.sub.2-P complex was calculated to be zero, as the fluorescence emission of tyrosine was totally quenched in the complex.

(26) For titrations in the presence of calcium, the conditional dissociation constants (K.sub.d) were determined by fitting the difference between fluorescence intensities measured in the presence (F) and in the absence (F0) of calcium, according to a one site saturation model: ΔF=(F.sub.max×[Ca])/(Kd+[Ca]) using SigmaPlot 10.0 software (Systat Software). In this equation, F.sub.max corresponds to the maximum of fluorescence determined by the software.

(27) 1.3 Exposure of E. coli Cells Producing or not the CaMΔ Peptide to Uranyl Toxicity

(28) E. coli cells were grown overnight in LB-MES 100 mM, pH 5.5. The cells where transferred to a LB-glucose (4 g/L) medium at pH 4.5, inoculated at 1/100 volume. IPTG was added at a OD.sub.600nm=0.4. After 30 minutes, 50 μM uranyl(acetate).sub.2 or 100 μM Na-acetate was added to the medium. Cell growth was followed by measuring the absorption at 600 nm.

(29) 2. Results

(30) 2.1 Binding Affinity of the CaMΔ, CaMΔ-WT and CaMΔ3 Peptides for Uranyl

(31) The peptides were prepared at a 10 μM concentration in 20 mM MES pH 6 or Tris pH 7, with 0.1 M KCl and 100 μM IDA. Increasing concentrations of uranyl nitrate were added to the peptide solution, until the peptide to uranyl ratio was approximately 1:4. By using this stoichiometric ratio, the protein samples were not affected by uranyl addition (as monitored by UV-Vis absorption), which is crucial for the interpretation of the results. Addition of uranyl nitrate decreased the fluorescence signal emitted by the single tyrosine present in the peptides at position 7 of the metal-binding loop (FIG. 3). Tyrosine fluorescence quenching by uranyl has been reported in the literature for other proteins such as transferrin.

(32) Conditional dissociations constants of the peptide—uranyl complexes (Kd) resulting from the competition experiments with IDA were determined at pH 6 and pH 7 for the CaMΔ peptide and at pH 6 for the CaMΔWT and CaMΔ3 peptides.

(33) Conditional dissociation constants of 1.8 (±0.5) 10.sup.−10 M and 2 (±0.1) 10.sup.−10 M were calculated at pH 6 and pH 7 for the CaMΔ—uranyl complex. There is no significant effect of pH on the affinity of the peptide CaMΔ for uranyl. The affinity of the CaMΔ peptide is two orders of magnitude greater than that of the CaM 1 peptide, possessing a 12 amino acid long binding loop, which has a Kd of 25 10.sup.−9 M at pH 6 (Pardoux et al., PLoS One, 2012, 7, e41922). Interestingly the peptide CaMΔ3, in which only one aspartate at position 3 of the loop has been deleted, has a much lower affinity for uranyl. A conditional dissociation constant of 130±10 10.sup.−9M was obtained for uranyl at pH 6. The affinity of this peptide for uranyl is 722 times lower than that of the CaMΔ peptide. It is also lower than the affinity of CaM1 for uranyl. This experiment demonstrates that it is not sufficient to suppress (at least) one of the aspartate ligands to increase the affinity for uranyl, but that structural factors significantly affect the affinity of the peptide binding loop for uranyl.

(34) Finally, a conditional dissociation constant of 2±0.1 10.sup.−10M was obtained for the CaMΔ-WT peptide, differing from the CaMΔ peptide by residues at positions 10 and 11 of the metal binding loop (numbering according to the native sequence of 12 AA). A threonine and a lysine are present in this peptide instead of two alanines in the CaMΔ peptide. The affinity for uranyl is equivalent to that of CaMΔ.

(35) The phosphorylated peptide CaMΔP presents similar binding affinities for uranyl, with conditional dissociation constants Kd=4 (±0.09) 10.sup.−10 M at pH 6 and Kd=1.3 (±0.3) 10.sup.−10 M at pH 7.

(36) Moreover, both peptides have a very low affinity for calcium. Conditional dissociation constants in the millimolar range were observed for the CaMΔ-Ca.sup.2+ complex at pH 6 (Kd>1 mM) and at pH 7 (Kd=8.7 mM). Similar dissociation constants were observed for the phosphorylated peptide CaMΔP with Kd of 7.8 mM at pH 6 and Kd=4.2 mM at pH 7.

(37) 2.2 Competition Experiments with Calcium

(38) The selectivity of the two peptides (CaMΔ and CaMΔP) for uranyl as compared to calcium is of the order of 10.sup.7. To check if this selectivity is actually observed in a medium containing both uranyl and calcium, the binding isotherm of uranyl was measured in the presence of 10 mM CaCl.sub.2 in the solution.

(39) FIG. 4 shows the superimposition of binding thermograms corresponding to Tyr fluorescence quenching (i.e. uranyl binding) in the absence and in the presence of 10 mM CaCl.sub.2. These results show that calcium has a very modest effect on uranyl titration.

(40) The results of this competition experiment show that these peptides can be used for uranyl detection in the presence of large concentrations of calcium.

(41) 2.3 the Expression of the CaMΔ Peptide in E. coli Cells Decreases Uranyl Toxicity to the Cells

(42) FIG. 5 shows growth curves recorded with E. coli cells exposed either to 50 μM uranyl acetate, or to 100 μM Na acetate as a control, in LB glucose medium at pH 4.5. Exposure conditions are detailed in the Methods. The FIG. 5 shows that uranyl exposure stops E. coli growth rapidly (full signs as compared to empty signs) except for the cells expressing the CaMΔ peptide. The growth of the cells expressing the CaMΔ peptide is lower than that of the other strains. This may be due to a too strong protein overexpression. However, addition of uranyl didn't affect anymore the growth of this strain. These results suggest that the chelation of uranyl by the CaMΔ peptide protect the whole cells by reducing uranyl toxicity.

(43) Conclusions

(44) CaMΔ peptide presents an affinity for uranyl in the subnanomolar range and a very high selectivity towards calcium. It is expressed in high quantities in E. coli cells.

(45) For these reasons, it is a promising tool for the development of biosensors (in vivo and in vitro) or efficient chelating systems in vitro.

