CALCIUM DEPENDENT PROTEIN KINASE CONSTRUCTS AND USES THEREOF

Abstract

The present invention relates to calcium dependent protein kinase (CORK) constructs comprising a pair of chromophores suitable for measuring real-time FRET occurrence upon conformational changes or activation of the CORK construct. Particularly, the present invention relates to CORK polypeptides comprising a variable domain (VD), a protein kinase domain (PKD), a pseudosubstrate segment (PS), and a calmodulin-like domain (OLD), said CLD comprising 4 EF-hand motifs EF1, EF2, EF3 and EF4, wherein a donor chromophore is inserted between PKD and PS and an acceptor chromophore is inserted C-terminally of EF4, or an acceptor chromophore is inserted between PKD and PS and a donor chromophore is inserted C-terminally of EF4, wherein said donor chromophore and said acceptor chromophore represent a Förster Resonance Energy Transfer (FRET) pair. The present invention further relates to polynucleotides encoding such polypeptides as well as vectors and host cells comprising such polypeptides and/or polynucleotides. Furthermore, the present invention relates to methods for measuring conformational change or activation status of a CDPK polypeptide by employing said polypeptides and to uses of said polypeptides for measuring conformational change or activation status of a CDPK polypeptide.

Claims

1. Calcium dependent protein kinase (CDPK) polypeptide comprising a variable domain (VD), a protein kinase domain (PKD), a pseudosubstrate segment (PS), and a calmodulin-like domain (CLD), said CLD comprising 4 EF-hand motifs EF1, EF2, EF3 and EF4, wherein (A) a donor chromophore is inserted between PKD and PS and an acceptor chromophore is inserted C-terminally of EF4, or (B) an acceptor chromophore is inserted between PKD and PS and a donor chromophore is inserted C-terminally of EF4, wherein said donor chromophore and said acceptor chromophore represent a Förster Resonance Energy Transfer (FRET) pair.

2. Polypeptide of claim 1, wherein said donor chromophore and/or said acceptor chromophore is a fluorescent protein (FP).

3. Polypeptide of claim 1 or 2, wherein (a) said donor chromophore is a cyan FP (CFP), and said acceptor chromophore is a yellow FP (YFP), (b) said donor chromophore is a green FP (GFP) or yellow FP (YFP), and said acceptor chromophore is a red FP (RFP), (c) said donor chromophore is a cyan FP (CFP), and said acceptor chromophore is a green FP (GFP), (d) said donor chromophore is a red FP (RFP), and said acceptor chromophore is a near-infrared FP, or (e) said donor chromophore is a non-naturally occurring amino acid and said acceptor chromophore is an FP.

4. Polypeptide of any one of claims 1 to 3, wherein (i) said donor chromophore is selected from the group consisting of mTurquoise (mT), eCFP, Aquamarine, mTurquoise2 (mT2), mCerulean3, mTFP1 and LUMP, and said acceptor chromophore is selected from the group consisting of cpVenus173, eYFP, mVenus, Venus, mCitrine, sEYFP and YPet, or (ii) said donor chromophore is selected from the group consisting of cpVenus173, eGFP, NowGFP, Clover, mClover3 and mNeonGreen, and said acceptor chromophore is selected from the group consisting of mRuby2, mRuby3, mRFP, nKOk, and mCherry.

5. Polypeptide of any one of claims 1 to 4, wherein said donor chromophore is mT or eCFP, and said acceptor chromophore is cpVenus173.

6. Polypeptide of any one of claims 1 to 5, wherein said donor chromophore is inserted directly at the C-Terminus of the PKD, directly at the N-Terminus of the PS, or between the C-Terminus of the PKD and the N-Terminus of the PS.

7. Polypeptide of any one of claims 1 to 6, wherein said acceptor chromophore is inserted directly at the C-Terminus of EF4, at the C-Terminus of the CDPK polypeptide, or between the C-Terminus of EF4 and the C-Terminus of the CDPK polypeptide.

8. Polypeptide of any one of claims 1 to 7, wherein the amino acid sequence comprising the PKD, the PS and the CLD of the CDPK is at least 37% identical to amino acid sequence 80 to 522 of SEQ ID NO: 1, 69-511 of SEQ ID NO: 2, 150-592 of SEQ ID NO: 3, 105-547 of SEQ ID NO: 4, 66-509 of SEQ ID NO: 5, 32-474 of SEQ ID NO: 6, 29-471 of SEQ ID NO: 7, 34-475 of SEQ ID NO: 8, 34-475 of SEQ ID NO: 9, 56-521 of SEQ ID NO: 10, or 51-507 of SEQ ID NO: 11.

9. Polypeptide of any one of claims 1 to 8, wherein the amino acid sequence comprising the PKD, the PS and the CLD of the CDPK is at least 54% similar to amino acid sequence 80-522 of SEQ ID NO: 1, 69-511 of SEQ ID NO: 2, 150-592 of SEQ ID NO: 3, 105-547 of SEQ ID NO: 4, 66-509 of SEQ ID NO: 5, 32-474 of SEQ ID NO: 6, 29-471 of SEQ ID NO: 7, 34-475 of SEQ ID NO: 8, 34-475 of SEQ ID NO: 9, 56-521 of SEQ ID NO: 10, or 51-507 of SEQ ID NO: 11.

10. Polypeptide of any one of claims 1 to 9, wherein (1) the amino acid sequence comprising the PKD is at least 44% identical to the amino acid sequence 80 to 338 of SEQ ID NO: 1, 69-327 of SEQ ID NO: 2, 150-408 of SEQ ID NO: 3, 105-363 of SEQ ID NO: 4, 66-324 of SEQ ID NO: 5, 32-290 of SEQ ID NO: 6, 29-287 of SEQ ID NO: 7, 34-292 of SEQ ID NO: 8, 34-292 of SEQ ID NO: 9, 56-325 of SEQ ID NO: 10, or 51-308 of SEQ ID NO: 11, or 60% similar to the amino acid sequence 80 to 338 of SEQ ID NO: 1, 69-327 of SEQ ID NO: 2, 150-408 of SEQ ID NO: 3, 105-363 of SEQ ID NO: 4, 66-324 of SEQ ID NO: 5, 32-290 of SEQ ID NO: 6, 29-287 of SEQ ID NO: 7, 34-292 of SEQ ID NO: 8, 34-292 of SEQ ID NO: 9, 56-325 of SEQ ID NO: 10, or 51-308 of SEQ ID NO: 11, (2) the amino acid sequence comprising the PS is at least 23% identical to the amino acid sequence 343 to 373 of SEQ ID NO: 1, 332-362 of SEQ ID NO: 2, 414-444 of SEQ ID NO: 3, 368-398 of SEQ ID NO: 4, 330-360 of SEQ ID NO: 56, 296-326 of SEQ ID NO: 6, 293-323 of SEQ ID NO: 7, 298-328 of SEQ ID NO: 8, 298-328 of SEQ ID NO: 9, 334-364 of SEQ ID NO: 10, or 317-347 of SEQ ID NO: 11, or 42% similar to the amino acid sequence 343 to 373 of SEQ ID NO: 1, 332-362 of SEQ ID NO: 2, 414-444 of SEQ ID NO: 3, 368-398 of SEQ ID NO: 4, 330-360 of SEQ ID NO: 56, 296-326 of SEQ ID NO: 6, 293-323 of SEQ ID NO: 7, 298-328 of SEQ ID NO: 8, 298-328 of SEQ ID NO: 9, 334-364 of SEQ ID NO: 10, or 317-347 of SEQ ID NO: 11, and/or (3) the amino acid sequence comprising the CLD is at least 27% identical to the amino acid sequence 374 to 522 of SEQ ID NO: 1, 363-511 of SEQ ID NO: 2, 445-592 of SEQ ID NO: 3, 399-547 of SEQ ID NO: 4, 361-509 of SEQ ID NO: 5, 327-474 of SEQ ID NO: 6, 324-471 of SEQ ID NO: 7, 329-475 of SEQ ID NO: 8, 329-475 of SEQ ID NO: 9, 365-521 of SEQ ID NO: 10, or 348-507 of SEQ ID NO: 11, or 50% similar to the amino acid sequence 374 to 522 of SEQ ID NO: 1363-511 of SEQ ID NO: 2, 445-592 of SEQ ID NO: 3, 399-547 of SEQ ID NO: 4, 361-509 of SEQ ID NO: 5, 327-474 of SEQ ID NO: 6, 324-471 of SEQ ID NO: 7, 329-475 of SEQ ID NO: 8, 329-475 of SEQ ID NO: 9, 365-521 of SEQ ID NO: 10, or 348-507 of SEQ ID NO: 11.

