Expression and Properties of theRecombinant Murine Golli-Myelin BasicProtein Isoform J37

Jaspreet Kaur,1 David S. Libich,1 Celia W. Campagnoni,2 D. Denise Wood,3

Mario A. Moscarello,3 Anthony T. Campagnoni,2 and George Harauz1*1Department of Molecular Biology and Genetics, and Biophysics Interdepartmental Group, University ofGuelph, Guelph, Ontario, Canada2Mental Retardation Research Center, Neuropsychiatric Institute and Hospital, UCLA School of Medicine, LosAngeles, California3Department of Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, Ontario, Canada

A recombinant form of the murine Golli-myelin basicprotein (MBP) isoform J37 (rmJ37) has been expressed inEscherichia coli and isolated to 95% purity via metalchelation and ion exchange chromatography. The pro-tein did not aggregate lipid vesicles containing acidicphospholipids, unlike the 18.5 kDa isoform of MBP. Thisresult is consistent with J37 having a functional role priorto the assembly of compact myelin. Circular dichroicspectroscopy showed that rmJ37 had a large proportionof random coil in aqueous solution but gained �-helixand �-sheet in the presence of monosialogangliosideGM1 and PI(4)P. Thus, like “classic” MBP, J37 is intrinsi-cally unstructured, and its conformation depends on itsenvironment and bound ligands. Analyses of the aminoacid sequence of rmJ37 predicted an N-terminal cal-modulin (CaM)-binding site. It was determined via a gel-shift assay and fluorescence spectroscopy that rmJ37and CaM interacted in a 1:1 ratio in a Ca2�-dependentmanner. However, the interaction was weak comparedwith 18.5 kDa MBP. © 2003 Wiley-Liss, Inc.

Key words: myelin basic protein; MBP; J37; genes ofoligodendrocyte lineage (Golli); mass spectrometry; lipidaggregation; circular dichroism; calmodulin; fluores-cence spectroscopy

The myelin basic proteins (MBPs) are a family ofmajor structural molecules of the myelin sheath of thecentral nervous system (Baumann and Pham-Dinh, 2001).The 18.5 kDa isoform of MBP has been the most inten-sively studied, especially regarding its high degree of post-translational modification and role in multiple sclerosis(Wood and Moscarello, 1997). There are several otherMBP isoforms that arise from differential splicing of the“classic” MBP gene and more from different transcriptionstart sites of the larger genetic unit called Golli (for genesof the oligodendrocyte lineage; Pribyl et al., 1993; Cam-pagnoni et al., 1993; Givogri et al., 2002). In contrast to

the 18.5 kDa isoform, these other isoforms are expressedin early oligodendrocyte development or differentiation,are also expressed in the immune system (spleen andthymus), and are localized in the nucleus as well as thecytoplasm (Pedraza et al., 1997; Givogri et al., 2000;Reyes and Campagnoni, 2002). New structure-functionrelationships have been suggested for the MBP proteinfamily, including regulation of transcription and signaltransduction (Staugaitis et al., 1996; Lintner and Dyer,2000; Campagnoni and Skoff, 2001).

In the mouse, three Golli-MBP isoforms have beenidentified: BG21, J37, and TP8 (Campagnoni et al., 1993).Both BG21 and J37 have segments in common with theclassic 18.5 kDa isoform of MBP (Fig. 1), but TP8 doesnot because of a frameshift mutation. None of the Golli-MBP isoforms has yet been purified to homogeneity froma natural source, and little is known about their physico-chemical properties. In the present study, we focused onthe purification and characterization of the recombinantmurine (rm) Golli-MBP isoform J37. We refer to thenatural form of this protein as mJ37 and to the recombi-nant (r) form as rmJ37, following the convention used forthe 18.5 kDa isoform of murine MBP (mMBP andrmMBP; Bates et al., 2000, 2002). Here, we describeoptimization of yield and purity and characterization bypolyacrylamide gel electrophoresis (PAGE), mass spec-trometry, and circular dichroic (CD) spectroscopy. Inaddition, we have investigated this protein’s interactionswith Ca2�-calmodulin (CaM), complementing parallelstudies with the 18.5 kDa isoform of MBP (Libich andHarauz, 2002a,b).

*Correspondence to: George Harauz, Department of Molecular Biologyand Genetics, University of Guelph, Guelph, Ontario, N1G 2W1 Canada.E-mail: gharauz@uoguelph.ca

Received 24 September 2002; Revised 18 November 2002; Accepted 21November 2002

Journal of Neuroscience Research 71:777–784 (2003)

© 2003 Wiley-Liss, Inc.