(46) The use of the binding loop sequence of CaMΔ for the other sites of calmodulin may not be as efficient for selecting uranyl binding at these sites as at site one. Therefore, structural parameters that could increase the affinity of site 2 toward uranyl as well as its uranyl/calcium specificity were explored using molecular dynamics. Two indicative informations can be obtained using molecular dynamic simulations; the first one concerns the structural model; the second one concerns the stabilisation energy associated with uranyl binding on the protein. The structural models of the Ef-hand2 obtained by molecular dynamics simulations show that uranyl coordination may be efficiently achieved in shorter binding loops, i.e. using deletion of at least two residues (FIG. 10).

EXAMPLE 2

Construction and Characterization of Calmodulin Derived Cameleon Biosensors

(47) 1. Methods

(48) 1.1 Construction of Expression Vector for Cameleon Biosensors

(49) Cloning steps were made with standard methods using XL1Blue cells as E. coli strain. All mutations were made using a QuickChange site-directed mutagenesis kit (Stratagene) and specific primer pairs according to the manufacturer.

(50) The gene coding for the cameleon biosensor WT (denoted eCFP-CaM-Linker-M13-eYFP) was constructed in three steps. The gene encoding the wild-type CaM from A. thaliana fused with a linker and the CaM-binding peptide of myosin light-chain kinase (M13) was synthetized by Eurofins MWG and cloned into the pQE30 plasmid (QIAGEN) between Sac I and Sal I restriction sites.

(51) Then, the enhanced Cyan Fluorescent Protein (eCFP) gene containing the TEV protease recognition site upstream of the coding sequence of eCFP was PCR-amplified using the S-TEV-eCFP-BamHI (SEQ ID NO: 43) and AS-eCFP-SacI (SEQ ID NO: 44) primers and cloned upstream the CaM-linker-M13 gene, between BamH I and Sac I restriction sites.

(52) Finally, the enhanced Yellow Fluorescent Protein (eYFP) was PCR-amplified using the S-eYFP-SalI (SEQ ID NO: 45) and AS-eYFP-HindIII (SEQ ID NO: 46) primers and cloned downstream the CaM-linker-M13 gene, between the Sal I and Hind III restriction sites. Both genes contained no stop codon except for the eYFP gene. The cameleon biosensor WT corresponds to the cDNA of SEQ ID NO: 47 and the protein of SEQ ID NO: 48.

(53) The construction of expression vector for the cameleon biosensor Δ was made by using the cameleon biosensor WT gene as a template and primers S-Δ (SEQ ID NO: 49) and AS-Δ (SEQ ID NO: 50). The constructions of expression vectors for the cameleon biosensor WT-S2M or for the cameleon biosensor A-S2M (S2M corresponding to the inactivation of site 2 of the domain 1) were made using as a template the cameleon biosensor WT gene or the cameleon biosensor Δ gene respectively and primers S-S2M (SEQ ID NO: 51) and AS-S2M (SEQ ID NO: 52). The cameleon biosensor Δ corresponds to the cDNA of SEQ ID NO: 34 and the protein of SEQ ID NO: 35. The cameleon biosensor WT-S2M corresponds to the cDNA of SEQ ID NO: 53 and the protein of SEQ ID NO: 54. The cameleon biosensor Δ-S2M corresponds to the cDNA of SEQ ID NO: 36 and the protein of SEQ ID NO: 37.

(54) 1.2 Expression of the Cameleon Biosensors

(55) The recombinant vectors pQE30 containing the biosensor genes were introduced in the E. coli strain M15Rep4. Recombinant fusion proteins were expressed as follows: the overexpression strain was grown at 37° C. in LB medium containing ampicillin (50 μg/mL) and kanamycin (50 μg/mL) until OD.sub.600 reached 0.5. Expression was then induced by addition of 0.1 mM isopropyl-D-thiogalactoside (IPTG) and the cultures were further incubated for 20 h at 17C. Cells were collected by centrifugation 20 min at 5000 rpm, and the bacterial pellet was frozen and stored at −80° C.

(56) 1.3 Purification of the Cameleon Biosensors

(57) Bacteria were resuspended in buffer A (50 mM Tris-HCl, 0.5 M NaCl, 25 mM imidazole pH 7.5) containing 1 mM 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF)+15 g/mL DNAse1+30 mM MgSO.sub.4. The cellular extracts were obtained by French Press lysis and a centrifugation step of 30 min at 15 000 rpm. The cellular extracts were applied on a 5 mL HiTrap™ Column (GE Healthcare) in buffer A at 1 mL/min flow rate. The proteins were eluted from the nickel resin at 4 mL/min flow rate using an imidazole gradient. The proteins were dialyzed against buffer A and the His-Tags were removed by incubation overnight at 4° C. in presence of the TEV protease followed by separation using HiTrap Chelating Column. Gel Filtration was performed for further purification of the proteins using a 26/600 Superdex 200 column (GE HealthCare) and 50 mM Tris-HCl buffer, pH 7.5, supplemented with 150 mM NaCl. The protein concentrations were measured according to the BC Assay from Uptima with bovine serum albumin as standard. The proteins were concentrated using Microcon® filtration system (Amicon Millipore®, with a cut off of 10 kDa).

(58) 1.4 FRET Measurement

(59) Fluorescence experiments were performed using an Infinite 1000 (TECAN). Cameleon biosensor WT proteins were first incubated with an excess of ethylenediaminetetraacetic acid (EDTA) and dialyzed overnight against 50 mM Tris-Cl pH 7 containing Chelex® resin. This step is used to remove calcium likely to be present in the different CaM-binding sites. For each measurement, 1 μM of protein was mixed in 200 μL of 50 mM Tris-CI pH 7 buffer (treated with Chelex®) at 25° C. CaCl.sub.2 or uranyl nitrate were added at varying concentrations between 0 and 10 μM. Excitation was performed at 440 nm and the emission spectrum recorded between 450 and 570 nm.

(60) 2. Results

(61) The results obtained with the cameleon biosensor WT protein (FIGS. 6 and 7, the spectra were normalized at 476 nm) show that this biosensor is able to do some FRET in presence of calcium, and that the maximum of FRET is obtained at 8 μM of calcium and above. Similar results are obtained with 8 μM of uranyl nitrate, showing that the cameleon biosensor obtained with the WT calmodulin shows similar sensitivities for calcium and uranyl.