11. Polypeptide of any one of claims 1 to 12, wherein said polypeptide comprises an amino acid sequence which is at least 70% identical to any one of SEQ ID Nos. 1 to 11, or 85% similar to any one of SEQ ID Nos. 1 to 11.

12. Polynucleotide encoding the polypeptide of any one of claims 2 to 11.

13. Vector comprising the polynucleotide of claim 12.

14. Host cell comprising the polypeptide of any one of claims 1 to 11, the polynucleotide of claim 12, and/or the vector of claim 13.

15. Method for measuring conformational change or activation status of a CDPK polypeptide, comprising the following steps: (1) cultivating the host cell of claim 14 comprising the polypeptide of any one of claims 1 to 11 under suitable conditions, (2) optionally adding calcium and/or applying stress treatment to the host cell, and (3) measuring FRET occurrence.

Description

[0082] The Figures show:

[0083] FIG. 1 Kinase activity of CPK21 and CPK23 plotted against Ca.sup.2+-concentrations (R.sup.2.sub.CPK21=0.91, R.sup.2.sub.CPK23=0.76). Activity is expressed as percentage of full Ca.sup.2+-saturation (means±SEM n=2-3).

[0084] FIG. 2 Comparison of FRET-recorded conformational change of CPK21D204A and CPK23D193A plotted against [Ca.sup.2+] (R.sup.2.sub.CPK21D204A=0.98, R.sup.2.sub.CPK23D193A=0.69). FRET efficiency change is given as percentage of increase in ratio over the baseline FRET signal (means±SEM n=3-6).

[0085] FIG. 3 Mean±SEM of half-maximal kinase activity (K50) or half-maximal effective concentration (EC50) of conformational change in n independent experiments. The Hillslope was fitted as shared value for all data sets of the same enzyme.

[0086] FIG. 4 Kinase activity of CPK21 PS variants carrying amino acid substitution (CPK21I373S, CPK21I373D) plotted against [Ca.sup.2+] (R.sup.2.sub.CPK21=0.85, R.sup.2.sub.CPK21I373S=0.97, R.sup.2.sub.CPK21I373D=0.87). Activity is expressed as percentage of full Ca.sup.2+-saturation (means±SEM n=2-3).

[0087] FIG. 5 FRET-recorded conformational change of CPK21D204A-1373S, CPK21D204A-1373D plotted against [Ca.sup.2+] (R.sup.2.sub.CPK21D204A=0.96, R.sup.2.sub.CPK21D204A-I373S=0.99, R.sup.2.sub.CPK21I3204A-I373D=0.88). FRET efficiency change is determined as in (FIG. 2) (means±SEM n=4-6).

[0088] FIG. 6 Kinase activity of CPK23 PS variants carrying amino acid substitution (CPK23S362I, CPK23S362D) plotted against [Ca.sup.2+] (R.sup.2.sub.CPK23=0.84, R.sup.2.sub.CPK23S362I=0.8). Activity is expressed as percentage of full Ca.sup.2+-saturation (means±SEM n=2-3). CPK23S362D activity indicated in counts per minute (cpm).

[0089] FIG. 7 Mean±SEM for K50 of activity or EC50 of conformational change and shared Hillslope in n independent experiments.

[0090] FIG. 8 Kinase activity of CPK23CLD21 and CPK23S362I-CLD21 plotted against Ca.sup.2+-concentrations (Kinase activity: R.sup.2.sub.CPK21=0.85, R.sup.2.sub.CPK23=0.89, R.sup.2.sub.CPK23CLD21=0.69, R.sup.2.sub.CPK23S362I-CLD21=0.92). Kinase activity (means±SEM n=2-4) is determined as described in FIG. 1.

[0091] FIG. 9 FRET efficiency change of CPK23D193A-CLD21 and CPK23D193A-S362I-CLD21 plotted against Ca.sup.2+-concentrations (Conformational change: R.sup.2.sub.CK21D204A=0.99, R.sup.2.sub.CPK23D193A=0.81, R.sup.2.sub.CPK23D193A-CLD21=0.91, R.sup.2.sub.CPK2D193A3-S362I-CLD21=0.98). FRET-imaged conformational change (means±SEM n=3-6) are determined as described in FIG. 2.

[0092] FIG. 10 Mean±SEM for K50 of activity or EC50 of conformational change and shared Hillslope in n independent experiments.

[0093] FIG. 11 Ca.sup.2+-dependent conformational change of CPK21D204A linker variants F1-F4 (right panel). V, variable domain; Kinase, kinase domain; PS, pseudosubstrate segment; CLD, calmodulin-like domain containing four EF-hand motifs (bright boxes); mT, mTurquoise and cpV173, cpVenus173. Numbers indicate the insertion or the deletion of amino acids (aa) with the linker aa sequence shown (left panel). FRET efficiency change is determined as in (FIG. 2) (R.sup.2.sub.F1=0.99, R.sup.2.sub.F2=0.98, R.sup.2.sub.F3=0.98, R.sup.2.sub.F4=0.99; means±SEM n=3). Variant F4 shows the highest change in emission ratio and was used as matrix for all further FRET analyses and is denominated as CPK21D204A. The CPK21D204A EC50 value is shown in (FIG. 3).

[0094] FIG. 12 Emission spectra of CPK21D204A (excited at 435 nm) at the indicated Ca.sup.2+-concentrations.

[0095] FIG. 13 Emission spectra of CPK21D204A (excited at 435 nm) at the indicated Mg.sup.2+ concentrations. For Mg.sup.2+ the experiment was repeated three times with similar results.

[0096] FIG. 14 pH-dependency of CPK21D204A FRET efficiency. FRET-recorded conformational change of CPK21D204A (left axis) plotted against pH concentrations in the absence of Ca.sup.2+ (EGTA, zero free Ca.sup.2+, means±SEM n=5) and in presence of saturating [Ca.sup.2+]-concentration (means±SEM n=5). The ratio FRET (Ca.sup.2+)/FRET (EGTA) is indicated in red (right axis). The experiment was repeated two times with similar results.

[0097] FIG. 15 Comparison of FRET-recorded conformational change of CPK21D204A with either mTurquoise (CPK21D204A-FRET) or CFP (CPK21D204A-FRET-CFP) as FRET donor (R.sup.2.sub.CPK21D204A-FRET=0.94, R.sup.2.sub.CPK21D204A-FRET-CFP=0.97; means±SEM n=4).

[0098] FIG. 16 Kinase activity of recombinant CPK21 and CPK21-FRET plotted against Ca.sup.2+-concentrations (R.sup.2.sub.CPK21=0.94; means±SEM n=2-3).

[0099] FIG. 17 FRET-recorded conformational change of CPK21-FRET compared with the respective kinase-deficient variants (CPK21D204A-FRET). FRET efficiency change is determined as in (FIG. 2) (R.sup.2.sub.CPK21D204A=0.96, R.sup.2.sub.CPK21=0.9, R.sup.2, means±SEM n=5-6).

[0100] FIG. 18 FRET-recorded conformational change of CPK23-FRET compared with the respective kinase-deficient variants (CPK23D193A-FRET). FRET efficiency change is determined as in (FIG. 2) (R.sup.2.sub.CPK23D193A=0.71, R.sup.2.sub.CPK23=0.91; means±SEM n=5-6).

[0101] FIG. 19 Mean±SEM for K50 of activity or EC50 of conformational change and shared Hillslope in n independent experiments.

[0102] FIG. 20 FRET-recorded conformational change of CPK23, CPK23S362I and CPK23S362D plotted against Ca.sup.2+-concentrations (R.sup.2.sub.CPK23=0.84, R.sup.2.sub.CPK23S362I=0.84, R.sup.2.sub.CPK23S362D=0.56). FRET efficiency change is determined as in (FIG. 3) (means±SEM n=3-6).

[0103] FIG. 21 Mean±SEM for K50 of activity or EC50 of conformational change and shared Hillslope in n independent experiments.

[0104] FIG. 22 FRET-recorded conformational change of TgCDPK1-FRET plotted against [Ca.sup.2+] (Toxoplasma gondii CDPK; cf. Table 1, SEQ ID NO: 11 herein). FRET efficiency change is given as percentage of increase in ratio over the baseline FRET signal (means±SEM n=3, EC50=1029 nM, ΔFRET.sub.max=13%).

