starmaker exhibits properties of an intrinsically disordered protein

Starmaker Exhibits Properties of an Intrinsically DisorderedProtein

Tomasz M. Kapłon,† Grzegorz Rymarczyk,† Małgorzata Nocula-Ługowska,† Michał Jakob,†

Marian Kochman,† Marek Lisowski,‡ Zbigniew Szewczuk,‡ and Andrzej Ozyhar*,†

Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, WybrzezeWyspianskiego 27, 50-370 Wrocław, Poland, and Faculty of Chemistry, University of Wrocław,

F. Joliot-Curie 14, 50-383 Wroclaw, Poland

Received February 8, 2008; Revised Manuscript Received May 6, 2008

Fish otoliths composed of calcium carbonate and an organic matrix play a primary role in gravity sensing and theperception of sound. Starmaker (Stm) was the first protein found to be capable of influencing the process ofbiomineralization of otoliths. Stm dictates the shape, size, and selection of calcium carbonate polymorphs in aconcentration-dependent manner. To facilitate exploration of the molecular basis of Stm function, we have developedand optimized a protocol for efficient expression and purification of the homogeneous nontagged Stm. Thehomogeneous nontagged Stm corresponds to its functional form, which is devoid of a signal peptide. Acomprehensive biochemical and biophysical analysis of recombinant Stm, along with in silico examinations, indicatefor the first time that Stm exhibits the properties of intrinsically disordered proteins. The functional significanceof Stm having intrinsically disordered protein properties and its possible role in controlling the formation ofotoliths is discussed.

Introduction

Otoliths in bony fishes and otoconia in mammals arecomposite crystals consisting of calcium carbonate and anorganic fraction composed mostly of proteoglycans and glyco-proteins. These biominerals are part of the gravity and linearacceleration detection system of the inner ear. They are involvedin the perception of balance, as well as in the reception ofsound.1 Several human disorders are associated with otoconiadeficiency, otoconia abnormality, or age-related otoconiadegeneration.2,3 Although otoliths and otoconia are indispensablefor proper postural function and orientation in the environment,only limited knowledge has been available to date about themolecular mechanisms involved in the biogenesis of thesecomposite crystals. In zebrafish (Danio rerio) the biomineral-ization of otoliths was shown to be strictly controlled by aStarmaker protein (Stm). Stm was the first protein found to becapable of influencing the process of biomineralization. UsingRNA antisense technology, it has been shown that Stm acts asa crystal growth inhibitor regulating the growth, shape, andcrystal lattice in a concentration-dependent manner.4 Withrespect to these features, Stm is akin to human Dentinsialophosphoprotein (DSPP), a key factor required for properteeth mineralization and also expressed in the human ear.2,3 BothStm and DSPP seem to have analogous functions despite theirrelatively low sequence homology.4,5 The expression of Stm isrestricted to cells from the sensory epithelium, includingneuroepithelial patches within the inner ear and clusters of thesensory hair cell epithelium of the lateral line organ. Stm wasalso found in the pineal gland.4 Because otoliths are extracel-lularly formed in the lumen of the otic cavity, all elementsnecessary for their formation need to be secreted to thiscompartment. In view of this fact, the computationally predicted

signal peptide of Stm was postulated to be active within Stm,and Otopetrin 1, a transmembrane protein located in the haircells of neuroepithelium, was suggested to be involved in thesecretion of Stm.6 Further analysis of Stm amino acid sequencedemonstrated some additional structural elements like fourhighly conserved 13-amino acid repeats, the function of whichis still unknown, and a 60 amino acid-long region rich inalternating D and S residues.4 A similar region rich in S and Dresidues arranged in a specific pattern, defined as a repeatingDSS motif, was found in DSPP. As was postulated by George,7

such an arrangement, combined with the phosphorylation ofabundant S residues, could create a spatial structure capable ofbinding high amounts of calcium ions, which is probablyessential for bridging inorganic crystals and the proteinaceousskeletons of biominerals.

Despite the identification of Stm and other proteins involvedin the formation of otoliths,8 the molecular basis of their functionin biomineralization remains unknown, possibly due to thedifficulty of obtaining pure preparations of these proteins,resulting from the their low concentration in natural sources.To further detail biochemical studies aimed at understandingmechanisms by which Stm acts as a key factor regulating otolithformation, we have developed and optimized a protocol for theefficient expression and purification of Stm. A comprehensivebiochemical and biophysical analysis along with in silicoexaminations importantly indicate for the first time that Stmexhibits features typical of intrinsically disordered proteins. Thefunctional significance of this structural feature of Stm has beenanalyzed and its possible role in the mineralization of otolithsis discussed.

Materials and Methods

Buffers. All buffers were prepared at 24 °C. Buffer A was 50 mMNa2HPO4, 150 mM NaCl, 10% (v/v) glycerol, 1 mM 2-mercapto-ethanol, pH 7.0. Buffer B was 10 mM Tris, 100 mM NaCl, 10% (v/v)glycerol, pH 7.0. Buffer L was 10 mM Na2HPO4, 2 mM KH2PO4,

* To whom correspondence should be addressed. Phone: 004871 32063 33. Fax: 004871 320 63 37. E-mail: [email protected].

† Wrocław University of Technology.‡ University of Wrocław.

Biomacromolecules 2008, 9, 2118–21252118

10.1021/bm800135m CCC: $40.75 2008 American Chemical SocietyPublished on Web 07/18/2008

137 mM NaCl, 6 mM (NH4)2SO4, 3 mM KCl, 1 mM 2-mercapto-ethanol, pH 7.0.