EXAMPLE 3

Uranyl Chelation by CaMΔ Immobilized on a Metallic Surface

(62) This example illustrates the possibility to use peptides derived from calmodulin immobilized on a solid support to chelate uranyl from a solution.

(63) 1. Methods

(64) The peptide CaMΔ was grafted on a gold strip surface modified by a 5 to 200 nm thick layer of polyacrylic acid, using activation via succinimide esters. A solution of CaMΔ at 100 g/mL in MES buffer 20 mM was used. Stable grafting of the protein was verified by Fourier transform infrared spectroscopy monitoring of the presence of the two amide I and amide II bands characteristic for proteins (FIG. 8). The protein was grafted on one half of the gold strip, the other half being used as a control area to identify possible nonspecific adsorption of uranyl. The interaction with uranyl was performed by a 2 hours dipping in an uranyl chloride solution (in ultrapure water) followed by thorough rinsing with ultrapure water (18 MΩ). X-ray photoelectron spectrometry (XPS) was used to identify the presence of nitrogen and uranyl at the gold strip surface.

(65) 2. Results

(66) FIG. 8 shows the FTIR transmission spectra recorded with the gold strip covered either with the polyacrylic acid alone (middle spectrum) or with protein grafted to polyacrylic acid layers of 5 nm (upper spectrum) and 50 nm (lower spectrum). The bands at 1673 and 1555 cm.sup.−1 are representative of the presence of the protein. These bands are observed in the upper and lower spectra. They are more intense in the lower spectrum, showing the impact of the polyacrylic layer thickness on the protein load onto the gold strip. These data show that CaMΔ was successfully grafted onto the gold strip. FIG. 9 shows the XPS spectra recorded on the gold strip: the upper spectrum was recorded at a spot containing protein and the lower spectrum was recorded on a spot corresponding to the gold strip without protein. The band at ˜382.5 eV corresponds to the presence of nitrogen, while the bands at ˜394 eV and 401 eV correspond to the presence of uranium. The presence of uranium is only observed concomitant to the presence of nitrogen (upper spectrum), indicating that uranyl is immobilized by the protein. These results show that specific uranyl adsorption by CaMΔ occurs when CaMΔ is immobilized on a solid metal support. These results show that CaMΔ may be used for uranyl chelation from water, for depollution applications.