[0105] The following sequences are provided herein:

TABLE-US-00005 Bold: PDK Underlined: PS Bold and underlined: CLD Protein Arabidopsis thaliana CPK21 SEQ ID NO: 1 MGCFSSKHRKTQNDGGEKSIPINPVQTHVVPEHRKPQTPTPKPMTQPIHQQISTPSSNPVS VRDPDTILGKPFEDIRKFYSLGKELGRGQFGITYMCKEIGTGNTYACKSILKRKLISKQDKED VKREIQIMQYLSGQPNIVEIKGAYEDRQSIHLVMELCAGGELFDRIIAQGHYSERAAAGIIRSI VNVVQICHFMGVVHRDLKPENFLLSSKEENAMLKATDFGLSVFIEEGKVYRDIVGSAYYVA PEVLRRSYGKEIDIWSAGVILYILLSGVPPFWAENEKGIFDEVIKGEIDFVSEPWPSISESAK DLVRKMLTKDPKRRITAAQVLEHPWIKGGEAPDKPIDSAVLSRMKQFRAMNKLKKLALKVIA ESLSEEEIKGLKTMFANIDTDKSGTITYEELKTGLTRLGSRLSETEVKQLMEAADVDGNGTI DYYEFISATMHRYKLDRDEHVYKAFQHFDKDNSGHITRDELESAMKEYGMGDEASIKEVIS EVDTDNDGRINFEEFCAMMRSGSTQPQGKLLPFH Protein Arabidopsis thaliana CPK23 SEQ ID NO: 2 MGCFSSKHRKTQNDGGGERSIPIIPVQTHIVDQVPDHRKPQIPSPSIPISVRDPETILGKPFED IRKFYSLGRELGRGGLGITYMCKEIGTGNIYACKSILKRKLISELGREDVKTEIQIMQHLSGQ PNVVEIKGSYEDRHSVHLVMELCAGGELFDRIIAQGHYSERAAAGTIKSIVDVVQICHLNGVI HRDLKPENFLFSSKEENAMLKVTDFGLSAFIEEGKIYKDVVGSPYYVAPEVLRQSYGKEIDI WSAGVILYILLCGVPPFWADNEEGVFVEILKCKIDFVREPWPSISDSAKDLVEKMLTEDPKR RITAAQVLEHPWIKGGEAPEKPIDSTVLSRMKQFRAMNKLKKLALKVSAVSLSEEEIKGLKT LFANMDTNRSGTITYEQLQTGLSRLRSRLSETEVQQLVEASDVDGNGTIDYYEFISATMHR YKLHHDEHVHKAFQHLDKDKNGHITRDELESAMKEYGMGDEASIKEVISEVDTDNDGKINF EEFRAMMRCGTTQPKGKQYPFH Protein Arabidopsis thaliana CPK1 SEQ ID NO: 3 MGNTCVGPSRNGFLQSVSAAMWRPRDGDDSASMSNGDIASEAVSGELRSRLSDEVQNKP PEQVTMPKPGTDVETKDREIRTESKPETLEEISLESKPETKQETKSETKPESKPDPPAKPKK PKHMKRVSSAGLRTESVLQRKTENFKEFYSLGRKLGQGQFGTTFLCVEKTTGKEFACKSIA KRKLLTDEDVEDVRREIQIMHHLAGHPNVISIKGAYEDVVAVHLVMECCAGGELFDRIIQRG HYTERKAAELTRTIVGVVEACHSLGVMHRDLKPENFLFVSKHEDSLLKTIDFGLSMFFKPD DVFTDVVGSPYYVAPEVLRKRYGPEADVWSAGVIVYILLSGVPPFWAETEQGIFEQVLHGD LDFSSDPWPSISESAKDLVRKMLVRDPKKRLTAHQVLCHPWVQVDGVAPDKPLDSAVLSR MKQFSAMNKFKKMALRVIAESLSEEEIAGLKEMFNMIDADKSGQITFEELKAGLKRVGANL KESEILDLMQAADVDNSGTIDYKEFIAATLHLNKIEREDHLFAAFTYFDKDGSGYITPDELQ QACEEFGVEDVRIEELMRDVDQDNDGRIDYNEFVAMMQKGSITGGPVKMGLEKSFSIALKL Protein Solanum lycopersicum CPK1 SEQ ID NO: 4 MGGCFSKKYTQQDANGHRAGRRVNQAYQKPPQPQPERPYQPQPQQERPYQPPPQPAYQ PPPQPKPQPQPHPVPVTVQSGQPQDQMQGPHMNNILGKPFEEIRKLYTLGKELGRGQFG VTYYCTENSTGNPYACKSILKRKLVSKNDREDMKREIQIMQHLSGQPNIVEFKGAYEDRQS VHLVMELCAGGELFDRIIARGYYSEKDAAEIIRQIVNVVNICHFMGVMHRDLKPENFLLTSK DENAMLKATDFGLSVFIEEGKVYRDIVGSAYYVAPEVLRRSYGKEADVWSAGVILYILLSG VPPFWAETEKGIFNTILKGEIDFQSDPWPSISNSAKDLIRKMLTQEPRKRITSAQVLEHPWL RLGEASDKPIDSAVLSRMKQFRAMNKLKKLALKVIAENLSEEEIKGLKAMFHNIDTDNSGTIT YEELKSGLARLGSKLTETEVKQLMEAADVDGNGSIDYIEFITATMHRHRLERDEHLFKAFQ HFDKDHSGFITRDELENAMKEYGMGDEATIKEIIAEVDTDNDGRINYEEFCAMMRSGTTQP QQKLF Protein Oryza sativa CPK1 SEQ ID NO: 5 MGNRTSRHHRAAPEQPPPQPKPKPQPQQQQQQWPRPQQPTPPPAAAPDAAMGRVLGRP MEDVRATYTFGRELGRGQFGVTYLVTHKATGKRFACKSIATRKLAHRDDIEDVRREVQIM HHLTGHRNIVELRGAYEDRHSVNLIMELCEGGELFDRIIARGHYSERAAAALCREIVAVVHS CHSMGVFHRDLKPENFLFLSKSEDSPLKATDFGLSVFFKPGEHFKDLVGSAYYVAPEVLK RNYGAEADIWSAGVILYILLSGVPPFWAESEDGIFDAVLRGHIDFSSEPWPSISNGAKDLVK KMLRQDPKERLTSAEILNHPWIREDGEAPDKPLDITVISRMKQFRAMNKLKKVALKVVAENL SDEEITGLKEMFRSLDTDNSGTITLEELRSGLPKLGTKISESEIRQLMEAADVDGNGTIDYA EFISATMHMNRLEKEDHILKAFEYFDKDHSGYITVDELEEALKKYDMGDDKTIKEIIAEVDTD HDGRINYQEFVAMMRNNNPEIAPNRRRMF Protein Selaginella moellendorffii 99178 SEQ ID NO: 6 MKAAGGIAASTPRSAITASVLGRSTESVRELYTLGRKLGQGQFGVTYLCVEKSSGKQYACK TIPKRKLISQEDVDDVRREIQIMHHLAGQPNVVQIKGAYEDAGSVHLVMELCAGGELFDRII QRGHYSERKAAELIRVIVGVVQACHSLGVMHRDLKPENFLLLSKHEDSLMKATDFGLSVF FKPGEVFTDVVGSPYYVAPEVLRKKYGPEADVWSAGVILYILLSGVPPFWAETEKGIFEQV LKGEIDFESHPWPVISQDAKDLIRKMLCPVPANRLKAHEVLGHPWARADGVAPDKPLDSA VLSRMKQFSAMNKIKKIALRVIAESLSEEEIAGLKEMFKMMDTDNSGSITFDELKAGLERVG SNLVESEIRDLMAAADVDNSGTIDYKEFITATLHLNKIEREEHLLAAFAYFDKDNSGYITKDE LQQVCAENHMGDEVIEEMMREADQDNDGRIDYSEFVTMMRKGAGGIGRKTMRNSLSITFR DLLTV Protein Physcomitrella patens 1s49_208V6 PpCPK1 SEQ ID NO: 7 MRRGVNLVPGQSFTHSVLQRNTENLKDLYTLGRKLGQGQFGTTYLCVEKTTGKEYACKSI AKRKLISQEDVDDVRRELHIMHHLSGHPNIVTIKGAYEDQVSVHLVMELCAGGELFDRIIQR GHYSEAQAAELCRVIVGVVETCHSLGVMHRDLKPENFLLSDPSENAALKTTDFGLSVFFK PGEVFTDVVGSPYYVAPEVLRKHYGPEADVWSAGVILYILLSGVPPFWAETEQGIFEQVLA GELDFVSEPWPSISESAKDLIRRMLDPVAKRRLKAHQVLSHPWIREAGVAPDRPMDPAVQ SRLKQFSAMNKLKKVAIRVIAEFLSEEEIAGLREMFKMIDTDHSGSITFEELKSGLERVGSNL VESEIRQLMDAADVDQNGTIDYGEFLAATLHLNKIEREENLFAAFSWLDKDHSGYLTVDEL QHACSEYNIGDTSIEELIREVDQDNDGRIDYNEFVTMMRKGNGTVGRATLRNSLSLSDALM HTN Protein Chlamydomonas reinhardtii 33.g782750 SEQ ID NO: 8 MGNCSSQDNTVPAGYKALKVQQVVQQSGDVRDFYTFDKQLGKGNFGIVHLVFDKKTNEK YACKSISKRKLVTPEDVEDVRREIQIMNHLAGHKNVVNIRGTYEDKNFIHIVMEVGAGGELF DRIAEAGHFSERRAAEVMRTIVSVVHHCHTMNVVHRDLKPENFLLTERGPGGVIKATDFGL SRFFKEGNQLDEIVGSPFYVAPEVLKRSYGKEADIWSCGVILYILLCGWPPFHGDSTQAIFK NILSAPLDLKTEPWSRVSADAKDCVRRMLARDPRKRLTAEQVLNHPWMRENGAAPDEAF VPEILIRMRQFTKMNMLKREALKVIARSLPHMELAGMREMFQEMDEDGSATITVDELREGL RRKGAEIALGEVQRILNDIDLDGNSKIDYEEFLAATMHLNKLSREENMIAAFEYFDKDKSGF ITRDELMNAMKDIDAEVDVDAILAQVDQNGDGRIDYEEFCAMMRATDLDVLKSAHEALKTK VVVKSVLARVQAEPMREDSITDMSRKSSRAMATAESRKQRGASATPANAEEGEQ Protein Volvox carteri 20014333m SEQ ID NO: 9 MGSCASTENQVPQGYKVLKVQAVVQQQGDVRDYYTFDKQLGKGNFGIVHLVYDKKTNEK FACKSISKRKLVTSEDVEDVRREIQIMNHLAGHKNIVSIRGTFEDKNFIHIVMELCSGGELFD RIAEAGHFSERRAAEVMRTIVSVVHHCHTMNVVHRDLKPENFLLTERGPGGVIKATDFGLS RFFKEGSSLDEIVGSPFYVAPEVLKRAYGKEADIWSCGVILYILLCGWPPFHGDSTQAIFKN ILSAPLDLKSEPWPRVSPDAKDCVRRMLARDPRKRLTAEQVLNHHWMRENGAAPDEAFV PEILIRMRQFTKMNLLKREALKVIARSLPHMELAGMREMFHDMDEDGSGTITVDELREGLR RKGAEIALSEVQRILNDIDLDGNSKIDYEEFLAATMHLNKLSREENMMAAFEYFDKDKSGFI TRDELVTAMRDIDQEVDVDALLRQVDKNGDGRIDYEEFCLMMRASDLDVLKCAHEVRILEG Protein Plasmodium falciparum CDPK1 SEQ ID NO: 10 MGCSQSSNVKDFKTRRSKFTNGNNYGKSGNNKNSEDLAINPGMYVRKKEGKIGESYFKVR KLGSGAYGEVLLCREKHGHGEKAIKVIKKSQFDKMKYSITNKIECDDKIHEEIYNEISLLKSL DHPNIIKLFDVFEDKKYFYLVTEFYEGGELFEQIINRHKFDECDAANIMKQILSGICYLHKHNI VHRDIKPENILLENKHSLLNIKIVDFGLSSFFSKDNKLRDRLGTAYYIAPEVLRKKYNEKCDV WSCGVILYILLCGYPPFGGQNDQDIIKKVEKGKYYFDFNDWKNISEEAKELIKLMLTYDYNK RITAKEALNSKWIKKYANNINKSDQKTLCGALSNMRKFEGSQKLAQAAILFIGSKLTTLEERK ELTDIFKKLDKNGDGQLDKKELIEGYNILRSFKNELGELKNVEEEVDNILKEVDFDKNGYIE YSEFISVCMDKQILFSEERLRDAFNLFDTDKSGKITKEELANLFGLTSISEQMWNEVLGEAD KNKDNMIDFDEFVNMMHKICDNKSS Protein Toxoplasma gondii CDPK1 SEQ ID NO: 11 MGQQESTLGGAAGEPRSRGHAAGTSGGPGDHLHATPGMFVQHSTAIFSDRYKGQRVLGK GSFGEVILCKDKITGQECAVKVISKRQVKQKTDKESLLREVQLLKQLDHPNIMKLYEFFED KGYFYLVGEVYTGGELFDEIISRKRFSEVDAARIIRQVLSGITYMHKNKIVHRDLKPENLLLE SKSKDANIRIIDFGLSTHFEASKKMKDKIGTAYYIAPEVLHGTYDEKCDVWSTGVILYILLSG CPPFNGANEYDILKKVEKGKYTFELPQWKKVSESAKDLIRKMLTYVPSMRISARDALDHE WIQTYTKEQISVDVPSLDNAILNIRQFQGTQKLAQAALLYMGSKLTSQDETKELTAIFHKMD KNGDGQLDRAELIEGYKELMRMKGQDASMLDASAVEHEVDQVLDAVDFDKNGYIEYSEF VTVAMDRKTLLSRERLERAFRMFDSDNSGKISSTELATIFGVSDVDSETWKSVLSEVDKNN DGEVDFDEFQQMLLKLCGN