Construction of Expression Vectors. Full-length Stm cDNA4 wasamplified using a PCR9 and the following primers 5′-GCCCGCG-GATCCctgtcccggacagtgtttg-3′ (forward) and 5′-GCCCGCAAGCTTg-gaaatcggcatagaagttttg-3′ (reverse). To amplify Stm cDNA devoid ofthe signal peptide (SP) coding sequence the forward and reverse primerswere 5′-GCCCGCGGATCCgcaccagtgagcaataacaatg-3′ and 5′-GCCCG-CAAGCTTggaaatcggcatagaagttttg-3′, respectively. Lowercase lettersin primer sequences represent sequences originating from the Stm gene,whereas uppercase letters represent nucleotides added for cloningpurposes. Each primer introduced a specific endonuclease recognitionsite (underlined sequences for BamHI and for HindIII), enablingsubsequent insertion of amplified fragments into linearized plasmid.PCR products were purified using a NucleoTraPCR kit (Macherey-Nagel, Germany), digested by BamHI and HindIII endonucleases andsubcloned into a set of pQE-80L vector (Qiagen, Germany) derivativesobtained in our laboratory (not shown). Each of the constructed plasmidsencoded one of the four recombinant proteins (Stm-His, His-Stm∆SP, Strep-Stm∆SP-His, and Stm∆SP) schematically depicted inFigure 1A. The presence of the inserts within each construct wasconfirmed by restriction analysis (data not shown) and the purifiedconstructs were verified by sequencing, using a CycleReader Auto DNASequencing Kit (MBI Fermentas, Lithuania) and the Pharmacia BiotechALF express analyzer.

Expression Analysis. Prior to elaboration of the purificationprocedure the expression of each recombinant Stm variant wasevaluated. Briefly, BL21(DE3) or BL21(DE3)pLysS E. coli cells(Novagen, Germany) were transformed with 2 ng of the respectiverecombinant pQE-80L derivative that encoded the desired Stm protein(Figure 1A) and plated on LB-agar plates, containing appropriateantibiotic (35 µg/mL chloramphenicol and/or 50 µg/mL carbenicillin).E. coli cells BL21(DE3) (Novagen, Germany) and carbenicillin at 50µg/mL were used for Stm-His. After incubation for 16 h at 37 °C, oneindividual colony was picked for each construct and used to inoculate10 mL of LB medium in a 100 mL shaker flask. After overnightincubation (29 °C, 182 rpm), each of the starting cultures was used toinoculate 100 mL of LB media in a 500 mL Erlenmeyer flask that wasplaced in a rotary shaker operated at 182 rpm at 29 °C. At an OD600

of 0.60, 1 mL samples were collected, centrifuged for 2 min at 14100× g, and the resulting bacterial pellets were resuspended in 75 µL ofthe 2 × SDS sample buffer (124 mM Tris-HCl, 4% (w/v) SDS, 10%(v/v) 2-mercaptoethanol, 20% (v/v) glycerol, 0.005% (w/v) bromphenolblue, pH 6.8)10 and stored at -80 °C. Because the pQE-80L expressionvector contains the phage T5 promoter and two lac operator sequencesisopropyl-�-thiogalactopyranoside (IPTG), a synthetic analog of lactosewas then added to the rest of each culture to a final concentration of0.25 mM. Then recombinant protein synthesis was carried out for 3 hat 29 °C, 182 rpm. Next, 1 mL culture samples were collected andprepared, as described above, and stored at -80 °C for subsequentSDS/polyacrylamide gel electrophoresis analysis (see below).

Purification of Stm∆SP and Strep-Stm∆SP-His. Glycerol stockcultures of host cells containing respective expression vectors were usedto inoculate 130 mL of LB medium containing chloramphenicol (35µg/mL) and carbenicillin (50 µg/mL). After overnight incubation at29 °C and 182 rpm the starting culture was used to inoculate 4 L ofLB medium, and the resulting suspension was divided into thirteen 1L Erlenmeyer flasks and placed in a rotary shaker operated at 29 °Cand 182 rpm. When OD600 of the cultures reached a value of 0.6,synthesis of the recombinant protein was induced by the addition ofIPTG to a final concentration of 0.25 mM. After 3 h of incubation,bacterial cells were collected into four 500 mL centrifugal tubes with10 min of centrifugation at 10000 × g, 4 °C. The resulting sedimentwas washed with 12 mL of buffer L (for each tube), centrifuged for 10min at 10000 × g, 4 °C, and finally resuspended in 24 mL of buffer Lin a 50 mL Falcon tube and stored at -80 °C.

A total of two pellets of frozen cells, obtained as described abovefrom a total of 2 L of culture, were thawed in a 24 °C water bath andplaced on ice. A single freezing and thawing step was sufficient forthe bacterial strain resident T7 lysozyme, provided by pLysS plasmid,11

to lyse the cells efficiently. Then DNase I and RNase A were added tothe final concentration of 10 µg/mL of each enzyme and the resultingsuspension was incubated on ice until there was a loss of viscosity.Proteolytic activity was partially inhibited by the addition of phenyl-methylsulfonyl fluoride (PMSF) to the final concentration of 0.5 mg/mL. The cell extract was subsequently clarified with a 30 mincentrifugation at 18500 × g, 4 °C, and the supernatant (45 mL) wasfractionated on ice with solid (NH4)2SO4. Proteins from the 55-75%