(67) TABLE-US-00001 TABLE Amino acid and nucleotide sequences Name Sequence SEQ ID NO: 1 Arabidopsis ATGGCGGATCAGCTCACCGACGATCAGATCTCTGAGTTTAA thaliana CaM3 GGAAGCTTTCAGCTTATTCGACAAGGATGGTGATGGTTGCA TTACCACCAAGGAGCTGGGTACTGTGATGCGTTCCCTTGGA CAAAACCCAACCGAAGCAGAGCTTCAAGACATGATCAACGA AGTGGATGCTGATGGTAACGGTACCATTGATTTCCCAGAGT TCTTGAACCTTATGGCTCGTAAGATGAAGGACACCGACTCT GAGGAAGAGCTCAAGGAAGCATTCCGGGTTTTCGACAAGGA CCAGAACGGTTTCATCTCAGCAGCTGAGCTCCGCCATGTGA TGACAAACCTTGGCGAGAAGCTTACTGATGAAGAAGTTGAT GAGATGATCAAGGAAGCTGATGTTGATGGTGATGGTCAGAT TAACtACGAAGAGTTTGTTAAGGTCATGATGGCTAAGTGAC T SEQ ID NO: 2 Arabidopsis MADQLTDDQISEFKEAFSLFDKDGDGCITTKELGTVMRSLG thaliana CaM3 QNFTEAELQDMINEVDADGNGTIDEPEFLNLMARKMKDTDS EEELKEAFRVFDKDQNGFISAAELRHVMTNLGEKLTDEEVD EMIKEADVDGDGQINYEEFVKVMMAK SEQ ID NO: 3 CaM3 EF-hand1 loop DKDGDGCITTKE SEQ ID NO: 4 CaM3 EF-hand2 loop DADGNGTIDFPE SEQ ID NO: 5 CaM3 EF-hand3 loop DKDQNGFISAAE SEQ ID NO: 6 CaM3 EF-hand4 loop DVDGDGQINYEE SEQ ID NO: 7 EF-hand1 loop  DGDGCITTKE ΔK2D3/ΔD1K2 SEQ ID NO: 8 EF-hand1 loop  DGDGYITTKE ΔK2D3/ΔD1K2 + C7Y SEQ ID NO: 9 EF-hand1 loop  DGDGYITAAE ΔK2D3/ΔD1K2 + C7Y + T10A + K11A SEQ ID NO: 10 EF-hand2 loop DGNGTIDFPE ΔA2D3/ΔD1A2 SEQ ID NO: 11 EF-hand2 loop DGNGYIDFPE ΔA2D3/ΔD1A2 + T7Y SEQ ID NO: 12 EF-hand2 loop DGDGTIDFPE ΔA2D3/ΔD1A2 + N5D SEQ ID NO: 13 EF-hand2 loop DGDGYIDFPE ΔA2D3/ΔD1A2 + T7Y + N5D SEQ ID NO: 14 EF-hand3 loop DQNGFISAAE ΔK2D3/ΔD1K2 SEQ ID NO: 15 EF-hand4 loop DGDGQINYEE ΔV2D3/ΔD1V2 SEQ ID NO: 16 CaMΔ TCC ATG GCG GAT CAG CTC ACC GAC GAT CAG ATC TCT GAG TTT AAG GAA GCT TTC AGC TTA TTC GAC GGT GAT GGT TaC ATT ACC GCC GCG GAG CTG GGT ACT GTG ATG CGT TCC CTT GGA CAA AAC CCA ACC GAA GCA GAG CTT CAA GAC ATG ATC AAC GAA GTG GcT GCT GcT GGT AAC GGT ACC ATT GAT TTC CCA GAG TTC TTG AAC CTT ATG GCT CGT AAG TGA SEQ ID NO: 17 CaMΔ SMADQLTDDQISEFKEAFSLEDGDGYITAAELGTVMRSLGQ NPTEAELQDMINEVAAAGNGTIDEPEFLNLMARK SEQ ID NO: 18 Calmodulin variant MADQLTDDQISEFKEAFSLEDGDGCITTKELGTVMRSLGQN from Cameleon PTEAELQDMINEVDADGNGTIDEPEFLNLMARKMKDTDSEE biosensor Δ ELKEAFRVFDKDQNGFISAAELRHVMTNLGEKLTDEEVDEM IKEADVDGDGQINYEEFVKVMMAK SEQ ID NO: 19 Calmodulin variant MADQLTDDQISEFKEAFSLFDGDGCITTKELGTVMRSLGQN from Cameleon PTEAELQDMINEVAAAGNGTIDEPEFLNLMARKMKDTDSEE biosensor Δ-S2M ELKEAFRVFDKDQNGFISAAELRHVMTNLGEKLTDEEVDEM IKEADVDGDGQINYEEFVKVMMAK SEQ ID NO: 20 Calmodulin variant MADQLTDDQISEFKEAFSLFDGDGCITTKELGTVMRSLGQN (Δ sites  PTEAELQDMINEVDGNGTIDFPEFLNLMARKMKDTDSEEEL 1, 2, 3, 4) KEAFRVFDQNGFISAAELRHVMTNLGEKLTDEEVDEMIKEA DGDGQINYEEFVKVMMAK SEQ ID NO: 21 M13 AAACGTCGCTGGCTTTATTGCGGTGAGCGCGGC CAACCGCTTTAAAAAAATTAGCTCGAGCGGCGCGCTG SEQ ID NO: 22 M13 KRRWKKNFIAVSAANRFKKISSSGAL SEQ ID NO: 23 skMLCK KRRWKKNFIAVSAANRFKKISSSGA SEQ ID NO: 24 MLCKp RRKWQKTGHAVRAIGRL SEQ ID NO: 25 smMLCK ARRKWQKTGHAVRAIGRLSS SEQ ID NO: 26 wasp venom VNWKKIGQHILSV SEQ ID NO: 27 p21 KRRQTSMTDFYHSKRRLIFSKRKP SEQ ID NO: 28 melittin QQRKRKIWSILAPLGTTLVKLVAGIG SEQ ID NO: 29 spectrin KTASPWKSARLMVTIVATENSIKE SEQ ID NO: 30 CaMKI AKSKWKQAFNATAVVRHMRKLQ SEQ ID NO: 31 CaMKII LKKFNARRKLKGAILTTMLATRNFS SEQ ID NO: 32 CaMKK RFPNGFRKRHGMAKVLILTDLRPIRRV SEQ ID NO: 33 peptidel LKWKKLLKLLKKLLKLG SEQ ID NO: 34 Cameleon TCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGT biosensor Δ GCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACA GGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCT GCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTGGG GCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAG CACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCA GGAGCGTACCATCTTCTTCAAGGACGACGGCAACTACAAGA CCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAAC CGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACA ACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAG GCCCACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGT GCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCG ACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACC CAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA CATGGTCCTGCTGGAGTTCGTGACCGCCGCCGAGCTCATGG CGGATCAGTTGACCGACGATCAGATCTCTGAATTTAAGGAA GCCTTCAGCTTATTCGACGGTGATGGTTGCATTACCACCAA GGAACTGGGTACTGTGATGCGTTCCCTGGGCCAAAACCCGA CCGAAGCAGAGCTGCAAGACATGATCAACGAAGTGGATGCG GATGGTAACGGTACCATTGATTTCCCGGAATTCTTGAACCT GATGGCCCGTAAGATGAAAGACACCGACAGCGAGGAAGAGC TGAAAGAAGCCTTCCGCGTTTTCGACAAAGACCAGAACGGT TTCATCAGCGCAGCGGAACTGCGCCATGTGATGACCAACCT GGGCGAAAAACTGACGGATGAAGAAGTTGATGAGATGATCA AAGAAGCGGATGTGGATGGTGATGGTCAGATTAACTACGAA GAGTTTGTTAAGGTGATGATGGCGAAAGGCGGTGGCGGTAG CAAACGTCGCTGGAAAAAAAACTTTATTGCGGTGAGCGCGG CCAACCGCTTTAAAAAAATTAGCTCGAGCGGCGCGCTGGTC GACATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGT GCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACA AGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCT GCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACG GCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAG CACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCA GGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGA CCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAAC CGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACA ACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAG GTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGT GCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCG ACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTAC CAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA CATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTC TCGGCATGGACGAGCTGTACAAGTAA SEQ ID NO: 35 Cameleon SMVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATY biosensor Δ GKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQ HDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLST QSALSKDPNEKRDHMVLLEFVTAAELMADQLTDDQISEFKE AFSLEDGDGCITTKELGTVMRSLGQNPTEAELQDMINEVDA DGNGTIDFPEFLNLMARKMKDTDSEEELKEAFRVFDKDQNG FISAAELRHVMTNLGEKLTDEEVDEMIKEADVDGDGQINYE EFVKVMMAKGGGGSKRRWKKNFIAVSAANRFKKISSSGALv dMVSKGEELFTGVVPILVELDGDVNGHKESVSGEGEGDATY GKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQ HDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSY QSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 36 Cameleon TCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGT biosensor GCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACA Δ-S2M GGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCT GCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTGGG GCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAG CACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCA GGAGCGTACCATCTTCTTCAAGGACGACGGCAACTACAAGA CCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAAC CGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACA ACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAG GCCCACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGT GCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCG ACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACC CAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA CATGGTCCTGCTGGAGTTCGTGACCGCCGCCGAGCTCATGG CGGATCAGTTGACCGACGATCAGATCTCTGAATTTAAGGAA GCCTTCAGCTTATTCGACGGTGATGGTTGCATTACCACCAA GGAACTGGGTACTGTGATGCGTTCCCTGGGCCAAAACCCGA CCGAAGCAGAGCTGCAAGACATGATCAACGAAGTGGCTGCG GCTGGTAACGGTACCATTGATTTCCCGGAATTCTTGAACCT GATGGCCCGTAAGATGAAAGACACCGACAGCGAGGAAGAGC TGAAAGAAGCCTTCCGCGTTTTCGACAAAGACCAGAACGGT TTCATCAGCGCAGCGGAACTGCGCCATGTGATGACCAACCT GGGCGAAAAACTGACGGATGAAGAAGTTGATGAGATGATCA AAGAAGCGGATGTGGATGGTGATGGTCAGATTAACTACGAA GAGTTTGTTAAGGTGATGATGGCGAAAGGCGGTGGCGGTAG CAAACGTCGCTGGAAAAAAAACTTTATTGCGGTGAGCGCGG CCAACCGCTTTAAAAAAATTAGCTCGAGCGGCGCGCTGGTC GACATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGT GCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACA AGTTCAGCGTGTCOGGCGAGGGCGAGGGCGATGOCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCT GCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACG GCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAG CACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCA GGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGA CCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAAC CGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACA ACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAG GTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGT GCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCG ACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTAC CAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA CATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTC TCGGCATGGACGAGCTGTACAAGTAA SEQ ID NO: 37 Cameleon SMVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATY biosensor GKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQ Δ-S2M HDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLST QSALSKDPNEKRDHMVLLEFVTAAELMADQLTDDQISEFKE AFSLFDGDGCITTKELGTVMRSLGQNPTEAELQDMINEVAA AGNGTIDEPEFLNLMARKMKDTDSEEELKEAFRVEDKDQNG FISAAELRHVMTNLGEKLTDEEVDEMIKEADVDGDGQINYE EFVKVMMAKGGGGSKRRWKKNFIAVSAANRFKKISSSGALv dMVSKGEELFTGVVPILVELDGDVNGHKESVSGEGEGDATY GKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQ HDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTFIGDGPVLLPDNHYLSY QSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 38 Cameleon SMVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATY biosensor GKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQ ΔΔΔΔ HDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLST QSALSKDPNEKRDHMVLLEFVTAAELMADQLTDDQISEFKE AFSLEDGDGCITTKELGTVMRSLGQNPTEAELQDMINEVDG NGTIDEPEFLNLMARKMKDTDSEEELKEAFRVEDQNGFISA AELRHVMTNLGEKLTDEEVDEMIKEADGDGQINYEEFVKVM MAKGGGGSKRRWKKNFIAVSAANRFKKISSSGALvdMVSKG EELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTEGYGVQCFARYPDHMKQHDFFKS AMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKG IDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIR HNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSK DPNEKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 39 CaM1 TCC ATG GCG GAT CAG CTC ACC GAC GAT CAG ATC TCT GAG TTT AAG GAA GCT TTC AGC TTA TTC GAC AAG GAT GGT GAT GGT TaC ATT ACC GCC GCG GAG CTG GGT ACT GTG ATG CGT TCC CTT GGA CAA AAC CCA ACC GAA GCA GAG CTT CAA GAC ATG ATC AAC GAA GTG GcT GCT GcT GGT AAC GGT ACC ATT GAT TTC CCA GAG TTC TTG AAC CTT ATG GCT CGT AAG TGA SEQ ID NO: 40 CaM1 SMADQLTDDQISEFKEAFSLFDKDGDGYITAAELGTVMRSL GQNPTEAELQDMINEVAAAGNGTIDFPEFLNLMARK SEQ ID NO: 41 Primer DGD S GAAGCTTTCAGCTTATTCGACGGTGATGGTTACATTACCGC CGCG SEQ ID NO: 42 Primer DGD AS CGCGGCGGTAATCTAACCATCACCGTCGAATAAGCTGAAAG CTTC SEQ ID NO: 43 Primer GAGA GGATCC GAG AAC CTG TAC TTC CAG TCC S-TEV-eCFP-BamHI ATG GTG AGC AAG GGC GAG GAG SEQ ID NO: 44 Primer TAAA GAGCTC GGCGGCGGTCACGAACTCCAGCA AS-eCFP-SacI SEQ ID NO: 45 Primer TATA GTCGAC ATG GTG AGC AAG GGC GAG GAG S-eYFP-SalI SEQ ID NO: 46 Primer GGGC AAGCTT TTA CTT GTA CAG CTC GTC CAT AS-eYFP-HindIII GCC G SEQ ID NO: 47 Cameleon TCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGT biosensor GCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACA WT GGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCT GCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTGGG GCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAG CACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCA GGAGCGTACCATCTTCTTCAAGGACGACGGCAACTACAAGA CCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAAC CGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACA ACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAG GCCCACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGT GCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCG ACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACC CAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA CATGGTCCTGCTGGAGTTCGTGACCGCCGCCGAGCTCATGG CGGATCAGTTGACCGACGATCAGATCTCTGAATTTAAGGAA GCCTTCAGCTTATTCGACAAAGATGGTGATGGTTGCATTAC CACCAAGGAACTGGGTACTGTGATGCGTTCCCTGGGCCAAA ACCCGACCGAAGCAGAGCTGCAAGACATGATCAACGAAGTG GATGCGGATGGTAACGGTACCATTGATTTCCCGGAATTCTT GAACCTGATGGCCCGTAAGATGAAAGACACCGACAGCGAGG AAGACCTGAAACAAGCCTTCCGCGTTTTCGACAAAGACCAG AACGGTTTCATCAGCGCAGCGGAACTGCGCCATGTGATGAC CAACCTGGGCGAAAAACTGACGGATGAAGAAGTTGATGAGA TGATCAAAGAAGCGGATGTGGATGGTGATGGTCAGATTAAC TACGAAGAGTTTGTTAAGGTGATGATGGCGAAAGGCGGTGG CGGTAGCAAACGTCGCTGGAAAAAAAACTTTATTGCGGTGA GCGCGGCCAACCGCTTTAAAAAAATTAGCTCGAGCGGCGCG CTGGTCGACATGGTGAGCAAGGGCGAGGAGCTGTTCACCGG GGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACG GCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCC ACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGG CAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCG GCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATG AAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTA CGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACT ACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTG GTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGA CGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACA GCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGC ATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGG CAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCA TCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTG AGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCG CGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGA TCACTCTCGGCATGGACGAGCTGTACAAGTAA SEQ ID NO: 48 Cameleon SMVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATY biosensor GKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYTDHMKQ WT HDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN RIELKGIDEKEDGNILGHKLEYNYISHNVYITADKQKNGIK AHEKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLST QSALSKDPNEKRDHMVLLEFVTAAELMADQLTDDQISEFKE AFSLFDKDGDGCITTKELGTVMRSLGQNPTEAELQDMINEV DADGNGTIDEPEFLNLMARKMKDTDSEEELKEAFRVEDKDQ NGFISAAELRHVMTNLGEKLTDEEVDEMIKEADVDGDGQIN YEEFVKVMMAKGGGGSKRRWKKNFIAVSAANRFKKISSSGA LvdMVSKGEELFTGVVPILVELDGDVNGHKESVSGEGEGDA TYGKLTLKFICTTGKLPVPWPTLVTTEGYGVQCFARYPDHM KQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNG IKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYL SYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDYK SEQ ID NO: 49 Primer S-Δ GGAAGCCTTCAGCTTATTCGACGGTGATGGTTGCATTACC SEQ ID NO: 50 Primer AS-Δ GGTAATGCAACCATCACCGTCGAATAAGCTGAAGGCTTCC SEQ ID NO: 51 Primer S-S2M GGT ACC GTT ACC AGC AGC AGC CAC TTC GTT GAT C SEQ ID NO: 52 Primer AS-S2M GAT CAA CGA AGT GGC TGC TGC TGG TAA CGG TAC C SEQ ID NO: 53 Cameleon TCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGT biosensor GCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACA WT-S2M GGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCT GCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTGGG GCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAG CACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCA GGAGCGTACCATCTTCTTCAAGGACGACGGCAACTACAAGA CCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAAC CGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACA ACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAG GCCCACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGT GCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCG ACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACC CAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA CATGGTCCTGCTGGAGTTCGTGACCGCCGCCGAGCTCATGG CGGATCAGTTGACCGACGATCAGATCTCTGAATTTAAGGAA GCCTTCAGCTTATTCGACAAAGATGGTGATGGTTGCATTAC CACCAAGGAACTGGGTACTGTGATGCGTTCCCTGGGCCAAA ACCCGACCGAAGCAGAGCTGCAAGACATGATCAACGAAGTG GCTGCGGCTGGTAACGGTACCATTGATTTCCCGGAATTCTT GAACCTGATGGCCCGTAAGATGAAAGACACCGACAGCGAGG AAGAGCTGAAAGAAGCCTTCCGCGTTTTCGACAAAGACCAG AACGGTTTCATCAGCGCAGCGGAACTGCGCCATGTGATGAC CAACCTGGGCGAAAAACTGACGGATGAAGAAGTTGATGAGA TGATCAAAGAAGCGGATGTGGATGGTGATGGTCAGATTAAC TACGAAGAGTTTGTTAAGGTGATGATGGCGAAAGGCGGTGG CGGTAGCAAACGTCGCTGGAAAAAAAACTTTATTGCGGTGA GCGCGGCCAACCGCTTTAAAAAAATTAGCTCGAGCGGCGCG CTGGTCGACATGGTGAGCAAGGGCGAGGAGCTGTTCACCGG GOTGOTGCCCATCCTGOTCGAGCTGGACGGCGACGTAAACG GCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCC ACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGG CAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCG GCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATG AAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTA CGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACT ACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTG GTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGA CGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACA GCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGC ATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGG CAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCA TCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTG AGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCG CGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGA TCACTCTCGGCATGGACGAGCTGTACAAGTAA SEQ ID NO: 54 Cameleon SMVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATY biosensor GKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQ WT-S2M HDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLST QSALSKDPNEKRDHMVLLEFVTAAELMADQLTDDQISEFKE AFSLFDKDGDGCITTKELGTVMRSLGQNPTEAELQDMINEV AAAGNGTIDEPEFLNLMARKMKDTDSEEELKEAFRVEDKDQ NGFISAAELRHVMTNLGEKLTDEEVDEMIKEADVDGDGQIN YEEFVKVMMAKGGGGSKRRWKKNFIAVSAANRFKKISSSGA LvdMVSKGEELFTGVVPILVELDGDVNGHKESVSGEGEGDA TYGKLTLKFICTTGKLPVPWPTLVTTEGYGVQCFARYPDHM KQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDEKEDGNILGHKLEYNYNSHNVYTMADKQKNG IKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLFTNHYL SYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 55 CaMΔ3 TCC ATG GCG GAT CAG CTC ACC GAC GAT CAG ATC TCT GAG TTT AAG GAA GCT TTC AGC TTA TTC GAC AAG GGT GAT GGT TaC ATT ACC GCC GCG GAG CTG GGT ACT GTG ATG CGT TCC CTT GGA CAA AAC CCA ACC GAA GCA GAG CTT CAA GAC ATG ATC AAC GAA GTG GcT GCT GcT GGT AAC GGT ACC ATT GAT TTC CCA GAG TTC TTG AAC CTT ATG GCT CGT AAG TGA SEQ ID NO: 56 CaMΔ3 SMADQLTDDQISEFKEAFSLFDKGDGYITAAELGTVMRSLG QNPTEAELQDMINEVAAAGNGTIDEPEFLNLMARK SEQ ID NO: 57 Primer S-Δ3Y TTCAGCTTATTCGACAAGGGTGATGGTTACATTACC SEQ ID NO: 58 Primer AS-Δ3Y GGTAATGTAACCATCACCCTTGTCGAATAAGCTGAA SEQ ID NO: 59 CaMΔ-WT TCC ATG GCG GAT CAG CTC ACC GAC GAT CAG ATC TCT GAG TTT AAG GAA GCT TTC AGC TTA TTC GAC GGT GAT GGT TaC ATT ACC ACC AAG GAG CTG GGT ACT GTG ATG CGT TCC CTT GGA CAA AAC CCA ACC GAA GCA GAG CTT CAA GAC ATG ATC AAC GAA GTG GcT GCT GcT GGT AAC GGT ACC ATT GAT TTC CCA GAG TTC TTG AAC CTT ATG GCT CGT AAG TGA SEQ ID NO: 60 CaMΔ-WT SMADQLTDDQISEFKEAFSLFDGDGYITTKELGTVMRSLGQ NPTEAELQDMINEVAAAGNGTIDEPEFLNLMARK SEQ ID NO: 61 Cameleon SMVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATY Biosensor GKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQ Δ1 Δ3 HDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLST QSALSKDPNEKRDHMVLLEFVTAAelMADQLTDDQISEFKE AFSLEDGDGCITTKELGTVMRSLGQNPTEAELQDMINEVDA DGNGTIDEPEFLNLMARKMKDTDSEEELKEAFRVEDQNGFI SAAELRHVMTNLGEKLTDEEVDEMIKEADVDGDGQINYEEF VKVMMAKGGGGSKRRWKKNFIAVSAANRFKKISSSGALvdM VSKGEELFTGVVPILVELDGDVNGHKESVSGEGEGDATYGK LTLKFICTTGKLPVPWPTLVTTEGYGVQCFARYPDHMKQHD FFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRI ELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQS ALSKDPNEKRDBMVLLEFVTAAGITLGMDELYK SEQ ID NO: 62 Cameleon TCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGT Biosensor GCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACA Δ1 Δ3 GGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCT GCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTGGG GCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAG CACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCA GGAGCGTACCATCTTCTTCAAGGACGACGGCAACTACAAGA CCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAAC CGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACA ACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAG GCCCACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGT GCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCG ACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACC CAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA CATGGTCCTGCTGGAGTTCGTGACCGCCGCCGAGCTCATGG CGGATCAGTTGACCGACGATCAGATCTCTGAATTTAAGGAA GCCTTCAGCTTATTCGACGGTGATGGTTGCATTACCACCAA GGAACTGGGTACTGTGATGCGTTCCCTGGGCCAAAACCCGA CCGAAGCAGAGCTGCAAGACATGATCAACGAAGTGGATGCG GATGGTAACGGTACCATTGATTTCCCGGAATTCTTGAACCT GATGGCCCGTAAGATGAAAGACACCGACAGCGAGGAAGAGC TGAAAGAAGCCTTCCGCGTTTTCGACCAGAACGGTTTCATC AGCGCAGCGGAACTGCGCCATGTGATGACCAACCTGGGCGA AAAACTGACGGATGAAGAAGTTGATGAGATGATCAAAGAAG CGGATGTGGATGGTGATGGTCAGATTAACTACGAAGAGTTT GTTAAGGTGATGATGGCGAAAGGCGGTGGCGGTAGCAAACG TCGCTGGAAAAAAAACTTTATTGCGGTGAGCGCGGCCAACC GCTTTAAAAAAATTAGCTCGAGCGGCGCGCTGGTCGACATG GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCAT CCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCA GCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAG CTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGT GCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCGTGC AGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGAC TTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCG CACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCG CCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATC GAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCT GGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCT ATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAAC TTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCT CGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCC CCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCC GCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGT CCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCA TGGACGAGCTGTACAAGTAA SEQ ID NO: 63 Cameleon SMVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATY Biosensor GKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQ Δ1 Δ2 Δ3 HDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLFTNHYLST QSALSKDPNEKRDHMVLLEFVTAAelMADQLTDDQISEFKE AFSLFDGDGCITTKELGTVMRSLGQNPTEAELQDMINEVDG NGTIDFPEFLNLMARKMKDTDSEEELKEAFRVFDQNGFISA AELRHVMTNLGEKLTDEEVDEMIKEADVDGDGQINYEEFVK VMMAKGGGGSKRRWKKNFIAVSAANRFKKISSSGALvdMVS KGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT LKFICTTGKLPVPWPTLVTTEGYGVQCFARYPDHMKQHDFF KSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL KGIDEKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNEK IRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSAL SKDPNEKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 64 Cameleon TCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGT Biosensor GCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACA Δ1 Δ2 Δ3 GGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCT GCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTGGG GCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAG CACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCA GGAGCGTACCATCTTCTTCAAGGACGACGGCAACTACAAGA CCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAAC CGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACA ACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAG GCCCACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGT GCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCG ACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACC CAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA CATGGTCCTGCTGGAGTTCGTGACCGCCGCCGAGCTCATGG CGGATCAGTTGACCGACGATCAGATCTCTGAATTTAAGGAA GCCTTCAGCTTATTCGACGGTGATGGTTGCATTACCACCAA GGAACTGGGTACTGTGATGCGTTCCCTGGGCCAAAACCCGA CCGAAGCAGAGCTGCAAGACATGATCAACGAAGTGGATGGT AACGGTACCATTGATTTCCCGGAATTCTTGAACCTGATGGC CCGTAAGATGAAAGACACCGACAGCGAGGAAGAGCTGAAAG AAGCCTTCCGCGTTTTCGACCAGAACGGTTTCATCAGCGCA GCGGAACTGCGCCATGTGATGACCAACCTGGGCGAAAAACT GACGGATGAAGAAGTTGATGAGATGATCAAAGAAGCGGATG TGGATGGTGATGGTCAGATTAACTACGAAGAGTTTGTTAAG GTGATGATGGCGAAAGGCGGTGGCGGTAGCAAACGTCGCTG GAAAAAAAACTTTATTGCGGTGAGCGCGGCCAACCGCTTTA AAAAAATTAGCTCGAGCGGCGCGCTGGTCGACATGGTGAGC AAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGT CCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACC CTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTG GCCCACCCTCGTGACCACCTTCGGCTACGGCGTGCAGTGCT TCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTC AAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCAT CTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGG TGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTG AAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCA CAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCA TGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAG ATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGA CCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGC TGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTG AGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCT GGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACG AGCTGTACAAGTAA SEQ ID NO: 65 Cameleon SMVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATY Biosensor GKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQ N-ter HDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLST QSALSKDPNEKRDHMVLLEFVTAAelpMADQLTDDQISEFK EAFSLFDKDGDGCITTKELGTVMRSLGQNPTEAELQDMINE VDADGNGTIDEPEFLNLMARKpvdMVSKGEELFTGVVPILV ELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPW PTLVTTEGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTI FFKDDGNYKTRAEVKFEGDTLVNRIELKGIDEKEDGNILGH KLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLAD HYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK SEQ ID