[0106] The invention is further illustrated by the following examples, however, without being limited to the example or by any specific embodiment of the examples.

EXAMPLES

[0107] CPK21 Evaluation

[0108] On the basis of Toxoplasma gondii CDPK1 structure (Ojo et al., Nat Struct Mol Biol (2010), 17: 602-607; Wernimont et al., Nat Struct Mol Biol (2010), 17: 596-601), a FRET approach was developed where the PS and CLD of CDPKs is sandwiched between a donor (mTurquoise, mT)—and acceptor (Venus circularly permutated at amino acid 173, cpV173)—fluorescence protein pair. The FRET donor was inserted between kinase (PKD) and PS because the kinase domain stabilizes the inactive enzyme conformation via interaction with the PS. Kinase activity measurements with purified CPK21 and a peptide substrate encompassing CPK21 in vivo phosphorylation site of slow anion channel-associated 1 (SLAC1) S59 (Geiger (2010), loc. cit.; Brandt et al., eLife (2015), 4: e03599) confirmed a high Ca.sup.2+-dependent activity with a half maximal activity (K50) of 449+/−90 nM Ca.sup.2+ (FIG. 1). All constructs sandwiching CPK21 PS and CLD displayed enhanced FRET efficiency with increasing [Ca.sup.2+], and with similar half maximal effective concentration (EC50) for Ca.sup.2+ (FIG. 11). Variant F4 was used as matrix for all further FRET analyses. The deduced EC50 value for the conformational change was ˜832 nM Ca.sup.2+ (FIG. 3). Differences between Ca.sup.2+-binding-induced conformational change and Ca.sup.2+-inducible kinase activity indicate that substrate affinity influences Ca.sup.2+-dependency of activity.

[0109] Ca.sup.2+-induced FRET changes of CPK21D204A were stable over a pH range of ˜7.2-8.2 (FIG. 14). Next, it was tested if intrinsic insertion of mT influences CPK21 activity. CPK21-FRET variant F4 was generated with the active enzyme sequence. CPK21-FRET displayed low constitutive auto- and trans-phosphorylation activity lacking a Ca.sup.2+-inducible component (FIG. 16). FRET efficiency is similar for active and kinase-deficient CPK21-FRET variants, but activity correlates with an increase of EC50 from 832 nM (CPK21D204A) to 1517 nM Ca.sup.2+ (FIG. 17).

[0110] CPK23 Evaluation

[0111] CPK23 activity measurements revealed a composite kinetic with a Ca.sup.2+-independent core activity of 42% and a residual Ca.sup.2+-dependent increase with low affinity (K50˜1864 nM Ca.sup.2+) (FIG. 1). Conformational change measurements with kinase deficient CPK23D193A-FRET or native CPK23-FRET showed a weak Ca.sup.2+-induced alteration in FRET efficiency of 10 or 9% with a low Ca.sup.2+]-affinity (EC50˜1391 or ˜1533 nM) (FIG. 2, FIG. 18). This demonstrates that differences in CPK21 and CPK23 Ca.sup.2+ sensitivity of kinase activity are reflected by conformational change. The high percentage of identical amino acids in CPK21 and CPK23 further allows to identify sites that may be responsible for Ca.sup.2+-dependency. The PS stabilizes CPK conformations through intra-domain interactions. Evaluation of the PS from the A. thaliana CPK gene family of 34 members revealed that a conserved isoleucine at consensus PS position 31 is uniquely replaced in CPK23 by serine (S362).