Figure 1. Recombinant derivatives of Stm. (A) The schematicrepresentation of Stm derivatives used in this study. Stm, wild-typefull-length Stm containing the signal peptide4 (SP) sequence andconsisting of 613 amino acid residues; Stm-His, full-length StmC-terminally tagged with an affinity tag consisting of the sequenceKLHHHHHHHH; His-Stm∆SP, Stm devoid of SP, where ∆SP meansdeletion of the SP sequence, and N-terminally tagged with an affinitytag consisting of the sequence MRGSHHHHHHGS; Strep-Stm∆SP-His, Stm devoid of SP, and C-terminally tagged with the sequenceKLHHHHHHHH and N-terminally with the Strep-tag II peptide WSH-PQFEK21 by a GAGS spacer and, simultaneously, MAS residueswere placed at the Strep-tag II N-terminus; Stm∆SP, Stm devoid ofSP and containing three vector-derived amino acid residues addedat the N-terminus (MGS) and C-terminus (KLN). (B) RepresentativeSDS/polyacrylamide gel electrophoresis analysis of Stm derivativesexpression. Whole cell extracts obtained from the bacterial expressionof the Stm derivatives indicated at the top of the gel were electro-phoresed on a 12% SDS/polyacrylamide gel and stained with Stains-All dye as described in Materials and Methods. Lane 1, molecularmass standards; lanes 2, 4, 6, and 8, bacterial cell extracts beforeinduction (NI) of the synthesis of the appropriate Stm derivative; lanes3, 5, 7, and 9, bacterial cell extracts 3 h after induction (3 h) with theIPTG of synthesis; the arrow indicates that recombinants of Stm arestained in blue.

Starmaker Exhibits Properties of Disordered Protein Biomacromolecules, Vol. 9, No. 8, 2008 2119

(NH4)2SO4 saturation fraction were dissolved in 5 mL of buffer A anddialyzed extensively against buffer A. The resulting solution wasconcentrated to a volume of 1 mL using an Amicon Ultra-4 centrifugalfilter unit (Millipore, Poland) and applied to the HiLoad 16/60 Superdex200 pg (Amersham Biosciences) column equilibrated with buffer A.The column was run on the AKTAexplorer (Amersham Biosciences)system at a flow rate of 0.5 mL/min at room temperature. Fractionscontaining Stm were combined and dialyzed against buffer B. A totalof 1 mL of Bio-Gel DNA-GRADE hydroxyapatite resin (HTP; Bio-Rad Laboratories, Germany), incubated overnight at 4 °C in buffer Bcontaining 1 M CaCl2, was packed into a 1 mL HR5/5 column(Amersham Biosciences), connected to the AKTAexplorer system, andequilibrated with buffer B. Then 10 mL of the preparation obtainedfrom the HiLoad 16/60 Superdex 200 pg column was applied to theHTP column and unbound proteins were washed out with 5 mL ofbuffer B. We experimentally elaborated the step gradient of 0 mM to300 mM phosphate, which was used to elute bound proteins at a flowrate of 0.25 mL/min. Fractions containing pure Stm derivatives werecombined, concentrated using an Amicon Ultra-4 centrifugal filter unit(Millipore, Poland) and stored at -80 °C. The concentration of purifiedproteins was determined using the biuret method.12

ESI Mass Spectrometry. Purified Stm∆SP and Strep-Stm∆SP-Hiswere dialyzed against 1% acetic acid and concentrated to 1 mg/mLusing an Amicon Ultra-4 centrifugal filter unit (Millipore, Poland).High-resolution mass spectra were obtained on a Bruker Microtof-Qspectrometer (Bruker Daltonik, Germany), equipped with an Apollo IIelectrospray ionization source with an ion funnel. The protein solutionwas infused at a flow rate of 3 µL/min. The mass spectrometer wasoperated in the positive ion mode. The mass resolution was 15000fwhm. The instrument parameters were as follows: a scan range ofm/z 400-2300; dry gas, nitrogen; and temperature, 180 °C. Beforeperforming each measurement the instrument was calibrated externallywith the Tunemix mixture (Bruker Daltonik, Germany) in the quadraticregression mode.

The acquisition of data was performed using micrOTOFcontrol 2.0software and data analysis was performed using DataAnalysis softwarefrom Daltonik GmbH (Bremen, Germany).

Analytical Gel Filtration. The amounts (indicated in the legend toFigure 4) of the purified Stm∆SP were loaded with a total volume of100 µL onto a Superdex 200 10/300 GL column (Amersham Bio-sciences), connected to the AKTAexplorer system and equilibrated withbuffer A at a flow rate of 0.5 mL/min. The column was calibrated usingthe following standard proteins: thyroglobulin (85 Å), apoferritin (67Å), catalase (52 Å), bovine serum albumin (35.5 Å), ovalbumin (30.5Å), chymotrypsinogen (20.9 Å), mioglobin (20.2 Å), and cytochromec (17 Å). The elution volume of each protein was used to calculateKAV factors,13 which were then plotted against corresponding Stokesradii (Figure 4, inset). Calculated KAV was then fitted to the standardcurve, and the Stokes radius of Stm∆SP was estimated.13

Circular Dichroism Spectra. Circular dichroism (CD) spectra wererecorded using a Jasco J-720 spectropolarimeter. The final spectraresulted from averaging five measurements performed at a temperatureof 20 °C, a scanning speed of 20 nm/min, data resolution of 0.2 nmand a bandwidth of 1.0 nm. A 2.15 µM solution of Stm∆SP was usedfor each measurement performed in 0.1 cm path length quartz cuvette.The contribution of buffer B was subtracted from the original data,which were then smoothed and converted to molar ellipticity. Denatur-ing conditions were obtained by the addition of an appropriate amountof 6 M GdmCl to the Stm∆SP solution and subsequent incubation atroom temperature for 2 h.

