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Amino acid substitutions around the chromophore of the chromoprotein

Rtms5 influence polypeptide cleavage q

Kristina Turcic b, Anne Pettikiriarachchi b, Jion Battad b, Pascal G. Wilmann a,Jamie Rossjohn a, Sophie G. Dove c, Rodney J. Devenish b, Mark Prescott b,*

a The Protein Crystallography Unit, Monash Centre for Synchrotron Science, Monash University, Clayton Campus, Vic. 3800, Australiab Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton Campus, Vic. 3800, Australia

c Centre for Marine Studies, University of Queensland, St. Lucia, Qld 4072, Australia

Received 15 December 2005Available online 28 December 2005

Abstract

Extension of the conjugated p-system of many all-protein chromophores with an acylimine bond is the basis for their red-shifted opti-cal properties. The presence of this post-translational modification is evident in crystal structures of these proteins. Harsh denaturationof proteins containing an acylimine bond results in partial polypeptide cleavage. For the red fluorescent protein DsRed, the extent ofcleavage is quantitative. However, this is not the case for the blue non-fluorescent chromoprotein Rtms5, even though all chromophoresin tetrameric Rtms5 contain an acylimine bond. We have identified two positions around the chromophore of Rtms5 where substitutionscan promote or suppress the extent of cleavage on harsh denaturation. We propose a model in which cleavage of Rtms5 is facilitated by atrans to cis isomerisation of the chromophore. 2006 Elsevier Inc. All rights reserved.

Keywords: All-protein chromophores; Rtms5; Chromoprotein; Structure; Acylimine bond

All-protein chromophores, including highly fluorescentproteins (FPs) and the intensely coloured, but non-fluores-cent chromoproteins (CPs) are found in a variety of marineorganisms and possess spectral properties covering theentire visual range of wavelengths. The chromophoreresponsible for light absorption and the fluorescence prop-erties in FPs and CPs arises from an extended conjugatedp-system that comprises a cyclic tri-peptide structure. This

chromophore forms inside the characteristic 11-strandedb-barrel as a result of the covalent rearrangement of threeconsecutive amino acids (XXG). The maturation of thechromophore is autocatalytic being solely dependent onthe presence of molecular oxygen. In FPs, such as DsRedand EqFP611, and the CP Rtms5 [14], which exhibit

red-shifted spectral properties compared to GFP, the con-jugated p-system is further extended through the formationof an acylimine bond. It has been proposed that an acyli-mine may represent an intermediate in the formation ofalternative chromophore structures such as those foundin zFP538 and asCP [1,57].

The presence of an acylimine bond in the chromophoreof DsRed was first deduced from mass spectrometry anal-

yses [8] and later observed in the crystal structures [2,9].The glutamine a-carbon of the chromophore (QYG) orig-inally sp3 hybridised appears sp2 hybridised. Refinement ofthe structure indicated that two of the four protomersbecame trapped as an immature green emitting anionic spe-cies [9]. Crystal structures of the blue tetrameric chromo-protein Rtms5 indicate the presence of sp2 hybridisationonly, suggesting that all chromophores or close to all(>90%) contain an acylimine bond [3,10]. Yet, when fullymature, Rtms5 contains no evidence of intermediates inthe maturation pathway [11].

0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2005.12.118

q Abbreviations: CP, chromoprotein; FP, fluorescent protein; QY,quantum yield.* Corresponding author. Fax: +61 3 9905 3726.

E-mail address: Mark.Prescott@med.monash.edu.au (M. Prescott).

www.elsevier.com/locate/ybbrc

Biochemical and Biophysical Research Communications 340 (2006) 11391143

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The acylimine linkage in all-protein chromophores canundergo irreversible hydration under various denaturationregimes. Harsh denaturation such as boiling in 0.1 N HClresults in cleavage of the polypeptide at the position ofthe acylimine bond which can be readily visualised bySDSPAGE. In the absence of crystallographic data, the

presence of the acylimine bond in members of the FP/CPfamily of proteins is often inferred from evidence of poly-peptide cleavage upon harsh denaturation as shown bySDSPAGE [8]. Pre-boiling of DsRed in 0.1 N HCl or0.1 N NaOH reveals approximately 50% cleavage of theprotein consistent with two of the four protomers in eachtetramer containing an acylimine [8,9]. Similar harsh dena-turation of Rtms5 produced only limited cleavage [3,12](and this manuscript). In the light of the correlation ofthe extent of cleavage with crystallographic data, reportedfor DsRed, this is a somewhat surprising result, as com-plete cleavage of the CPs such as Rtms5 upon acid/alkalitreatment is to be expected. By comparison gtCP, a CP

closely related to Rtms5, showed $80% cleavage afterharsh denaturation [13].