MATERIALS AND METHODS

Materials

Electrophoresis-grade acrylamide, ultrapure Tris base, andultrapure Na2EDTA were purchased from ICN Biomedicals(Costa Mesa, CA). Electrophoresis-grade sodium dodecyl sulfate(SDS) was obtained from Bio-Rad Laboratories (Mississauga,Ontario, Canada). The Ni2�-NTA (nitrilotriacetic acid) agarosebeads were purchased from Qiagen (Mississauga, Ontario, Can-ada). Other chemicals were of reagent grade and were acquiredfrom either Fisher Scientific (Unionville, Ontario, Canada) orSigma-Aldrich (Oakville, Ontario, Canada). Acetonitrile andtrifluoroacetic acid (TFA; both of HPLC grade) were obtainedfrom Fisher Scientific and Sigma-Aldrich, respectively.

Phosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylserine (PS), phosphatidylinositol (PI), cholesterol(CHL), sphingomyelin (SPM), and phosphatidylinositol-4-phosphate [PI(4)P] were purchased from Avanti Polar Lipids(Alabaster, AL). Monosialoganglioside GM1 was purchased fromSigma-Aldrich.

The natural C1 charge isomer of the 18.5 kDa isoform ofbovine MBP (bMBP/C1; Beniac et al., 1997) and rmMBP(Bates et al., 2000, 2002) were purified as previously describedand used as controls. Purified bovine brain calmodulin waspurchased from Calbiochem (La Jolla, CA). Protein concentra-tions were determined either by the Bradford assay withbMBP/C1 as the standard or by measuring the absorbance at280 nm.

Purification and Characterization of rmJ37

The cloning of the mJ37 gene has been previously de-scribed (Campagnoni et al., 1993). The gene was contained in apET-22b(�) vector with which competent Escherichia coli BL21-CodonPlus(DE3)-RP cells (Stratagene, La Jolla, CA) weretransformed. Cells were grown in LB ampicillin (amp)�-chloramphenicol (cam)� (50 �g/ml and 33 �g/ml, respectively)until A600 � 0.5–0.6. The cultures were then induced with

1 mM isopropyl-�-D-thiogalactopyranoside (IPTG) and grownfor a further 3 hr. Cells were harvested by centrifugation, andthe pellet was frozen at –20°C until use.

The frozen pellet was suspended in 25 ml lysis buffer(20 mM Tris-HCl, pH 7.9, 5 mM imidazole, 0.5 M NaCl, 6 Murea) and stirred vigorously overnight at 4°C. The suspensionwas centrifuged, and the supernatant was collected and filteredthrough a 5 �m syringe filter. An Ni2�-NTA column (1 ml of50% w:v slurry per 1 liter of culture) was equilibrated with 10column volumes of binding buffer, and the protein was loadedonto the column at a rate of 1 ml/min. The column was washedwith 25 column volumes of binding buffer, and rmJ37 waseluted with 10 ml of 20 mM Tris-HCl, pH 7.9, 1 M imidazole,0.5 M NaCl, 6 M urea.

Protein-containing fractions from the Ni2�-NTA columnwere dialyzed into 50 mM sodium acetate, pH 5.0, and loadedonto a CM52 cation exchange column. The CM52 column waswashed with 3.3 column volumes of each of 1) 50 mM sodiumacetate, pH 5.0; 2) 50 mM MOPS, pH 7.0; and 3) 50 mMTris-HCl, pH 8.5. The final wash with (4) 50 mM glycine, pH10.0, eluted rmJ37, which was subsequently dialyzed into dis-tilled, deionized (dd)H2O.

Protein preparations were evaluated by discontinuousSDS-polyacrylamide gel electrophoresis (SDS-PAGE; 5% stack-ing, 14% separating) along with low-range molecular massmarkers (MBI-Fermentas, Burlington, Ontario, Canada) andstaining with Coomassie brilliant blue R250 (Fisher Scientific).Western blotting using rabbit anti-bovine MBP polyclonal IgGwas performed as previously described (Beniac et al., 2000).