Fluorescence Measurements. Fluorescence measurements wereperformed using a FLUOROLOG-3 fluorometer (Spex, Jobin Yvon Inc.,France). Data acquisition was performed in buffer B, using a 115F-QSquartz cuvette (Hellma GmbH & Co. KG, Germany) with an excitationwavelength λEX ) 280 nm. Collected data were corrected for the buffercontribution. All measurements were performed at 25 °C.

SDS/Polyacrylamide Gel Electrophoresis. Proteins were separatedon SDS/polyacrylamide gels according to Laemmli10 and stained witheither Coomassie Brilliant Blue R 25014 or carbocyanine (Stains-All;Sigma, Poland) dye.15,16 Molecular mass protein standards fromFermentas (Lithuania) were used.

Disorder Predictions. In silico analysis of the naturally disorderedregionswerecarriedoutusingaPONDRVL-XT(http://www.pondr.com),17,18

and DISOPRED 2 (http://bioinf.cs.ucl.ac.uk),19 neural network predic-tors with the default settings and the full-length Stm sequence fromthe NCBI Database (NP_942112).

Results

Expression and Purification. The full-length Stm, C-terminally tagged with an affinity tag consisting of six histidineresidues (Stm-His, Figure 1A) was expressed in E. coli. Wetried to use Coomassie Brilliant Blue R-250 stained SDS/polyacrylamide gels in our preliminary experiments of theexpression analysis. Unfortunately, it was very hard to observeany expression of Stm-His using this detection method. It is awell-known fact that binding Coomassie Brilliant Blue R-250dye depends on a positively charged residue with adjoininghydrophobic residues.20 This was probably the reason why Stm,a protein of extremely low hydrophobicity, exhibited loweredCommassie-binding. As indicated in the introduction, Stm isextremely hydrophilic; in total, 35% of Stm is composed ofacidic residues.4 Therefore, to detect Stm, we decided to usecarbocyanine (Stains-All). The Stains-All dye stains blue acidicand calcium-binding proteins, whereas all other proteins arestained red.16 As shown in Figure 1B, Stm-His, as well as otherderivatives of Stm described below, are easily stained by theStains-All dye. In particular, for Stm-His, two blue bands, notobserved before the IPTG induction, could be detected (Figure1B, compare lanes 3 and 2). At its N-terminus, the Stm-Hisprotein contains a 20-residue long fragment, which was predictedin silico to be a signal peptide (SP).4 This is justified insofar asStm is active in the extracellular compartment, which is an oticcavity6 and, thus, most probably is devoid of SP. Because ofthis, we assumed that the observed heterogeneity of Stm-Hismay reflect incomplete digestion of SP during the export of Stm-His to the bacterial periplasma. Therefore, in our furtherexperiments, we constructed, expressed, and studied derivativesof Stm, which were completely devoid of the SP sequence. Thetruncation of SP apparently stabilized the recombinant Stm,because for all subsequently studied Stm derivatives (Figure1A), only a single blue-stained band with a mobility corre-sponding to the expected molecular mass could be observed(Figure 1B, lanes 5, 7, and 9). Initially, we decided to obtainan SP-devoid recombinant Stm, tagged with eight His residuesat the N-terminus (His-Stm∆SP, Figure 1A). Surprisingly,preliminary purification experiments using a Talon-Co2+ affinityresin showed that, under the standard conditions recommendedby the manufacturer (Clontech, U.S.A.), most of the His-Stm∆SP protein remained unbound by this resin (data notshown). This could be due to either proteolytic digestion or thehiding of the N-terminal region of His-Stm∆SP, which wouldpreclude interaction with Co2+ ions. And so the Stm derivativewith eight His residues placed at the C-terminus of therecombinant protein was produced. Simultaneously, a nine-amino acid peptide (Strep-tag II,21), which had been developedas an affinity tool for the purification of corresponding fusionproteins with streptavidin columns (Strep-Tactin,21), was at-tached to the N-terminus (Strep-Stm∆SP-His, Figure 1A). It isgenerally accepted that a double-tag strategy facilitates thepurification of full-length proteins and increases protein purity