The stable acylimine bond, despite variation in theextent of cleavage of denatured proteins, appears to repre-sent a natural endpoint in the maturation of DsRed,EqFP611, and CPs such as Rtms5. However, for a numberof other proteins the acylimine bond may represent anintermediate in the formation of the respective chromo-phore structures [5]. Examples include the three ring struc-ture identified as the chromophore in the structure ofzFP538 [7] and the cleavage of the polypeptide backboneleading to the kindling chromophores found in the CP

isolated from Anemonia sulcata [14], now commerciallyavailable as the variant, KFP1 [15].

We show that substitutions at two positions close to thechromophore are able to significantly influence the level ofcleavage without markedly affecting the absorption spectraof the protein. We conclude that the level of cleavage upondenaturation in CPs is not necessarily a reflection of the

amount of acylimine present in the protein but ratherreflects the differential reactivity of the acylimine bond.We propose that the ease with which the trans non-copla-nar chromophore can access the cis configuration duringdenaturation may influence the efficiency of cleavage(Fig. 1).

Materials and methods

Cloning. DNA cassettes encoding Rtms1, Rtms5, and Rtms5H146S wereretrieved by PCR and cloned into the BamHI/NotI site of pQE9N orpQE10N [12,16]. Site-directed mutagenesis was performed using theQuikchange Kit (Stratagene, USA). pQE30 encoding the open-readingframe of gtCP was a gift from Dr. K. Lukyanov (Russian Academy ofSciences, Moscow, Russia).

Protein expression and purification. Escherichia coli (Nova Blue) cellsfreshly transformed with a vector encoding a CP were inoculated fromsingle colonies into LB medium and incubated overnight with orbitalshaking (200 rpm) at 28 C. Two millilitres of the overnight culture wastransferred to 250 ml LB medium and after 4 h incubation IPTG wasadded to a final concentration of 0.2 mM. Incubation was continued

overnight after which cells were harvested. Recombinant protein wasisolated and purified by Ni-NTA [4]. Protein solutions were bufferexchanged by exhaustive dialysis against 20 mM TrisHCl, pH 8.0,300 mM NaCl and concentrated with a centrifugal ultrafiltration device(Millipore Corp., MWCO 10000). Proteins were stored at room temper-ature for several days to ensure maturation was complete.

Cleavage analysis. Proteins were denatured by boiling in 0.1 N HCl for5 min. Solutions were neutralised by the addition of NaOH before sub-jecting proteins to analysis by SDSPAGE. Coomassie-stained gels wereimaged and then integrated density values (IDV) were calculated for eachpolypeptide and the extent of cleavage was estimated using the followingrelationship:

%cleavage IDV18 kDa IDV11 kDa=IDV18 kDa IDV11 kDa

IDV29 kDa 100;

where 18 and 11 kDa represent the two cleavage products and 29 kDa thenon-fragmented protein.

Spectrometry. Absorbance spectra were determined at 24 C using aVarian Cary 50 spectrophotometer.

Results and discussion

Polypeptide cleavage into $18 and $11 kDa fragmentsupon denaturation occurs to different extents for a numberof all-protein chromophores [3,8,12,13]. In order to investi-gate the structural basis for this seemingly unusual behav-iour, we compared the amino acid sequence of three closelyrelated CPs that show significantly different degrees ofcleavage; gtCP ($80%), Rtms5 ($20%), and Rtms1($80%, this report). These proteins differed at 10 aminoacid positions (Table 1). We reasoned that a major influ-ence upon cleavage is likely to be those amino acid sidechains relatively close to the position of the chromophoreand acylimine bond. Two positions, 65 and 146, are locatedclose to the chromophore in the crystal structure of Rtms5[3]. Position 65 immediately precedes the chromophore tri-peptide sequence, Q66Y67G68. The acylimine linkage islocated between Ca and N of Q66. Position 146 is posi-tioned close to the tyrosyl moiety. Substitutions at position

146 are key for enhancing the fluorescence QY of CPs

Fig. 1. The chromophore environment of Rtms5. The trans non-coplanarchromophore (dark grey) is shown with the acylimine linkage highlighted(arrow). Side chains contributing to the hydrophobic pocket in whichthe C65 side-chain resides are shown. An H146S substitution close to thetyrosyl moiety is believed to promote trans-cis isomerisation of the

chromophore.

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through a mechanism involving trans-cis isomerisation ofthe chromophore [3,17,18].