[0112] Next, mutually swapped amino acid substitutions at PS position 31 were assessed for their impact on Ca.sup.2+-dependency of kinase activity and conformational change. In CPK21 substitutions at I373 for S or D, mimicking CPK23, caused an increase of the K50 Ca.sup.2+ in kinase assays from 449 nM (WT) to 1111 nM (I373S) and 1622 nM (I373D) and an increase of the EC50 Ca.sup.2+ for the corresponding CPK21D204A-FRET conformational change from 832 nM (D204A) to 925 (D204A-I373S) and 1701 nM (D204A-I373D) (FIGS. 4 & 5). These data indicate that the highly Ca.sup.2+-responsive CPK21 can be turned into the Ca.sup.2+-insensitive CPK23 upon a single amino acid exchange. In CPK23, the substitution S362I, mimicking CPK21, renders the enzyme in kinase assays more sensitive toward Ca.sup.2+, and S362D results in constitutive Ca.sup.2+-independent kinase activity (FIG. 6). Interestingly, when introduced in CPK23-FRET, both amino acid substitutions showed a similar weak conformational change as native CPK23 protein (FIG. 20). But the low FRET efficiency of CPK23 conformational change, compared to CPK21, could mask subtle changes. Therefore, a CLD domain swap was generated. Chimeric CPK23D193A-CLD21-FRET reveals an increase in maximal FRET efficiency whereas the EC50 Ca.sup.2+ for the conformational change was retained (FIG. 9), indicating that the CLD contributes to the structure of the Ca.sup.2+-bound form. The combination of both, CLD21 swap and S362I amino acid substitution, in CPK23D193A-CLD21-S362I-FRET, lead to a Ca.sup.2+-dependency of conformational change indistinguishable from CPK21D204A-FRET (FIG. 9). With respect to kinase activity, CPK23CLD21- and CPK23CLD21-S362I chimeras display increased Ca.sup.2+-dependency in comparison to native CPK23 (FIG. 8). The substitution at PS position 31 from CPK consensus hydrophobic to polar amino acid (I to S) may impact protein surface properties especially in case of phosphorylated S (mimicked by D). This indicated that ABA-dependent CPK23 auto-phosphorylation alters the Ca.sup.2+-inducible component of CPK23 activity. These results identify intramolecular FRET as a tool suitable to determine in vivo activation/Ca.sup.2+-binding/conformational changes of CDPKs.

[0113] To evaluate the applicability of CDPK-FRET, in vivo plant pollen tubes were used which are characterized by their continuous tip-focussed Ca.sup.2+ gradient essential to maintain polar cell growth. CPK-FRET variants were transiently expressed in pollen tubes that carry the Ca.sup.2+-sensor R-GECO1 (red fluorescent genetically encoded Ca.sup.2+-indicator for optical imaging) (Zhao et al., Science (2011), 333: 1888-1891) as stable transgene. To increase the brightness of the FRET donor fluorescence, mT was substituted by CFP (cyan fluorescent protein) without altering the kinetics of conformational change (FIG. 15). To focus on cytoplasmic Ca.sup.2+ concentrations, CPK variants lacking their myristoylation (G2A)- and palmitoylation (C3V)-sites were generated (Simeunovic et al., J Exp Bot (2016), 67: 3855-3872). Pollen tubes display a robust tip-based [Ca.sup.2+] oscillation pattern when grown on medium containing 10 mM Cl.sup.−. Time-lapse imaging of transfected CPK21G2A-C3V-D204A-FRET-CFP displayed not only a pollen tube tip-focussed FRET signal but recorded the cytosolic [Ca.sup.2+] oscillation pattern reported by R-GECO1 simultaneously (data not shown). Thus, reversible CPK21 enzyme activation and inactivation, imaged by CPK-intrinsic FRET, was recorded in real time in its dependency of cytosolic [Ca.sup.2+].

[0114] TgCDPK1 (Toxoplasma Gondii) Evaluation

[0115] In addition to the existing ratiometric FOrster resonance energy transfer (FRET)-based reporter for plant CDPK conformation changes, this approach was extended to the protist CDPK Toxoplasma gondi TgCDPK1; cf. also FIG. 22.

[0116] A construct was created with the donor chromophore (eCFP) inserted C-terminally to amino acid position 308 of SEQ ID NO: 11. The acceptor chromophore was inserted C-terminally of EF4 corresponding to the amino acid position 507 of SEQ ID NO: 11.

[0117] The TgCDPK1-FRET fusion protein showed a FRET efficiency change in dependency of Ca.sup.2+ concentrations. Based on literature data for Ca.sup.2+-dependent TgCDPK1 kinase activity (Ingram et al., PNAS (2015), E4675-E4984), this shows that TgCDPK1-FRET reflects the conformation change leading to Ca.sup.2+ dependent activation.

[0118] Methods

[0119] Mutagenesis and Cloning of CPK21 and CPK23 Enzyme Variants

[0120] Expression vector pGEX-6P1 (GE Healthcare)-based recombinant synthesis of CPK21 and CPK23 constructs carrying a C-terminal polyhistidine-tag and N-terminal GST-tag has been described previously (Geiger (2010), loc. cit.). CPK21 and CPK23 in pGEX-6P1 were used as a template for PCR-based site-directed mutagenesis with specific primers for the variants CPK21I373S, CPK21I373D, CPK23S362I, CPK23S362D (for primer sequence see Table 4).

[0121] CPK23CLD21-S362I chimeric construct was generated by replacing a part of CPK23 pseudosubstrate segment (from aa 353), the CLD23 and a part of pGEX-6P1 vector backbone in pGEX-6P1-CPK23 with the homolog sequence from pGEX-6P1-CPK21 via HindIII and PstI. To create the pGEX-6P1-CPK23CLD21 construct amino acid substitution I362S was introduced by site-specific mutagenesis primers CPK21I373S-F and CPK21I373S-R with CPK23CLD21-S362I in pGEX-6P1 as PCR template. CPK23CLD21 and CPK23CLD21-S362I chimeric constructs carry the A358 codon from CPK21 instead of CPK23 as a result from cloning strategy.

[0122] The pXCS-CPK23-HA-Strep in vivo expression plasmid used in this study has been described previously (Geiger (2010), loc. cit.). To create a CPK23 kinase-deficient variant, the amino acid substitution D193A was introduced by site-specific mutagenesis primers CPK23D193A-F and CPK23D193A-R.

[0123] Mutated coding sequences were verified by sequencing and transferred via restriction sites into the CPK sequence containing vectors.

[0124] Generation of CPK FRET Sensors

[0125] CPK21 and CPK23 variable and kinase domain coding sequences were re-amplified from plasmids pXCS-CPK21D204A-HA-Strep (Geiger (2011), loc. cit.) and pXCS-CPK23D193A-HA-Strep using the forward primer CPK21-VK XbaI EcoRI-F or CPK23-VK XbaI EcoRI-F and the reverse primer CPK21/23-VK XbaI-R (for primer sequence see Table 4). Fragments were transferred via the N- and C-terminal introduced XbaI site into pUC-F3-II (Waadt et al., eLife (2014), 3: e01739) resulting in pUC-F3-II-CPK21D204A-VK (variable and kinase domain) or pUC-F3-II-CPK23D193A-VK. PUC-F3-II contain the FRET donor (mTurquoise, mT) (Goedhardt et al., Nat Methods (2010), 7: 137-139) and the FRET acceptor (Venus circularly permutated at amino acid 173, cpV173) (Nagai et al., PNAS (2004), 101: 10554-10559).

[0126] CPK21 pseudosubstrate segment and CLD were isolated by PCR, using for linker variant F1 the primers CPK21-PSCLD ApaI-F and CPK21-PSCLD SmaI-R, for linker variant F2 CPK21-PSCLD SpeI-F and CPK21-PSCLD KpnI-R, or for linker variant F3 CPK21-PSCLD BamHI-F and CPK21-PSCLD SalI-R and inserted between ApaI/SmaI (F1), SpeI/KpnI (F2) and BamHI/SalI (F3) in pUC-F3-II-CPK21D204A-VK resulting into pUC-F3-II-CPK21D204A-FRET (F1-F3), respectively. CPK21 and CPK23 coding sequence covering the pseudosubstrate segment and CLD domain until EF-hand 4 (CPK21 aa 522, CPK23 aa 511) was amplified with primers introducing ApaI (CPK21-PSCLD ApaI-F and CPK23-PSCLD ApaI-F) and SmaI (CPK21-PSCLD-EF4 SmaI-R or and CPK23-PSCLD-EF4 SmaI-R) restriction sites and the fragments were inserted between ApaI/SmaI in pUC-F3-II-CPK21D204A-VK to yield pUC-F3-II-CPK21D204A-FRET (F4), or in pUC-F3-II-CPK23D193A-VK to yield pUC-F3-II-CPK23D193A-FRET. CPK21 aa 523 and CPK23 aa 512 was identical to the first aa of SmaI restriction site thus in pUC-F3-II-CPK21D204A-FRET (F4), or in pUC-F3-II-CPK23D193A-FRET CPK aa sequence until aa 523 (CPK21) or 512 (CPK23) is present.