Reverse-phase HPLC was performed using a Waters ap-paratus with a Symmetry 300 C18, 5 �m, 4.6 � 250 mmcolumn. Detection was at 214 nm, the flow rate was 0.5ml/min, and the column was maintained at 30°C. Acetonitrileconstituted the mobile phase, and TFA was the ion-pairingagent. Elution gradients were begun at 80% solvent A (ddH2Owith 0.1% TFA) and 20% solvent B (acetonitrile with 0.1%TFA), and run at a rate of 1% solvent B per 1 min. Electrosprayionisation mass spectrometry was performed as previously de-scribed (Bates et al., 2000, 2002).

Lipid Vesicle Aggregation Assays

Lipid vesicle aggregation assays were adopted from pub-lished protocols (Jo and Boggs, 1995; Boggs et al., 1997, 2000).Lipids were combined to form large unilamellar vesicles (LUVs)comprising 92% PC, 8% PS (molar ratios), and also LUVs witha lipid composition similar to that of the cytoplasmic face ofmyelin (Cyt-LUVs): 44% CHL, 27% PE, 13% PS, 11% PC, 3%SPM, and 2% PI (molar ratios). The aggregation assay involvedtitrating the LUVs (in 20 mM HEPES-NaOH, pH 7.4, 10 mMNaCl) with either rmJ37 or bMBP/C1 (control) to give a finallipid:protein ratio (w:w) of 30:1, 60:1, 90:1, or 120:1. Theabsorbance at 450 nm (A450) was recorded after 15 min ofincubation. Lipid suspensions with no added protein served asthe blank.

Circular Dichroic Spectroscopy

CD spectroscopy was performed as previously described(Bates et al., 2000, 2002) with rmJ37 at 0.2 mg/ml in aqueoussolution (0.5 mM Tris-HCl, pH 7.4), and in the presence of

Fig. 1. Comparison of murine Golli-MBP (full translation), mBG21,rmJ37, and 18.5 kDa rmMBP. The numbers refer to amino acidsstarting with the N-terminal Gly (not the Met). The protein mBG21also has a Golli-specific pentapeptide sequence at its C-terminus(VSSEP). The C-terminal sequence segment KSAHK . . . SPMAR of18.5 kDa rmMBP is predicted to be a calmodulin-binding site and isshaded. Here, another CaM-binding site is predicted in the Golli-specific segment EIHRG . . . SQTAS, also shaded. The recombinantform of murine J37 (rmJ37) is missing a tetrapeptide MARR sequenceat its C-terminus. Both rmJ37 and rmMBP have a C-terminal tag(LEHHHHHH). The positions of tryptophanyl residues are also noted.

778 Kaur et al.

either monosialoganglioside GM1 or PI(4)P at molar lipid:pro-tein ratios of 3:1, 10:1, and 50:1. All spectra were collected usinga Jasco J-600 spectropolarimeter (Japan Spectroscopic Co., To-kyo, Japan). The data were smoothed using an inverse-squarealgorithm in the SigmaPlot (SPSS, Chicago, IL) computer pro-gram for presentation.

Interactions With CaM: Mobility Shift Assays andFluorescence Spectroscopy

The proteins rmJ37 and CaM were mixed in variousmolar ratios (2:1, 1:1, 0:1, 1:0, 1:2) in a buffer consisting of50 mM HEPES-NaOH, pH 7.4, 100 mM NaCl, 1 mM CaCl2,0.0035% dithiobis[succinimidylpropionate] (DSP). Sampleswere electrophoresed (12% SDS-PAGE) in either the absence orthe presence of reducing agents [14.4 mM �-mercaptoethanoland 10 mM dithiothreitol (DTT); Libich and Harauz, 2002b].

Changes in the inherent fluorescence of the single Trpresidue in rmJ37 (in 50 mM Tris-HCl, pH 7.4, 250 mM NaCl,1 mM CaCl2) were observed upon addition of CaM (whichdoes not contain Trp), as previously described (Libich andHarauz, 2002a). To ensure that the interaction with CaM was infact a Ca2�-dependent process under these conditions, controlexperiments were performed in the presence of chelating agents(8 mM EDTA, 2 mM EGTA) instead of 1 mM CaCl2. Anadditional control experiment was performed in 6 M guanidinehydrochloride (GdnHCl).

Accessibility of Trp Residue Via AcrylamideQuenching: Steady-State Measurements

The accessibility of the single Trp residue in rmJ37 wasassessed as previously described (Libich and Harauz, 2002a).Titrating the protein solution with the collisional quencheracrylamide yielded data that were fitted to the Stern-Volmerequation:

F0/F � (1 � Ksv[Q])exp(V[Q]), (1)

where F0 and F are the fluorescence intensities at 340 nm in theabsence and presence of acrylamide, respectively, [Q] is theconcentration of the acrylamide quencher, and Ksv and V aredynamic and static quenching constants, respectively (Lakowicz1999). The experiment was repeated in the presence of anequimolar amount of CaM. The soluble Trp analogueN-acetyltryptophanamide (NATA) was used as the reference.