2120 Biomacromolecules, Vol. 9, No. 8, 2008 Kapłon et al.

due to two independent and subsequent purification steps. Yetagain, purification experiments carried out according to standardprotocols resulted in a very poor yield of the double-tagged Stm.Approximately 100 µg of Strep-Stm∆SP-His was obtained from1 L of bacterial culture (data not shown). Because ESI massspectrometry analyses showed that both tags were present inthe purified Strep-Stm∆SP-His (data not shown and also seebelow), we assumed that for some reason the structure of Stmprevents tags from interacting with appropriate resins and, thus,precludes using an affinity chromatography technique based ontags placed either on the C- or N-termini of Stm. Therefore,our next purification strategy was based on standard proteinfractionation techniques and the nontagged, SP-devoid Stm(Stm∆SP, Figure 1A) was expressed (see Figure 1B, lane 9).To clarify the bacterial lysate and to remove contaminatingproteases, ammonium sulfate fractionation was applied as thefirst step of the purification procedure. Small-scale pilot experi-ments revealed that Stm∆SP is soluble in a fraction ofammonium sulfate saturated to 55%, whereas increasing theammonium sulfate saturation to 75% led to the almost completeprecipitation of Stm∆SP (data not shown). Thus, we decidedto use 55-75% ammonium sulfate lysate cut (Figure 2C,D,compare lanes 5 and 6) as starting material. Because the primarystructure analyses of Stm suggested that this protein might revealcharacteristics of an intrinsically disordered protein (IDP; seebelow), size-exclusion chromatography was chosen as the nextstep of the purification procedure. According to published data,IDPs exhibit a substantially increased hydrodynamic volumerelative to globular proteins, leading to a significant increase intheir apparent molecular mass. The molecular mass of Stm andits derivatives, calculated on the basis of their amino acidcomposition, is about 65 kDa. However, as a putative IDP familymember, Stm was expected to elute earlier as a protein with anapparently higher molecular mass, due to the increased hydro-dynamic volume relative to the globular protein of the samemolecular mass.22,23 Indeed, as shown on Figure 2A, Stm∆SPwas eluted in a volume close to the void volume (V0) of theHiLoad 16/60 Superdex 200 pg column, and consequently, itwas separated from many of the contaminating proteins presentin the ammonium sulfate fraction (Figure 2A, inset, alsocompare lanes 6 and 7 in Figure 2C).

As was shown by Sollner et al.,4 Stm plays a crucial role inthe biomineralization of otoliths, which are mostly composedof calcium carbonate. It has been suggested that repeating aseries of alternating S and D residues in Stm could serve as atemplate for binding calcium ions. Hydroxyapatite, analogicallyas calcium carbonate, should be effectively bound by Stm.Accordingly, chromatography on hydroxyapatite was selectedas the next step of purification. Because of the acidic characterof Stm, the surface of the hydroxyapatite was loaded withcalcium ions.24–26 Figure 2B illustrates the hydroxyapatitechromatography carried out according to the protocol especiallyelaborated for Stm in our laboratory. An appropriate stepwisegradient of phosphate ions allows effective separation ofStm∆SP from contaminating proteins. The purified Stm∆SPappeared as a single band on the 15% SDS/polyacrylamide gel(Figure 2C and D, lane 8). The densitometric analysis of theCoomassie Brilliant Blue R-250 stained gel revealed the purityof Stm∆SP at 94%. It is noteworthy that this value has beenunderestimated because, as discussed above, Stm∆SP poorlybinds Coomassie Brilliant Blue R-250. The final preparation ofStm∆SP was also devoid of significant amounts of truncatedStm∆SP, as judged by in-gel Stains-All detection (Figure 2D,lane 8). To confirm the identity of Stm∆SP, ESI mass

Figure 2. Purification of Stm∆SP. (A) Preparative gel filtration ofStm∆SP. The 55-75% (NH4)2SO4 fraction was obtained from a 2 Lculture of E. coli BL21(DE3)pLysS cells transformed with the appropriatederivative of the pQE-80L expression vector (see Materials and Meth-ods). After dialysis, the resulting solution, which was concentrated to avolume of 1 mL, was loaded onto the HiLoad 16/60 Superdex 200 pgcolumn equilibrated with buffer A and connected to the AKTAexplorer(Amersham Biosciences) system. Fractions of 1 mL were eluted at aflow rate of 0.5 mL/min and subsequently analyzed by CoomassieBrilliant Blue R 250- stained 10% SDS/polyacrylamide gel (inset, eachlane corresponds to the respective fraction, the arrow shows therecombinant Stm∆SP). Fractions 3-10, marked as hatched areas onthe chromatogram, were combined, dialyzed against buffer B, andapplied to the next step of the purification procedure (Figure 2B). (B)Hydroxyapatite chromatography. The sample (10 mL) from the HiLoad16/60 Superdex 200 pg column was applied onto a HR5/5 column filedwith 1 mL of HTP resin equilibrated with buffer B and connected to theAKTAexplorer system. The flow rate was 0.25 mL/min and 0.5 mLfractions were collected. Note that Stm∆SP is devoid of W and Yresidues and, hence, it reveals no significant absorption at 280 nm.Taking this into consideration, absorption at 220 was also monitoredduring the separation process. The experimentally elaborated stepgradient of phosphate was used to elute bound proteins. The hatchedarea indicates fractions containing pure Stm∆SP as judged by subse-quent SDS/polyacrylamide gel electrophoresis (see Figure 2C,D). (C)Representative Coomassie Brilliant Blue R 250, stained SDS/polyacry-lamide gel analysis of the expression and purification procedure ofStm∆SP. Lane 1, molecular mass standards; lanes 2 and 3, the wholebacterial cell extract before the induction of Stm∆SP with IPTG (TOTNI) and 3 h later (TOT 3 h; ca. 30 µg); lane 4, soluble protein fractionobtained after cell lysis (ca. 40 µg); lanes 5 and 6, proteins from 0-55%and 55-75% (NH4)2SO4 fractions, respectively (ca. 20 µg in lane 5 and40 µg in lane 6); lane 7, the combined gel filtration fractions 3-10 (Figure2A, hatched area; ca. 6 µg); lane 8, the purified Stm∆SP afterhydroxyapatite chromatography (ca. 6 µg). (D) The same analysis as in(C), but the SDS/polyacrylamide gel was stained using Stains-All dye.Note that amounts of the applied samples were halved. In (C) and (D),the position of molecular mass standards in kDa are indicated on theleft, and the arrows on the right mark positions of Stm∆SP.


spectrometry was applied, resulting in a value of 64497.3 ( 4Da, which differs from the expected value (64628.2 Da) by 131mass units. The observed difference was caused by the specificdigestion of N-terminal formylmethionine, as judged by theN-terminal protein sequence analysis (data not shown).