[0127] CPK21D204A full length sequence was amplified using primers CPK21-FL ApaI-F and CPK21-PSCLD SmaI-R, and the respective fragment was ligated into ApaI/SmaI of pUC-F3-II resulting in pUC-F3-II-CPK21D204A-FRET (F5).

[0128] Escherichia coli expression vectors were obtained by sub-cloning CPK21D204A-FRET (F1-F4) and CPK23D193A-FRET via EcoRI/SacI or CPK21D204A-FRET (F5) via NdeI/SacI into pET-30a (+) (Novagen). The resulting pET-30a-CPK21D204A-FRET (F1-F4) and pET-30a-CPK23D193A-FRET constructs carry a N-terminal polyhistidine-tag derived from pET-30a vector backbone and a C-terminal Strep-tag derived from pUC-F3-II-CPK2/D204A-FRET (F1-F4) or pUC-F3-II-CPK23D/93A-FRET constructs. pET-30a-CPK21D204A-FRET (F5) carries a Strep-tag derived from pUC-F3-II-CPK21D204A-FRET (F5).

[0129] For cloning of CPK21- and CPK23-FRET variants, mutation-containing CPK sequences or the CPK23CLD21 chimeric sequence were exchanged via restriction sites from pGEX-6P-1-CPK constructs into pET30a-CPK-FRET.

[0130] For in vivo transient expression CPK21D204A-FRET and CPK23D193A-FRET were sub-cloned via EcoRI/Ecl13611 and inserted between EcoRI/SfoI into pXCS-HA-Strep (Witte et al., Plant Mol Biol (2004), 55: 135-147) yielding pXCS-CPK21D204A- or pXCS-CPK23D193A-FRET. The p35S of pXCS-CPK-FRET was substituted with the ubiquitin4-2 promoter from parsley from V69-Ubi:Cas9-MCS-U6 (Kirchner et al., PLoS ONE (2017), 12: e0185429) via AscI/XhoI yielding pXC-Ubi-CPK21D204A-FRET or pXC-Ubi-CPK23D/93A-FRET. For cytosolic localization, pXC-Ubi-CPK-FRET construct was used as a template for PCR-based site-directed mutagenesis introducing G2A in a first mutagenesis reaction and G2A-C3V mutation in a second reaction (for primer sequence see Table 4) (Wener et al., Gene (1994), 151: 119-123). Mutated coding sequences were validated by sequencing and transferred via restriction sites within CPK-VK sequence into the pXC-Ubi-CPK-FRET construct.

[0131] The primers CFP NdeI-F and CFP ApaI-R (Table 4) were used to amplify CFP (cyan fluorescent protein) (Heim et al., Curr Biol (1996), 6: 178-182) with attached NdeI/ApaI sites and CFP was cloned into NdeI and ApaI linearized pXC-Ubi-CPK21G2A-C3V-FRET or pXC-Ubi-CPK23G2A-C3V-FRET resulting in pXC-Ubi-CPK21G2A-C3V-FRET-CFP or pXC-Ubi-CPK23G2A-C3V-FRET-CFP.

[0132] Expression in Escherichia Coli and Protein Purification

[0133] CPK constructs were in general synthesized as recombinant double-tagged fusion proteins in E. coli. For in vitro kinase assays expression vector pGEX-6P-1 was used and proteins were purified using the N-terminal His-Tag and a C-terminal GST-Tag. For in vitro FRET measurements, expression vector pET30a-CPK-FRET was used and proteins were purified using the N-terminal His-Tag and a C-terminal Strep-Tag.

[0134] To synthesize and purify proteins for kinase assays the pGEX-6P-1-CPK expression, vectors were introduced in E. coli BL21 (DE3) (Stratagene). Bacteria were grown at 37° C. in LB medium containing 100 μg/ml ampicillin and protein expression was induced at an OD.sub.600 of 0.4-0.6 with 0.3 mM isopropylthiol-β-galactoside (IPTG). Cells were incubated for additional 4 h at 28° C. and harvested by centrifugation. Cells were lysed in 4 ml histidine-lysis buffer (50 mM HEPES-KOH pH 7.4, 300 mM NaCl, 0.2% (v/v) Triton X-100, 1 mM DTT, 10 μl protease inhibitor cocktail for histidine tagged proteins (Sigma)/0.2 g weight of E. coli cells and 30 mM imidazole) using 1° mg/ml lysozyme and sonification and a centrifugation was performed to remove the cell debris. Supernatant was gently end-over-end rotated with 300-600 μl Ni sepharose 6 fast flow (GE Healthcare) at 4° C. for 1 h. Sample/Ni sepharose mix was loaded on empty columns and washed 1×10 ml histidine-washing buffer (50 mM HEPES-KOH pH 7.4, 300 mM NaCl) with 30 mM imidazole and 1×10 ml histidine-washing buffer with 40 mM imidazole. Proteins were eluted 3× in 500 μl histidine-elution buffer (50 mM HEPES-KOH pH 7.4, 300 mM NaCl, 500 mM imidazole). Eluate was incubated at 4° C. for 1 h with Glutathione sepharose. Eluate/Glutathione sepharose mix was loaded on empty columns washed 3×3 ml GST-wash buffer (50 mM Tris-HCl pH 8.0, 250 mM NaCl, 1 mM DTT) and eluted 3× with 300 μl GST-elution buffer (100 mM Tris-HCl pH 8.4 and 20 mM glutathione). Proteins were dialyzed using micro dialysis capsule QuixSep (Roth) and dialysis membrane with 6000-8000 Da cut off (Roth). Dialysis-buffer was composed of 30 mM MOPS pH 7.4 and 150 mM KCl.

[0135] To synthesize and purify proteins for FRET measurements, pET30a-CPK-FRET expression vectors were transformed into E. coli BL21 (DE3) pLySs strain (Stratagene). Bacteria were grown at 37° C. in TB medium containing 50 μg/ml kanamycin and 34 μg/ml chloramphenicol and protein expression was induced at an OD.sub.600 of 0.4-0.6 with 0.4 mM IPTG. Cells were incubated for additional 4 h at 22° C. and harvested by centrifugation. Cells were lysed in 6 ml histidine-lysis buffer (see above) using 1 mg/ml lysozym and sonification followed by centrifugation to remove cell debris. Supernatant was incubated with 300-600 μl Ni sepharose 6 fast flow (GE Healthcare) at 4° C. for 1 h. Sample/Ni sepharose mix was loaded on empty columns and washed 1×10 ml histidine-washing buffer with 30 mM imidazole and 1×10 ml histidine-washing buffer with 40 mM imidazole. Proteins were eluted 3× in 500 μl histidine-elution buffer. Eluate was incubated at 4° C. for 45 min with Strep-tactin macroprep (IBA). Strep-tagged recombinant proteins were purified as described by Schmidt and Skerra (Schmidt et al., Nat Protoc (2007), 2: 1528-1535) with the modification that EDTA was omitted from elution and wash buffer. Proteins were dialyzed using micro dialysis capsule QuixSep (Roth) and dialysis membrane with 6000-8000 Da cut off (Roth). Dialysis buffer was composed 30 mM Tris-HCl pH 7.4, 150 mM NaCl and 10 mM MgCl.sub.2.

[0136] The CPK21D204A-FRET variant containing the entire CPK flanked by the fluorescence proteins (F5) was affinity purified using the C-terminal Strep-Tag as described (Schmidt, loc. cit.) with the modification that EDTA was omitted from elution and wash buffer.

[0137] Protein purity of E. coli expressed proteins was confirmed by 10% SDS-PAGE and Coomassie staining. For in vitro analyses, protein concentrations were quantified based on the method of Bradford (Protein assay, Bio-Rad).

[0138] Protein Sequence Comparison

[0139] The analysis of the pseudosubstrate segment of the entire A. thaliana Col-0 CPK gene family was conducted on basis of protein sequences from uniport (The UniProt Consortium, Nucleic Acid Res (2017), 45: D158-D169) with the program Web Logo (Crooks et al., Genome Res (2004), 14: 1188-1190; Schneider et al., Nucleic Acid Res (1990), 18: 6097-6100).