RESULTS AND DISCUSSIONProtein Purification and Characterization

The recombinant form of murine J37 (rmJ37), witha C-terminal hexahistidine tag, was expressed in E. coliBL21-CodonPlus(DE3)-RP. The best yield of rmJ37 wasobtained with A600 � 0.5–0.6 at induction, and growthfor 3 hr afterwards at 37°C, in comparison with rmMBP,for which the optimal A600 � 0.3 and 2 hr growthpostinduction have been reported (Bates et al., 2000).

The first step of purification involved an Ni2�-NTAaffinity column, from which generally 1 mg of protein waseluted for 1 liter of cell culture. This eluant was found by14% SDS-PAGE to comprise the major species rmJ37

(27.6 kDa) and some lower molecular mass bands (lane 1in Fig. 2a). Removal of these contaminants by morestringent washes of the Ni2�-NTA column with higherconcentrations of imidazole resulted in excessive loss ofrmJ37 protein. Thus, an additional step of CM52 cationexchange chromatography was added. Most of the con-taminants were removed by washes with lower pH buffers,i.e., acetate (pH 5), MOPS (pH 7), and Tris (pH 8.5). Thefinal wash with a glycine buffer (pH 10) yielded an elec-trophoretically purer preparation of rmJ37 (lane 2 in Fig.2a), especially appreciable on Western blotting (Fig. 2b)and reverse-phase HPLC (Fig. 2c). The recovery of pro-tein from the CM52 column was 40%, so the final yield ofrmJ37 was 0.4 mg per 1 liter of cell culture. The HPLCprofile of the CM52 column eluant showed a major peakrepresenting rmJ37, with a small shoulder (Fig. 2c). Thepurity as assessed by integrating peak areas was at least 95%.The fraction representing the major peak in the HPLCelution profile (Fig. 2c) was collected and evaluated byelectrospray ionization mass spectrometry (Fig. 2d). Themajor peak represented 27,585.91 � 0.41 amu (average of25 runs), which corresponded to the expected mass of27,586.90 amu, well within the limits of experimentalerror expected for this technique (0.02%, or 5.5 amu).Thus, the rmJ37 preparation was not posttranslationallymodified and lacked the N-terminal fMet.

All rmJ37 preparations had a propensity to aggrega-tion, and this protein was far worse than MBP in this

Fig. 2. Purification of rmJ37 from E. coli BL21-CodonPlus(DE3)-RP.a: SDS-PAGE of eluants from Ni2�-NTA (lane 1) and CM52 (lane 2)columns, with molecular mass markers and 18.5 kDa bMBP/C1 (lane3). Note that rmJ37 appears to correspond to a molecular mass exceed-ing 36 kDa; this anomaly has been previously noted for highly basicproteins of the MBP family, including the Golli-MBP isoforms (Cam-pagnoni et al., 1993). b: Western blot of duplicate gel shown in a,probed with anti-MBP (18.5 kDa) polyclonal antibody. c: HPLCprofile of rmJ37-containing fraction from CM52 column. d: Repre-sentative electrospray ionisation mass spectra of peak fraction in c.

Properties of Recombinant Murine J37 779

regard. This problem was alleviated by maintaining theprotein concentration below 1 mg/ml and by workingwith small aliquots maintained at –80°C. The addition ofa small amount (0.001%) of Triton X-100 to all samplebuffers has previously been found to be beneficial inpreventing adsorption of MARCKS-related protein(“myristoylated alanine-rich C-kinase substrate”; Schleiffet al., 1996) or 18.5 kDa MBP (our unpublished obser-vations) to the sides of the tubes, and it is also suggestedhere.

Lipid Vesicle Aggregation AssaysOne of the major roles attributed to the 18.5 kDa

isoform of MBP is to hold together apposing cytoplasmicfaces of oligodendrocyte membranes in the myelin sheath(Staugaitis et al., 1996); thus, its ability to aggregate mem-brane vesicles serves as a functional assay (Jo and Boggs,1995; Boggs et al., 1997, 2000). Here, rmJ37 did notaggregate either simple PC:PS LUVs or Cyt-LUVs, incontrast to the bMBP/C1 control (Fig. 3). Given that J37

accounts for a considerable portion of classic MBP, espe-cially compared with BG21 (Fig. 1), this result could nothave been predicted. Although separate halves of the 18.5kDa isoform can bind lipid vesicles (Boggs et al., 1981,1999; Walker and Rumsby, 1985), the large classic MBPsegment in rmJ37 was here insufficient, or in the wrongstructural context, to effect their aggregation. These ob-servations suggest that membrane apposition cannot be afunction of J37 in vivo, which is consistent with theprotein being localized in the nucleoplasm (Reyes andCampagnoni, 2002).