Using the sequence comparison (Table 1) we reasonedthat cysteine 65 was in-part responsible for the reducedextent of cleavage for Rtms5. In order to test this idea,site-directed mutagenesis was used to generate the variantsRtms5C65Y and Rtms5C65S with the expectation that theseproteins would show increased cleavage. Serine and tyro-sine are present at position 65 in the highly fragmentingRtms1 and gtCP proteins, respectively. After pre-boilingsamples in 0.1 N HCl, cleavage of purified Rtms5C65Y,Rtms5C65S, and wtRtms5 was compared using SDSPAGE(Fig. 2). In addition to the full-length polypeptide having amobility corresponding to 29 kDa, two smaller polypep-tides corresponding to 18 and 11 kDa were observed. Thesize of the two smaller polypeptides is consistent with

cleavage at the position of the acylimine bond in the full-length polypeptide as reported previously [3]. Minoramounts of non-specific cleavage are apparent that pre-sumably are due to acid hydrolysis of peptide bonds atother positions in the polypeptide. These results show thatthe substitutions C65S and C65Y in Rtms5 significantlyincreased the extent of cleavage (Fig. 2A). Cleavage wasincreased from $20% to $73% for Rtms5C65Y andRtms5C65S (Table 2). DsRed showed approximately 50%

cleavage, while GFP which does not contain an acyliminebond showed no cleavage (Fig. 2E).

The absorption spectra for Rtms5C65Y, Rtms5C65S, andRtms5 were determined (data not shown). The absence ofmaturation intermediates at 410 nm confirmed that theproteins had reached an endpoint in their maturation[11,12]. A single major absorbance peak was observed foreach of the proteins. Extinction coefficients for each pro-tein were comparable (Table 2). The presence of an acyli-mine linkage is responsible for the red-shifted opticalproperties of Rtms5 proteins [8]. Since there were no majoralterations in the position of kmax or the extinction coeffi-cient, we conclude that each of these proteins contains sim-ilar amounts of acylimine. These results indicate thatincreased cleavage of Rtms5C65Y and Rtms5C65S does notresult from increased acylimine bond content.

In order to confirm the general importance of position65, in particular the presence of a cysteine at this position,we next investigated the effect of cysteine substitutions atposition 65 in the two highly fragmenting proteins Rtms1and gtCP (Table1). The variants Rtms1Y65C and gtCPS65C

were generated by site-directed mutagenesis and their prop-erties were compared to those of the parent proteins Rtms1and gtCP. The degree of cleavage of Rtms1Y65C (18%) wasdramatically reduced when compared to the cleavage of

Table 1Amino sequence differences for gtCP, Rtms5, and Rtms1

Position gtCP Rtms5 Rtms1

10 K T T30 Q E E65 S C Y91 Y F Y

106 T T A119 I T I121 N H H

146 N H N147 T S T154 D G D206 I K K

Key positions investigated in this study are indicated in boldface. Num-bering according to [3].

Fig. 2. Cleavage of all-protein chromophores upon denaturation analysed by SDSPAGE. Proteins were pre-boiled in 0.1 N HCl, was subjected toSDSPAGE and then polypeptides stained with Coomassie blue. (A) Lane 1, Rtms5; lane 2, Rtms5C65S; lane 3, Rtms5C65Y. (B) Lane 1, Rtms1; lane 2,Rtms1Y65C. (C) Lane 1, gtCP; lane 2, gtCPS65C. (D) Lane 1, Rtms5H146S; lane 2, Rtms5H146SC65Y. (E) Lane 1, DsRed; lane 2, GFP. The positions of

molecular weight standards are indicated at left.

Table 2Properties of Rtms5, Rtms1, gtCP, and their variants

Chromoprotein % cleavageafter pre-boilingin 0.1 N HCl

e

(M1 cm1)Absorbance(kmax, nm)

Rtms5 20.1 1.9 69,900 591Rtms5C65Y 73.1 7.1 71,000 593

Rtms5C65S 73.7 5.5 77,900 586Rtms1 78.1 3.7 70,000 586Rtms1Y65C 18.1 0.4 57,000 586gtCP 76.9 3.3 53,000 580gtCPS65C 63.5 3.4 58,800 586Rtms5H146S 57.5 2.3 66,700 589Rtms5H146S,C65Y 77.7 2.5 70,900 590

The extent of cleavage from three independent gels was determined usingthe relationship shown in Materials and methods.