[0140] Preparation of Calcium and Magnesium Buffers

[0141] For CPK protein kinase assays, calculated reciprocal dilutions of zero-Ca.sup.2+-buffer (10 mM EGTA 150 mM KCl, 30 mM MOPS pH 7.4) with high-Ca.sup.2+-buffer (10 mM CaCl.sub.2, 10 mM EGTA 150 mM KCl, 30 mM MOPS pH 7.4) were mixed. For the analysis of CPK-FRET conformational changes high-Ca.sup.2+-buffer (20 mM CaCl.sub.2; 20 mM EGTA, 150 mM NaCl, 10 mM MgCl.sub.2, 30 mM Tris-HCl pH 7.4) and zero-Ca.sup.2+-buffer (20 mM EGTA, 150 mM NaCl, 10 mM MgCl.sub.2, 30 mM Tris-HCl pH 7.4) were mixed accordingly, preceding a 1:1 dilution with CPKs. Correspondingly, buffer solutions for CPK-FRET analysis in a Mg.sup.2+ concentration gradient were prepared by a mixture of high-Mg.sup.2+-buffer (120 mM MgCl.sub.2, 20 mM EDTA, 150 mM NaCl, 30 mM Tris-HCl pH 7.4) and zero-Mg.sup.2+-buffer (20 mM EDTA, 150 mM NaCl, 30 mM Tris-HCl pH 7.4), followed by a 1:1 dilution with FRET protein in Mg.sup.2+-dialysis buffer (30 mM Tris-HCl pH 7.4, 150 mM NaCl) before data acquisition. The indicated free Ca.sup.2+ or Mg.sup.2+ concentrations were calculated with the WEBMXC extended website http://www.stanford.edu/-cpatton/maxc.html based on Patton et al., Cell Calcium (2004), 35: 427-431.

[0142] In Vitro Kinase Assays

[0143] In vitro kinase activity with recombinant purified proteins were conducted as described using a 20 aa peptide (41-RGPNRGKQRPFRGFSRQVSL-60; JPT Peptide Technologies) derived from the CPK21 and CPK23 in vivo phosphorylation substrate protein SLAC1 (slow anion channel-associated 1). For kinase reaction (30 μl) the enzyme (˜90 nM) was incubated in 25 mM MOPS pH 7.4, 125 mM KCl, 10 mM MgCl.sub.2, 10 μM ATP, 3 μCi [γ-.sup.32P]-ATP, 10 μM SLAC1 peptide, 6.67 mM EGTA and different concentration of CaCl.sub.2 for 20 min at 22° C. The reaction was stopped by adding 3 μl 10% phosphoric acid. Phosphorylation of the SLAC1 peptide was assessed after binding of phosphor-peptides to P81 filter paper and scintillation counting as described (Franz et al., Mol Plant (2011), 79: 803-820). 2-4 technical replicates were evaluated per sample. Ca.sup.2+-dependent kinase activity is indicated as percentage of maximal activity (best fit-value obtained for maximal activity). Kinase activities can be described by a four parameter logistic equation with a global model with shared Hillslope (GraphPad Prism Software). For comparison of enzyme variants data from different measurements were combined in one figure.

[0144] For analysis of autophosphorylation activities of protein kinases, the reaction was the same as above with the modifications that SLAC1 substrate peptide was omitted from kinase reaction and ˜255 nM enzyme was used. The reaction was stopped by adding 5× SDS-PAGE loading buffer and boiling for 5 min and samples were separated by 10% SDS-PAGE. Phosphorylation was determined by autoradiography and phospho-imaging (Typhoon FLA 9500, GE Healthcare).

[0145] In Vitro Analyses of CPK-FRET

[0146] CPK-FRET protein (˜415 nM) in dialysis buffer diluted 1:1 with Ca.sup.2+-buffers of defined concentrations (see above) was evaluated using the TECAN Infinite M200 PRO plate reader (TECAN). Excitation at 435 nm (bandwidth 5 nm) and emission within the range of 470-600 nm was monitored in 2 nm steps with 10 flashes of 20 μs and 400 Hz. To obtain optimal emission spectra the gain settings were calculated by the TECAN software from a CPK-FRET high-Ca.sup.2+-buffer sample. cpVenus173/mTurquoise FRET ratios were calculated on the basis of maximal values from emission bands of mTurquoise (470-490 nm) and cpVenus173 (518-538 nm). FRET ratios are plotted against increasing Ca.sup.2+ concentrations using GraphPad Prism 4 software and are described by a four parameter logistic equation with shared Hillslope value for all data sets of the same enzyme. The best fit-value obtained for bottom FRET ratio is used to calculate ΔFRET as the percentage change of emission ratios. ΔFRET is defined as:

[00001]$\Delta {\mathrm{FRET}\left[{}_{\mathrm{free}}\mathrm{Ca}^{2+}\right]}_{x\mu M}=\frac{\mathrm{FRET}{\mathrm{ratio}\left[{\mathrm{Ca}}^{2+}\right]}_{x\mu M}}{\mathrm{FRET}\mathrm{ratio}\mathrm{Bottom}}\times 100-100$

[0147] The percentage change of emission ratio ΔFRET is fitted by a four parameter logistic equation using graph GraphPad Prism 4 software. For comparison of enzyme variants, data from different measurements were combined in one figure.

[0148] The pH-dependency of a CPK-FRET sensor was assessed with CPK21D204A-FRET in dialysis buffers (30 mM Tris-HCl, 150 mM NaCl and 10 mM MgCl.sub.2) of different pH values (pH 5.0, 6.9 and 8.0). Dialysed proteins were diluted 1:1 with either high-Ca.sup.2+-buffer or zero-Ca.sup.2+-buffer (see above) within 30 mM Tris-HCl adjusted accordingly to a pH range between 5.0-8.4. Curves were fitted by a four parameter logistic equation and ratio change was calculated by dividing the Ca.sup.2+ through the EGTA value.

[0149] Protein Expression in Protoplast and Purification for MS Measurement

[0150] Preparation and transfection of Arabidopsis leaf mesophyll protoplasts to transiently express CPK23-HA-Strep and CPK23D193A-HA-Strep was conducted as described (Wu et al., Plant Methods (2009), 5: 16). Approximately 9.6×10.sup.5 protoplasts of cpk23 (SALK_007958) (Ma et al., Plant Mol Biol (2007), 65: 511-518) line were transfected with 96 μg of pXCS-CPK23-HA-Strep or pXCS-CPK23D193A-HA-Strep and incubated in the dark for 14 h. One transfection reaction was divided by two for ABA treatment and as control. Protoplasts were treated with 30 μM final ABA for 10 min at RT. Cells were collected by centrifugation (twice 10000×g, 2 s), frozen in liquid nitrogen, and pellets were stored at −80° C. Protoplasts were resuspended into 600 μl of extraction buffer (100 mM Tris-HCl pH 8.0, 100 mM NaCl, 5 mM EDTA, 5 mM EGTA, 20 mM DTT, 10 mM NaF, 10 mM NaVO.sub.4, 10 mM β-glycerole-phosphate, 0.5 mM AEBSF, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 100 μg/ml avidin, 0.2% NP-40, 1× phosphatase inhibitor mixture (Sigma) and 1× protease inhibitor mixture (Sigma) and centrifuged at 20000×g for 10 min at 4° C. Supernatant was incubated with 24 μl Strep-Tactin MacroPrep (IBA) beads on a rotation wheel for 45 min at 4° C. After centrifugation (500×g, 1 min) beads were solved in 100 μl 6 M urea, 2 M thiourea, pH 8.0 and incubated for 10 min at 4° C. on a rotation wheel. After centrifugation (500×g, 1 min) the protein containing supernatant was transferred to a new tube and reduction of disulfide bonds, alkylation of cysteines and tryptic digestion was conducted as described (Dubiella et al., PNAS (2013), 110: 8744-8749). Peptide containing reactions were vacuum-dried at 30° C. and stored at −20° C.

[0151] Targeted Analysis Phosphorylation by Directed MS

[0152] Samples were subsequently desalted through C18 tips. Digested protein mixtures were spiked with 500 fmol of 13C.sub.6-R/K mass-labelled standard peptide before mass spectrometry analysis. Tryptic peptide mixtures including the stable-isotope labelled standard peptides were analysed on a nano-HPLC (Easy nLC, Thermo Scientific) coupled to an Orbitrap mass spectrometer (LTQ-Orbitrap, Thermo Scientific) as mass analyser. Peptides were eluted from a 75 μm analytical column (Easy Columns, Thermo Scientific) on a linear gradient running from 10% to 30% acetonitrile in 120 min and were ionized by electrospray.

[0153] The target peptide V(pS)AVSLSEEEIK (m/z of doubly-charged ion for phosphopeptide 685.8262; non-phosphopeptide 645.8430) was analysed in its phosphorylated and non-phosphorylated state using the stable-isotope labelled synthetic standard peptide as an internal reference and for normalisation between samples. Standards carried a .sup.13C.sub.6-labeled amino acid (arginine or lysine) at their C-terminal ends.