Circular Dichroic SpectroscopyThe conformation of the 18.5 kDa MBP isoform is

predominantly a random coil in aqueous solution as as-sessed by CD, but the protein adopts some ordered sec-ondary structure (i.e., �-helix, �-sheet, and �-turn) in thepresence of detergents and lipids (Polverini et al., 1999).Lipids such as PI(4)P and monosialoganglioside GM1 areconvenient for such studies, because they do not precip-itate the MBP preparation (Bates et al., 2000, 2002; Ish-iyama et al., 2001). We use PI(4)P instead of PI(4,5)P2because it is considerably less expensive; the two lipidsappear to interact with 18.5 kDa MBP in essentially thesame way (Ishiyama et al., 2001, 2002).

Here, rmJ37 was also primarily disordered in aque-ous solution (33% random coil) but inherently less so than18.5 kDa rmMBP (45% random coil; Bates et al., 2000). Inthe presence of lipids, rmJ37 attained ordered secondarystructure, especially with PI(4)P (Fig. 4). The proportionof random coil decreased to 30% with GM1 and to 25%with PI(4)P. The spectra were suggestive of a mixture of�-helix and �-sheet, with the �-helical component in-creasing with increasing amounts of lipid. Thus, the con-formation of the protein depends on its environment, andit represents another example of the “intrinsically unstruc-tured” class of proteins that includes 18.5 kDa MBP(Dunker et al., 2002; Hill et al., 2002).

Interactions With CaMA search for CaM-binding motifs in rmJ37 was per-

formed using a target database (http://calcium.oci.utoron-to.ca; Yap et al., 2000). The predicted CaM-binding“Others” motif of the 18.5 kDa rmMBP isoform (Libichand Harauz, 2002a) was partly abolished here because ofthe loss of exon 10 (Fig. 1). However, a new CaM-binding site was suggested in the Golli-specific portion ofthe protein, matching either a “1–10” or a “1–8–14”motif. The new predicted site was rmJ37(19–39;EIHRGEAGKKRSVGKLSQTAS), comprising a tribasicKKR sequence that forms part of several CaM-bindingmotifs. Here, we tested the hypothesis that rmJ37 bindsCaM in two ways.

When rmJ37 and CaM were incubated at equimolarratios in the presence of the cross-linker DSP and analyzedby 12% SDS-PAGE, a higher order band was observedmigrating at roughly 47 kDa (Fig. 5). DSP is a homobi-functional cross-linking reagent, like glutaraldehyde, butwith a disulfide bridge, so that cross-linking can be re-

Fig. 3. Lipid vesicle aggregation assays using bMBP/C1, and rmJ37,with PC:PS LUVs (top) and Cyt-LUVs (bottom). Increasing amountsof bMBP/C1, but not rmJ37, resulted in an increased absorbance at 450nm, indicating aggregation.

780 Kaur et al.

versed under reducing conditions. The band disappearedwhen cross-linking was reversed under reducing condi-tions (not shown). Insofar as an rmJ37:CaM complexwould be 44.4 kDa in size, this result indicates that thermJ37:CaM stoichiometry was 1:1. Neither rmJ37 norCaM formed homomultimeric complexes when incu-bated with DSP alone. Moreover, only the equimolarassembly was formed, unlike the 18.5 kDa MBP isoform,for which multiple bands representing 1:2 or 2:1 com-plexes were observed (Libich and Harauz, 2002b). Thus,the rmJ37–CaM interaction involved only one bindingsite on rmJ37. Moreover, the rmJ37–CaM interactionappeared weaker than that of MBP–CaM: Both the MBPand CaM bands almost completely disappeared when in-cubated at equimolar ratios (Libich and Harauz, 2002b),whereas, here, a significant proportion of rmJ37 and CaMremained unassociated. In summary, the results presentedin Figure 5 support the prediction that J37 binds CaM andsuggest weak binding to a single target site on J37.