The elaborated purification procedure typically yielded about0.85 mg of Stm∆SP from 1 L of the culture medium. Moreover,this procedure seems to be universal for various Stm derivativesbecause it also allowed us to purify Strep-Stm∆SP-His with asimilar yield and purity (data not shown). It is noteworthy thatthe ESI mass spectrometry analysis of the purified Strep-Stm∆SP-His confirmed the presence of both peptide tags (datanot shown).

Stm Exhibits Properties of an Intrinsically DisorderedProtein. Both the extremely highly acidic character of Stm,which is probably crucial for its functioning as a calcium ion-binding protein,4 as well as the unusual properties observedduring elaboration of the purification procedure, inspired us totake a closer look at the amino acid composition of Stm. Tothat end, Composition Profiler was used,27 a web-based toolfor semiautomatic analysis of the enrichment or depletion ofamino acids. Figure 3A shows the composition profiles of Stmand Stm∆SP residues in comparison to the PDB Select 25 dataset, based on a subset of structures from the Protein Data Bank,biased toward the composition of proteins easy to crystallize,that is, by definition, proteins possessing an ordered structure.Our analysis revealed that both full-length Stm and Stm devoidof the SP sequence (Stm∆SP), thus better corresponding to thefunctional protein, have a distinctive amino acid composition.Generally, they are rich in polar and charged amino acids (E,D, S, K, T, and H) and simultaneously poor in nonpolar residues(V, L, I, F, M); some of them (W, Y, C) are even absent inboth proteins. According to Dunker et al.,29 such an amino acidcomposition is probably a symptom of an intrinsic disorder, adistinguishing feature of intrinsically disordered proteins (IDPs),also known as intrinsically unstructured or natively unstructuredproteins. Intrinsic disorder is essential for IDPs as their variousbiologically important functions stem either directly from thestructural disorder state or from local folding/ordering inmolecular recognition.23,28 Indeed, a more detailed analysis ofthe data presented in Figure 3A based on the classificationelaborated by Dunker et al.29 indicates that, among polar andcharged amino acids, three (S, E, K) can be categorized asdisorder-promoting residues and the other three (D, T, H) asdisorder-neutral residues. At the same time, the most seriouslydepleted nonpolar residues are either order-promoting (V, L, I,F) or disorder-neutral (M) residues. To determine whether thesequence attributes of Stm and Stm∆SP are similar to those ofIDPs, we also analyzed the fractional difference between IDPcompositions (DisProt 3.4 data set,30) and PDB S25 composi-tions. A comparison of these results with data obtained for Stmand Stm∆SP (Figure 3A) indicates that the overall trend forIDPs and Stm data sets is similar. Remarkably, the frequenciesof five disorder-promoting residues (A, R, Q, G, and P) aresignificantly lower than in the data set for IDPs. At the sametime, Stm and Stm∆SP are significantly rich in some otherdisorder-promoting residues (S, E, K) and considerably deficientin order-promoting residues (V, L, I, F). Three order-promotingresidues (W, C, Y) are completely missing from the Stmproteins. All these results together suggest that Stm and itsrecombinant derivatives belong to the class of IDPs. Thissupposition is further supported by the Uversky plot (Figure3B),22,29 which unquestionably classifies Stm and Stm∆SP asmembers of the IDP family. The data presented in Figure 3C

were obtained using DISOPRED19 and PONDR VL-XT,17,18,31

predictors, two of the algorithms that are widely used for theprediction of disordered regions within amino acid sequences.The data clearly indicate that Stm and Stm∆SP are highlydisordered, although a few potential regions of order werepredicted to exist within their sequences. In particular, oneregion corresponding to the SP sequence (positions 2-22) waspredicted by both algorithms to possess an ordered structure inthe full-length Stm (Figure 3C). Interestingly, according toPONDR VL-XT, a few potential regions of order werepredicted, two of them around residues 33-53 and 348-359in the wild-type Stm and in Stm∆SP. Unfortunately, becausethe DISOPRED classifies these regions to be ordered, theiractual character is ambiguous and needs to be experimentallystudied.