[0154] Information-dependent acquisition of fragmentation spectra for multiple-charged peptides was used with preferred precursor selection of the target peptides through implementation of an inclusion lists (Schmidt et al., Mol Syst Biol (2011), 7: 510). Full scans were obtained at a resolution of FWHM (full width at half maximum) of 60000, CID fragment spectra were acquired in the LTQ. Additional fragmentation though multistage activation (Schroeder et al., Anall Chem (2004), 76: 3590-3598) was used if peptides displayed a loss of phosphoric acid (neutral loss, 98 Da) upon MS/MS fragmentation.

[0155] Protein identification and intensity quantitation was performed as described (Menz et al., Plant J (2016), 88: 717-734). To allow robust identification and quantitation of the internal standard peptide, multiplicity was set to 2 and Lys6 and Arg6 were selected as stable isotope labels and in general, data analysis was focused on the target peptide sequences only.

[0156] Quantification of Target Peptide Abundance Changes

[0157] For quantitative analysis, the ion intensities of .sup.13C.sub.6-labeled standard peptides were used for normalisation between samples and replicates. Normalised ion intensities of phosphorylated and non-phosphorylated target peptides were averaged between replicates of the same treatments.

[0158] Transient Pollen Transformation

[0159] Nicotiana tabacum (cultivars Petit Havana SR1) plants were grown on soil with a day/night regime of 10 h/14 h, and a temperature of 22 to 24/20 to 22° C. provided by a 30 klx white light (SON-T Agro 400 W; Philips). Pollen of tobacco lines expressing the R-GECO1 calcium sensor (Zhao, loc. cit.) as a stable transgene under the control of a pollen-specific promoter (pLeLAT52::R-GECO1 line) was used from frozen stocks to perform transient transformation using a homemade particle bombardment device has been described in detail (Gutermuth et al., Plant Cell (2013), 25: 4525-4543). Biolistic transformation was performed with pXC-Ubi-CPK21D204A-G2A-C3V-CFP and pXC-Ubi-CPK23D193A-G2A-C3V-CFP on agar plates containing pollen tube growth medium (1 mM MES-Tris pH 5.8, 0.2 mM CaCl.sub.2, 9.6 mM HCl, and 1.6 mM H.sub.3BO.sub.3). Osmolarity of pollen media was adjusted to 400 mosmol kg.sup.−1 (Vapor Pressure Osmometer 5520) with D(+)-sucrose.

[0160] Live-Cell Fluorescence Imaging

[0161] The setup for wide-field live-cell imaging and the appropriate software to control sample acquisition has been described in detail (Guthermuth, loc. cit.). Images were recorded with a time interval of 5 s. For simultaneous CFP/YFP/RFP-imaging a triple-band dichroic mirror (Chroma #69008; ET-ECFP/EYFP/mCherry) was used to reflect excitation light on the samples. Excitation of CFP and R-GECO1 was performed with a VisiChrome High-Speed Polychromator System (Visitron Systems) at 420 nm and 550 nm, respectively. Optical filters (Chroma Technology Corporation) for CFP (ET 470/24 nm), YFP (ET 535/30 nm) and R-GECO1 (624/40) were used for fluorescence detection with a back-illuminated 512×512 pixel Evolve EMCCD camera (Photometrics). A high-speed 6-position filter wheel (Ludl Electronic Products Ltd.) ensured the quasi simultaneous imaging of all three channels with a lag-time of ˜0.1 sec. For image processing the following steps were conducted for R-GECO (R-GECO.sub.excitation/R-GECO.sub.emission), FRET (CFP.sub.excitation/YFP.sub.emission) and CFP (CFP.sub.excitation/CFP.sub.emission) channels using Fiji (National Institute of Health) background subtraction (same value for FRET and CFP channel), gaussian blur, 32-bit conversion, kymographs were generated, and threshold adjusted. A self-made script for the Octave 4.0.3 free software http://www.gnu.org/software/octave/) was used to quantify fluorescence intensities of each channel at ˜5-15 μm behind the tip of the growing pollen tubes over time. FRET-analysis was performed by dividing the FRET signal by the CFP signal. Correlation analyses for R-GECO and FRET channels signals were done using graph GraphPad Prism 4 software.

[0162] Statistics

[0163] Analyses of kinase activities and conformational changes were performed by a four parameter logistic equation with a global model with shared hillslope for all data sets of the same enzyme. A 95% confidence interval was used. Statistical analysis of MS-data was performed by one-way ANOVA (P=0.005, F=9.604, degrees of freedom=3 (between columns) and =8 (within columns)) followed by Tukey's multiple comparison test and P<0.05 was considered significant. For Pearson correlation a two-tailed P value, a 95% confidence interval and a significance level of 0.05 was used. All statistical analyses were performed using GraphPad Prism 4.

TABLE-US-00006 TABLE 4 Sequences of oligonucleotide primers SEQ Construct Sequence ID NO. site-directed mutagenesis primers CPK21D204A-F GGTGTGGTTCATCGAGCTCT 12 CAAGCCTGAG CPK21D204A-R CTCAGGCTTGAGAGCTCGAT 13 GAACCACACC CPK23D193A-F GTGTGATTCATCGAGCTCTC 14 AAGCCTGAG CPK23D139A-R CTCAGGCTTGAGAGCTCGAT 15 GAATCACAC CPK21I373S-F* GCTCTAAAGGTTAGCGCGGA 16 GAGTCTATC CPK21I373S-R* GATAGACTCTCCGCGCTAAC 17 CTTTAGAGC CPK21I373D-F GCTAGCTCTAAAGGTTGACG 18 CGGAGAGTCTATC CPK21I373D-R GATAGACTCTCCGCGTCAAC 19 CTTTAGAGCTAGC CPK23S362I-F GCCCTAAAGGTTATCGCGGT 20 GAGTCTATC CPK23S362I-R GATAGACTCACCGCGATAAC 21 CTTTAGGGC CPK23S362D-F GCCCTAAAGGTTGACGCGGT 22 GAGTCTATC CPK23S362D-R GATAGACTCACCGCGTCAAC 23 CTTTAGGGC CPK21G2A-F GATATCGAATTCATGGCTTG 24 CTTCAGCAGTAAACAC CPK21G2A-R GTGTTTACTGCTGAAGCAAG 25 CCATGAATTCGATATC CPK21G2AC3V-F GATATCGAATTCATGGCTGT 26 CTTCAGCAGTAAACAC CPK21G2AC3V-R GTGTTTACTGCTGAAGACAG 27 CCATGAATTCGATATC CPK23G2A-F GATATCGAATTCATGGCTTG 28 TTTCAGCAGTAAACAC CPK23G2A-R GTGTTTACTGCTGAAACAAG 29 CCATGAATTCGATATC CPK23G2AC3V-F GATATCGAATTCATGGCTGT 30 TTTCAGCAGTAAACAC CPK23G2AC3V-R GTGTTTACTGCTGAAAACAG 31 CCATGAATTCGATATC Cloning of CPK-FRET constructs CPK21-FL ApaI-F ATGGGCCCATGGGTTGCTTC 32 AGCAG CPK21-VK XbaI ATTCTAGA GAATTCATGGG 33 EcoRI-F TTGCTTC CPK23-VK XbaI ATTCTAGA GAATTCATGGG 34 EcoRI-F TTGTTTC CPK21/23-VK ATTCTAGATTCTCCCCCTTT 35 XbaI-R GATCCAAG CPK21-PSCLD ATGGGCCCGCACCAGACAAG 36 ApaI-F CCTATTG CPK21-PSCLD ATACTAGTGCACCAGACAAG 37 SpeI-F CCTATTG CPK21-PSCLD GGATCCGCACCAGACAAGCC 38 BamHI-F TATTG CPK21-PSCLD CCCGGGATGGAATGGAAGCA 39 SmaI-R GTTTC CPK21-PSCLD ATGGTACCATGGAATGGAAG 40 KpnI-R CAGTTTC CPK21-PSCLD ATGTCGACATGGAATGGAAG 41 SalI-R CAGTTTC CPK21-PSCLD- ATCCCGGGCTGCGTGCTGCC 42 EF4 SmaI-R ACTTC CPK23-PSCLD- ATCCCGGGTTGTGTGGTGCC 43 EF4 SmaI-R ACATC CFP NdeI-F ATCATATGGTGAGCAAGGGC 44 GAGG CFP ApaI-R ATGGGCCCCTTGTACAGCTC 45 GTCCATG * Used for mutagenesis of I362 in CPK23CLD21-S362I yielding in CPK23CLD21 F: forward, R: reverse