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wtRtms1 (78%) (Fig. 2B). Cleavage of gtCPS65C was alsoreduced compared to that of wtgtCP but to a lesser extent.The extinction coefficient for Rtms1Y65C was reduced by19% compared to that for wtRtms1. This change is not suf-ficient to explain the dramatic fall (60%) in the degree ofcleavage. These results confirm the importance of amino

acids at position 65 in influencing cleavage of CPs. Howev-er, limited cleavage produced by the introduction of a cys-teine at position 65 in gtCP suggests that other positionscontribute to the behaviour of the acylimine.

We next investigated the influence of substitutions atposition 146 on the extent of cleavage. The variantsRtms5H146S and Rtms5H146S,C65S were generated by site-di-rected mutagenesis and cleavage of the proteins evaluatedby SDSPAGE. The results show that Rtms5H146S frag-ments more readily (60%) compared to wtRtms5(Fig. 2D; Table 2). When combined with the substitutionC65Y cleavage was further increased (Fig. 2D; Table 2).Extinction coefficients determined for each of the variants

were found to be similar. These results indicate that thesubstitution H146S can enhance cleavage of Rtms5. It isnot clear from these results whether substitutions atpositions 146 and 65 act synergistically as cleavage forRtms5H146S,C65Y and Rtms5C65Y is similar.

Collectively these results show that cleavage of CPs canbe altered dramatically in the absence of significant chang-es in the acylimine content of the protein, as judged by theabsorbance spectra. It can be concluded that, unlikeDsRed, cleavage of these CPs upon harsh denaturationdoes not indicate the level of acylimine content and there-fore cannot be used as a measure of chromophore

maturation.It is clear that denaturation-induced cleavage of CPs is

significantly influenced by the nature of the amino sidechains close to the chromophore. How might the chromo-phore environment influence the extent of cleavage ofRtms5 upon denaturation? Recent structural evidence indi-cates that a number of CPs including Rtms5 contain a transnon-coplanar chromophore (Fig. 1) whereas DsRed con-tains only cis planar chromophores 50% of which containan acylimine linkage [13,5,9,12]. Although the crystalstructures Rtms1 and gtCP have not been determined,the similarity in spectral properties and amino acidsequence to Rtms5 suggests their chromophore conforma-tion is most likely to be trans non-coplanar. We proposethat reactions leading to cleavage are favoured by increasedmobility of the chromophore, a condition that favours a cisplanar chromophore conformation. We assume that thecorrect orientation of particular amino acids in the chro-mophore environment is required for cleavage and isomeri-sation of the chromophore takes place in the early stages ofprotein unfolding once a cis conformation is assumed.

What additional evidence is there to support this pro-posed mechanism? Trans-cis isomerisation is the basis ofthe photoswitch for AsCP, a photoconverting CP [19].Chromophore maturation includes cleavage of the poly-

peptide in the native structure at the position where the

acylimine bond would otherwise be located [1,5,19]. Molec-ular dynamic simulations suggest that mobility of the chro-mophore resulting from polypeptide cleavage facilitatesphotoinduced isomerisation of the chromophore [19]. ForCPs that do not undergo cleavage in the native structure,it is likely that substitutions at position 65 would affect

the mobility or positioning of the chromophore and, there-fore its ability to isomerise. The side chain of the aminoacid at position 65 projects into a hydrophobic pocket(Fig. 1). Preliminary data indicate that Rtms1 undergoesphotoconversion more readily than Rtms5.

Amino acid substitutions at H146 (Fig. 1) are key toincreasing the fluorescence QY of certain chromoproteinsby $100-fold [3,17,20]. Structural and optical data indicateincreased fluorescence in the engineered chromoprotein,HcRed, results from increased mobility of the chromo-phore favouring trans non-coplanar to cis coplanar isom-erisation [12]. Although the cis conformation was notobserved in Rtms5H146S, the increased QY compared to

Rtms5 indicates increased chromophore mobility.Finally, our results suggest that amino acid substitutions

at position 65 are worthy of further investigation.Increased mobility of the chromophore may alter the wayin which the fluorescence properties of these CPs respondto illumination and other external influences. It has beensuggested that the acylimine bond represents an intermedi-ate in the formation of other chromophore structures [7].Isomerisation of the chromophore during maturationmay have a role to play in polypeptide cleavage in thenative structure of particular APCs.

Acknowledgments

We thank Konstantin Lukyanov (Institute of Bioor-ganic Chemistry, Russian Academy of Sciences, Mos-cow, Russia) for providing a vector encoding gtCP.This work was supported by a Monash University smallgrant. P.W. is supported by a Monash University Ph.D.scholarship. J.R. is supported by a Wellcome Trust Se-nior Research Fellowship in Biomedical Science inAustralia.

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