Low mean hydrophobicity and a high net charge associatedwith a disordered state preclude the formation of the hydro-phobic cluster and promote an extended conformation byelectrostatic repulsion. Consequently, IDPs are characterized bya substantially larger hydrodynamic volume relative to globularproteins, leading to an increase in their apparent molecularmass.22,23 The first experimental hint that Stm might reveal suchcharacteristics was its atypical behavior during preparative gelfiltration (Figure 2A). However, because of contaminants stillpresent during preparative gel filtration, which may affect theobserved elution volume, purified Stm∆SP was used to ac-curately determine its Stokes radius using a gel filtrationtechnique. As shown in Figure 4, Stm∆SP was eluted from theSuperdex 200 10/300 GL column as a single peak, with anelution volume corresponding to a Stokes radius of 78.6 ( 3Å. This value is substantially higher than the 35.5 Å, the valuewhich should be observed assuming there is a globular shapefor the Stm∆SP molecule with a calculated molecular mass of64497.0 Da. Obviously, the high value of the estimated Stokesradius might also result from the oligomerization of globularStm∆SP. The symmetry of the Stm∆SP peak, the absence ofany additional peaks resulting from the dissociation of theputative Stm oligomer, and finally, the protein concentration-independence of the observed elution volume (Figure 4) allsuggest that, at least under conditions applied in this study,Stm∆SP occurs as a monomer with a substantially largerhydrodynamic volume. As can be inferred from the amino acidcomposition, one of the reasons for the increased hydrodynamicvolume of Stm may be its low hydrophobicity, resulting in alack of the hydrophobic core. To acquire evidence for thissupposition we analyzed the W residue fluorescence emissionspectrum, which is widely used as a tool to make inferencesregarding the local structure and dynamics of proteins. Becausethe wild-type Stm and Stm∆SP derivative does not contain anyW residue, the purified Strep-Stm∆SP-His protein, which hasone W residue within the Strep-tag II sequence, was used. Figure5A presents the fluorescence emission spectrum of the Strep-Stm∆SP-His measured with an excitation of 280 nm. Theemission spectrum has a maximum fluorescence (λmax) of 350nm, suggesting the occurrence of the W residue in a polarenvironment. This suggests that it is unlikely that Stm possessesan extensive hydrophobic core, otherwise, the W residue wouldprobably be engaged in its formation. However, this experimentcannot exclude the existence of hydrophobic clusters, whichmight be created without the participation of the N-terminal Wresidue. The presence of some residual hydrophobic clustersmight not interfere with the extended conformation of Stm.However, further experiments are needed to confirm suchassumptions.


The far-UV CD spectrum of Stm∆SP is typical for adisordered protein, as can be seen from its large negativeellipticity at 198 nm (Figure 5B). However, the observedellipticity values at 200 and 222 nm suggest the existence ofsome residual secondary structures. To acquire further evidencefor the occurrence of a secondary structure within Stm∆SP, we

Figure 3. Computational analysis of the Stm sequence. (A) Aminoacid composition of Stm. The amino acid composition of Stm,Stm∆SP, and IDPs (represented by the DisProt 3.4 data set,30) areshown relative to the ordered globular proteins PDB S25 data set.27

Analysis was accomplished using the Composition Profiler.27 Valuesbelow zero indicate depletion in a given residue, whereas positivevalues indicate the wealth of a given residue. Note that for W, C,and Y residues the largest possible magnitude (-1) for a negativepeak was observed for Stm and Stm∆SP, indicating that W, C, andY are completely missing from these proteins. The residue order isbased on the B-factor,43 which estimates the flexibility of each residuewith the most rigid residues on the left and the most flexible on theright. (B) Charge-hydropathy analysis - Uversky plot.22 A PONDR(http://www.pondr.com.) server was used for the analysis of the meanhydrophobicity and net charge of the wild-type Stm and the Stm∆SPrecombinant derivative. The results were compared with valuesobtained for proteins used originally by Uversky et al.44 The solid linerepresents the border between IDP family classified proteins (opentriangles) and globular proteins (solid triangles). (C) The predictionof disordered regions from an amino acid sequence. The predictionof the degree of disorder in wild-type Stm was calculated from itsprimary structure using PONDR VL-XT (dashed line) and DISOPRED2 (solid line) neural network predictors. For the PONDR predictor thescore of >0.5 (horizontal dashed line) indicated a high probability ofdisorder, whereas the boundary value for DISOPRED 2 was a scoreof 0.05 (horizontal solid line). Note, that the same predictions forStm∆SP yielded an essentially identical plot. The only difference wasobserved at the N terminus region, because Stm∆SP is devoid in 21amino acids of the SP sequence (not shown).

Figure 4. High value of the Stokes radius of Stm∆SP suggests itslow compactness. Analytical gel filtrations were performed on aSuperdex 200 10/300 GL column with a flow rate of 0.5 mL/min andan injection volume of 0.1 mL. The column was equilibrated with bufferB and the protein concentration of the samples was 1.57 mg/mL (solidline), 0.157 mg/mL (dashed line), and 0.0157 mg/mL (short dashedline). Based on the calibration curve (inset), Stm∆SP can becharacterized as a protein with an apparent Stokes radius of 78.6 (3 Å. Note that this value did not depend on the protein concentration.In the inset, the standard proteins are shown as open triangles andStm∆SP is shown as a black circle.

Figure 5. Spectroscopic analyses of the Stm. (A) The fluorescenceemission spectrum of Strep-Stm∆SP-His. The excitation wavelengthwas 280 nm and the protein concentration was 0.3 µg/µL. Dataacquisition was performed in buffer B at 25 °C. (B) CD spectrum ofStm∆SP. The far-UV CD spectrum of Stm∆SP was recorded in bufferB in 0, 1, and 5 M GdmCl. The protein concentration was 2.15 µM.


recorded the CD spectra in the presence of guanidium chloride(GdmCl) at 1 and 5 M concentrations. The comparison of thesespectra (Figure 5B) with the spectrum recorded in the absenceof the denaturant indicated that ellipticity at 220 nm increases,reflecting the denaturation of secondary structures.

The data presented above, taken together, strongly suggestthat full-length Stm and the recombinant Stm∆SP derivativebelong to a class of IDPs.