NO: 66 Cameleon TCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGT Biosensor GCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACA N-ter GGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCT GCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTGGG GCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAG CACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCA GGAGCGTACCATCTTCTTCAAGGACGACGGCAACTACAAGA CCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAAC CGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACA ACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAG GCCCACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGT GCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCG ACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACC CAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA CATGGTCCTGCTGGAGTTCGTGACCGCCGCCGAGCTCccgA TGGCGGATCAGTTGACCGACGATCAGATCTCTGAATTTAAG GAAGCCTTCAGCTTATTCGACAAGGATGGTGATGGTTGCAT TACCACCAAGGAACTGGGTACTGTGATGCGTTCCCTGGGCC AAAACCCGACCGAAGCAGAGCTGCAAGACATGATCAACGAA GTGGATGCGGATGGTAACGGTACCATTGATTTCCCGGAATT CTTGAACCTGATGGCCCGTAAGccgGTCGACATGGTGAGCA AGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTC GAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTC CGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCC TGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGG CCCACCCTCGTGACCACCTTCGGCTACGGCGTGCAGTGCTT CGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCA AGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATC TTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGT GAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGA AGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCAC AAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCAT GGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGA TCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGAC CACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCT GCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGA GCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTG GAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGA GCTGTACAAGTAA SEQ ID NO: 67 Cameleon SMVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATY Biosensor GKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQ Δ1 Δ2 HDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLST QSALSKDPNEKRDHMVLLEFVTAAELMADQLTDDQISEFKE AFSLEDGDGCITTKELGTVMRSLGQNPTEAELQDMINEVDG NGTIDFPEFLNLMARKMKDTDSEEELKEAFRVFDKDQNGFI SAAELRHVMTNLGEKLTDEEVDEMIKEADVDGDGQINYEEF VKVMMAKGGGGSKRRWKKNFIAVSAANRFKKISSSGALVDM VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGK LTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHD FFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRI ELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQS ALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 68 Cameleon TCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGT Biosensor GCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACA Δ1 Δ2 GGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCT GCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTGGG GCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAG CACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCA GGAGCGTACCATCTTCTTCAAGGACGACGGCAACTACAAGA CCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAAC CGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACA ACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAG GCCCACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGT GCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCG ACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACC CAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA CATGOTCCTGCTGGAGTTCGTGACCGCCGCCGAGCTCATGG CGGATCAGTTGACCGACGATCAGATCTCTGAATTtaAGGAA GCCTTCAGCTTATTCGACGGTGATGGTTGCATTACCACCAA GGAACTGGGTACTGTGATGCGTTCCCTGGGCCAAAACCCGA CCGAAGCAGAGCTGCAAGACATGATCAACGAAGTGGATGGT AACGGTACCATTGATTTCCCGGAATTCTTGAACCTGATGGC CCGTAAGATGAAAGACACCGACAGCGAGGAAGAGCTGAAAG AAGCCTTCCGCGTTTTCGACAAAGACCAGAACGGTTTCATC AGCGCAGCGGAACTGCGCCATGTGATGACCAACCTGGGCGA AAAACTGACGGATGAAGAAGTTGATGAGATGATCAAAGAAG CGGATGTGGATGGTGATGGTCAGATTAACTACGAAGAGTTT GTTAAGGTGATGATGGCGAAAGGCGGTGGCGGTAGCAAACG TCGCTGGAAAAAAAaCTTTATTGCGGTGAGCGCGGCCAACC GCTTTAAAAAAATTAGCTCGAGCGGCGCGCTGGTCGACAtg gtGAGCAAGGGCgaggagcTGtTCACCGGGgtggtgCCCAT CctggtCGAgctgGaCGGCGAcgtAAACGGCCACAagtTCA GcgtgTCCGGCgAGGGCgagGGCGatgCCAcCTACGGCAAG CTgaCCcTGAAGTTCATCTGCACCACCGGCAAGCTGCCCgt GCCctgGCCCACCCTcgtgaCCACCTTCGGCtACGGCgtGC AgtgCtTCGCCCGCTACCCCGACCACATGAAGCAGCACGAC TTCTTCAAGTCCGCCAtgCCCGAAGGCTACGTCCAGGAGCG CACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCG CCGAGGTGAAGTTCGAGGGCGACACCCTGgtGAACCGCATC GAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCT GGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCT ATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAAC TTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCT CGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCC CCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCC GCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGT CCTGCTGGAGTTCGTGACCGCCGCCGGGATCacTCTCGGCA TGGACgaGCTGTACAAGTAA SEQ ID NO: 69 EF-hand1 loop + DKDGDGYITAAE C7Y + T10A + K11A SEQ ID NO: 70 EF-hand1 loop KDGDGCITKE ΔD1T10 SEQ ID NO: 71 EF-hand1 loop  KDGDGCITTE ΔD1K11 SEQ ID NO: 72 EF-hand1 loop DKGDGCITKE ΔD3T10 SEQ ID NO: 73 EF-hand1 loop DKGDGCITTE ΔD3K11 SEQ ID NO: 74 EF-hand1 loop KDGDGCITE ΔD1T10K11 SEQ ID NO: 75 EF-hand1 loop DKGDGCITE ΔD3T10K11 SEQ ID NO: 76 EF-hand2 loop ADGNGTIDPE ΔD1F10 SEQ ID NO: 77 EF-hand2 loop ADGNGTIDFE ΔD1P11 SEQ ID NO: 78 EF-hand2 loop DAGNGTIDPE ΔD3F10 SEQ ID NO: 79 EF-hand2 loop DAGNGTIDFE ΔD3P11 SEQ ID NO: 80 EF-hand2 loop ADGNGTIDE ΔD1F10P11 SEQ ID NO: 81 EF-hand2 loop DAGNGTIDE ΔD3F10P11 SEQ ID NO: 82 EF-hand3 loop KDQNGFISAE ΔD1A10 SEQ ID NO: 83 EF-hand3 loop KDQNGFISAE ΔD1A11 SEQ ID NO: 84 EF-hand3 loop DKQNGFISAE ΔD3A10 SEQ ID NO: 85 EF-hand3 loop DKQNGFISAE ΔD3A11 SEQ ID NO: 86 EF-hand3 loop KDQNGFISE ΔD1A10A11 SEQ ID NO: 87 EF-hand3 loop DKQNGFISE ΔD3A10A11 SEQ ID NO: 88 EF-hand4 loop VDGDGQINEE ΔD1Y10 SEQ ID NO: 89 EF-hand4 loop VDGDGQINYE ΔD1E11 SEQ ID NO: 90 EF-hand4 loop DVGDGQINEE ΔD3Y10 SEQ ID NO: 91 EF-hand4 loop DVGDGQINYE ΔD3E11 SEQ ID NO: 92 EF-hand4 loop VDGDGQINE ΔD1Y10E11 SEQ ID NO: 93 EF-hand4 loop DVGDGQINE ΔD3Y10E11