Abstract
Near-infrared (NIR) fluorescent proteins (FPs) designed from PAS (Per-ARNT-Sim repeats) and GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA transcriptional activator) domains of bacterial phytochromes covalently bind biliverdin (BV) chromophore via one or two Cys residues. We studied BV interaction with a series of NIR FP variants derived from the recently reported BphP1-FP protein. The latter was engineered from a bacterial phytochrome RpBphP1, and has two reactive Cys residues (Cys15 in the PAS domain and Cys256 in the GAF domain), whereas its mutants contain single Cys residues either in the PAS domain or in the GAF domain, or no Cys residues. We characterized BphP1-FP and its mutants biochemically and spectroscopically in the absence and in the presence of denaturant. We found that all BphP1-FP variants are monomers. We revealed that spectral properties of the BphP1-FP variants containing either Cys15 or Cys256, or both, are determined by the covalently bound BV chromophore only. Consequently, this suggests an involvement of the inter-monomeric allosteric effects in the BV interaction with monomers in dimeric NIR FPs, such as iRFPs. Likely, insertion of the Cys15 residue, in addition to the Cys256 residue, in dimeric NIR FPs influences BV binding by promoting the BV chromophore covalent cross-linking to both PAS and GAF domains.
Highlights
The non-invasive imaging of biological processes in vivo is one of the most important problems of modern molecular and cell biology
BphPs are multi-domain proteins composed of a photosensory core module (PCM) and an output effector module
The PCM can be separated into the chromophore-binding domain (CBD), which is minimally required for chromophore binding and PHY domain, which is implicated in photoconversion of the chromophore between Pr and Pfr states and signal propagation to the effector module
Summary
The non-invasive imaging of biological processes in vivo is one of the most important problems of modern molecular and cell biology. Fluorescent NIR FPs [5,20,21,22,23,24] have been engineered on the basis of CBD of BphPs, photoactivatable NIR FPs [25]—on the basis of PCM of BphPs, and NIR reporters of protein-protein interaction [3,4,5]—on the basis of separate PAS and GAF domains of BphPs. Interestingly, the absorption/fluorescence spectra of NIR FPs of an Infra-red Fluorescent Protein (IFP) family (IFP1.4 [26], IFP2.0 [21], IFP1.4rev [22] and Wi-Phy [23]) engineered from Deinococcus radiodurans DrBphP differ no more than 16/14 nm, respectively. The blue-shift of these two BV based chromophores, both covalently bound to Cys256, originates from the smaller chromophore π-conjugated systems relative to a BV adduct bound to Cys in the PAS domain and found in natural BphPs and in common (red-shifted) NIR FPs, such as iRFP702, iRFP713 and iFRFP720 [27]. Insights into spectral characteristics of NIR FPs were obtained in studies of dimeric iRFPs and thIenirsimghuttsainnttsowspitehctCraylscrheasriadcuteesrisetiitchserofinNtIhReFPPAs Sw(eCreyso1b5t)aionredininthsetuGdAieFs dofomdiaminesric(CiRysF2P5s6a),nwdith theCiryms uretasindtus ewsiitnh CboytshreGsAidFueasneditPhAerSindothmeaPinAsS, (aCnyds1la5c)koinr gintthheeCGyAsFredsoidmuaeisns[2(8C]y. sIt25h6a)s, wbeitehnCfoyusnd restidhautesthine binottehraGcAtioFnanodf BPAVSwdiothmdaiinmse, raincdNlIaRckFinPgs tihsegCovyesrrneesdidubeysi[n2t8e]r.-mIt ohnaos mbeeernafnodunindttehr-adtothmeain intearllaocsttieornicoeffBfeVctws. iItnhddiimmeerriicciNRFIRP vFaPrsiaisntgsowveitrhnoeudtbCyyisn1t5e,ra-mcoovnaolmenetrbainnddiinngteorf-dBoVmtoaiСnyasl2lo5s6teinricone effemctosn. oInmdeirmaelrliocsitRerFiPcavllayriainnhtsibwitisthtohuet cCoyvsa1l5e,nat cboivnadleinngt boinf dBinVg toof BaVnottohCerysm25o6nionmoenre wmhoenroemaserthe allopsrteesreicnaclelyoifnChyibsi1t5s tahlleocsotevraicleanlltybpinrodminogteosf BBVV btoinadniontghteor Cmyosn2o5m6 einr wbohtehrmeaosnthoemperress[e2n8c]e. of Cys allosteriIcnaltlhyisprpoampeort,eswBeVpberinfodrimngedtosCpeycst2r5a6l cinhabroatchtemriozantoiomnerosf [B2p8]h.P1-FP and its mutants in buffered soIlnuttihoinsspaanpderi,nwtehepeprrfeosremnecde sopf edcetrnaaltuchrianrgacategreinzta,tigounaonfidBinpehPh1y-dFrPoachnldoriitds em(uGtdanntHs Cinl)b. uBfpfehrPe1d-FP solcuotinotnaisnasntdheinCtyhse rperseidseunecse(Cofyds1e5naatnudrinCgysa2g5e6n)ti,ngubaonthidtihnee PhAydSraonchdlothriedGe A(GFddnoHmCal)in. sBpwhhPe1re-FaPs its conmtauintasnttshehCavyes raesiCdyuses r(eCsiydsu1e5 aenitdheCr ysin256th) einPbAoSth (tBhpehPPA1-SFPa/nCd25th6Ie) GoAr FindotmheainGsAwFhedroemasain its (mBpuhtPan1-tFs Ph/Cav1e5Sa), Coyrs nroesiCduyes erietshiedrueins t(hBephPPA1S-FP(B/Cp1h5PS1/-CF2P5/6CI)2. 56AIb) soorrbianncteheanGdAFfludoormesaciennce (BpmhPea1s-uFrPe/mCe1n5tSs),ofotrhensoe NCyIRs FrePsivdaurieasnt(sBprehvPe1a-lFedP/tCha1t5Sth/eCs2p5e6cIt)r. aAl cbhsaorrabcatnerciestiacnsdofflBupohrePs1c-eFnPceand meBapsuhrPe1m-FePn/tCs 1o5fS thaersee NdeItRerFmPinveadriabnyts BreVveacloevdaltehnattlythebosupnedctratol chCayrsa2c5t6erisotniclsy oaf nBdphtPh1o-sFeP of andBpBhpPh1P-F1P-F/CP2/5C61I—5SbyarBeVdceotvearmlenintleydbobuynBdVto cCoyvsa1l5enotnlyly.bOouunr dobstoervCaytiso2n5s6 foornmlyonanomd etrhicosvearoiafnts BphoPf B1-pFhPP/1C-F2P56cIo—nbfiyrmBVthceocvoanlecnlutlsyiobnosumndadtoe fCoyrsd1i5moenrliyc. iORFuPr sob[2s8e]r.vations for monomeric variants of BphP1-FP confirm the conclusions made for dimeric iRFPs [28]
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