Discussion

Calcium carbonate biominerals are one of the most abundantminerals on the surface of the Earth.32 In eukaryotes, allbiologically controlled calcium carbonate minerals are associatedwith an organic matrix, which represents only a small fraction(from 0.1 wt % to a few wt %) of the total biomineral. Thematrix plays an essential role in mineralization, and its functionsare crystal nucleation, the control of crystal shape, and crystalgrowth and inhibition.32 Among the proteinaceous moiety, thekey components of the matrix are unusually acidic proteins, richin aspartic acid residues. Several acidic proteins have beenidentified, but none of them have been established in polymorphselection, possibly due to the difficulty of working with highlyacidic proteins.33 Stm, which also can be classified as a veryacidic protein (24.6% of D and 10.6% of E residues), was thefirst protein shown capable of influencing the process ofbiomineralization.

To lay the foundation for systematic studies on the molecularmechanism of Stm-dependent calcium carbonate biomineral-ization, we aimed at elaborating experimental conditions underwhich pure recombinant Stm could be obtained with a satisfyingyield. Because initial experiments demonstrated that full-lengthStm is prone to degradation, possibly due to the presence ofthe SP sequence, we decided to express Stm as a protein devoidof SP. Two Stm recombinants (His-Stm∆SP and Strep-Stm∆SP-His) were subjected to purification via affinity chromatographyunder standard conditions. Surprisingly, only a small fractionof the recombinant Stm molecules could be effectively boundby the affinity resins, despite the fact that mass spectrometryanalyses unambiguously confirmed the presence of the appropri-ate affinity tags. It is not clear why the tags are not able tointeract with affinity resins. One reasonable explanation for theseunexpected observations could be the involvement of the StmC- and N-termini in some interaction with other parts of theStm molecule or with other Stm molecules. The latter, veryattractive explanation, could indicate that Stm exists in anoligomeric state. Unfortunately, at the moment, we do not haveexperimental data supporting this hypothesis.

Finally, the purification procedure based on standard proteinfractionation techniques was elaborated for the nontagged Stm(Stm∆SP), with the amino acid sequence corresponding to thesequence of mature Stm, that is, devoid of the SP sequence.4

The protein can be obtained with a reasonable yield and purity.This lays the foundation for the systematic biochemical char-acterization of the Stm∆SP molecule. Because our procedureresults in a protein that completely lacks post-translationalmodifications, one of the future tasks could be analysis of thepossible role of post-translational modifications on Stm∆SPstructure and function. The post-translational modifications arebelieved to exert a significant effect on the structure of DSPP,a human homologue of Stm.34

Many of the observations and analyses presented aboveindicate that Stm∆SP appears to have properties characteristicof IDPs, a class of proteins that lack, at least in vitro under

physiological conditions, rigid tertiary structure, and exist insteadas highly dynamic ensembles of interconverting structures.22,23,29

IDPs carry out numerous important biological functions, whichare invariably associated with their structural disorder. Recently,it has been suggested that IDPs actually fall into five broadfunctional classes based on their mode of action.35 The firstclass is that of an entropic chain, with functions stemmingdirectly from protein disorder. IDPs in the other four categories,including the so-called effectors, scavengers, assemblers, anddisplay sites, function via molecular recognition. The majorfunctional asset of these IDPs is related to their significantdisorder-order transition, that is, induced local folding uponbinding to the respective target. Interestingly, due to their highlyflexible structure, IDPs or proteins having disordered regionsare typically involved in biological processes in which interac-tions with multiple partners are often involved.36,37 As notedrecently, many proteins involved in biomineralization exhibitstructural trends toward extended, random coil, or other unstablestructures and, thus, can be classified as IDPs.38

Which of the putative partners of Stm are able to induce adisorder-order transition? Some very recent data coming fromstudies on otolith formation suggest possible candidates. Theprocess of otolith formation can be separated into two stages.During the first stage seeding particles composed of glycogenand the otolith matrix protein-1 (OMP-1) form the nucleus ofthe otolith.8,39,40 Then the otoliths grow diurnally, resulting inthe formation of daily rings within their microstructure.41 A ringis composed of an incremental zone predominated by a calciumcarbonate and a discontinuous zone comprising the organicmatrix.42 Sollner et al.4 have demonstrated that successive ringswere no longer present when Stm expression was blocked usingantisense oligonucleotides. Simultaneously, a change in thecrystal lattice and, thus, a change in otolith morphology wasobserved. In the most severely affected otoliths, which weredevoid of Stm, the organic matrix did not run in radial directions.Instead, the organic matrix was excluded from a single, largeinorganic crystal. A simple interpretation of these findings isthat Stm is an indispensable component of the matrix involvedin its organization, most likely upon encountering a properproteinaceous partner. Very recently, immunohistological analy-sis indicated that layers in the organic matrix consist of twoproteins, OMP-1, and otolin-1. Because OMP-1 and otolin-1are localized at almost the same positions, it has been suggestedthat these proteins, which are necessary for normal otolithgrowth and their correct anchoring onto the sensory macule,are also likely to interact with each other in vivo.39,40 Immu-nochemistry has also shown that Stm is an integral componentof otoliths throughout all stages of otolith development.6 Thus,it is possible that all three proteins participate in a complex setof interactions that would represent the mechanistic foundationfor proper otolith formation and calcification.

Acknowledgment. We thank professor Teresa Nicolson(Oregon Hearing Research Center and Vollum Institute, OregonHealth and Science University, Portland, OR 97201) for thesupply of the Starmaker cDNA, cloned into pCR4-TOPOplasmid. This work was supported by the Polish Ministry ofScience and Higher Education Grant 2827/P01/2007/32 and bythe Wrocław University of Technology.

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BM800135M


starmaker exhibits properties of an intrinsically disordered protein

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