Further insight into the nature of the rmJ37–CaMinteraction was obtained via single Trp fluorescence spec-

troscopy (Fig. 6). The intrinsic Trp fluorescence spectrumof rmJ37 alone had a peak at about 350 nm (Fig. 6a). In thepresence of an equimolar amount of CaM and in 1 mMCa2�, there was a slight increase in peak fluorescenceemission, and a slight “blue shift” of the spectrum towardsmaller wavelengths. In a buffer lacking Ca2� but com-prising 8 mM EDTA and 2 mM EGTA, the fluorescenceemission spectrum of rmJ37 was the same as in Ca2�-containing buffer, but it remained unchanged upon theaddition of an equimolar amount of CaM (Fig. 6b). Thesame result was obtained in a buffer consisting of 1 mMCa2� and 6 M GdnHCl (Fig. 6c). These results togethersuggested that rmJ37 bound CaM in a Ca2�-dependentmanner and that the protein’s native structure was essentialfor recognition by CaM. However, in comparison withthe 18.5 kDa MBP isoform (Libich and Harauz, 2002a),the changes in fluorescence intensity upon titration withCaM were so small that the apparent dissociation constantscould not be obtained with confidence. This phenomenoncould be due simply to the Trp residue of rmJ37 being toofar away from the CaM-binding site or partially seques-tered within the protein, so that its environment did notchange significantly upon rmJ37’s interaction with CaM.Alternatively, the CaM target on J37 might not be con-ducive to strong binding, given that an �-helix formed bythe predicted segment would be unstable (because of theGly residues) and nonamphipathic, resulting in a largepopulation of unassociated protein. This latter explanationis supported by the gel-shift assays.

Fig. 4. CD spectroscopy of rmJ37 preparations under different condi-tions, in aqueous solution (aq) and in the presence of lipids GM1 (a) andPI(4)P (b) at molar lipid:protein ratios of 3:1, 10:1, and 50:1.

Fig. 5. Standard SDS-PAGE, under nonreducing conditions, of rmJ37(27.6 kDa) incubated with bovine brain CaM (16.8 kDa) for 1 hr in thepresence of the reversible homobifunctional cross-linker DSP. Thebuffer consisted of 50 mM HEPES-NaOH, pH 7.5, 100 mM NaCl,1 mM CaCl2, 0.0035% DSP. Lanes represent: molecular mass markersand 1, 2:1 rmJ37:CaM; 2, 1:1 rmJ37:CaM; 3, 1:2 rmJ37:CaM; 4, 0:1rmJ37:CaM (i.e., CaM alone); and 5, 1:0 rmJ37:CaM (i.e., rmJ37alone).

Properties of Recombinant Murine J37 781

Accessibility of Trp Residue Via AcrylamideQuenching: Steady-State Measurements

It has been shown by fluorescence studies and acryl-amide quenching that the single Trp fluorophore in the18.5 kDa MBP isoform is in a primarily aqueous environ-ment and that there are both dynamic and static compo-nents to the quenching mechanism that are adequatelymodelled using equation 1 (Nowak and Berman, 1991;Libich and Harauz, 2002a). The same experiments were

performed here to investigate the local environment of theTrp residue in rmJ37, in the presence and absence of CaM(Fig. 7). In comparison with that of the 18.5 kDa MBPisoform, the value of Ksv (representing dynamic quench-ing) for rmJ37 was lower, and V (representing staticquenching) was greater. Thus, dynamic quenching playeda lesser role in rmJ37 than in 18.5 kDa MBP. When CaMwas present in equimolar amounts, Ksv was not changedsignificantly, but V decreased. In summary, the acrylamidequenching data indicated that the Trp76 residue of rmJ37was partially sequestered within the structure and con-firmed the CaM-binding data. Further experiments usingfluorescence lifetime measurements will be required tounderstand better the behavior of the Trp residue in rmJ37(Lakowicz, 1999).

Potential Functions of Golli-MBP IsoformsProteins of the MBP family have been suggested to

have roles in signal transduction, probably in the phos-phoinositide signalling pathway that affects axonal cy-toskeletal organization (Wood and Moscarello, 1997;Lintner and Dyer, 2000; Reyes and Campagnoni, 2002).There is an intriguing comparison with other proteinsinvolved in signal transduction, such as MARCKS (Ar-buzova et al., 2002): MBP and MARCKS are intrinsicallyunstructured, are phosphorylated by protein kinase C andMAP kinases, bind acidic lipids strongly, and also bindCaM and actin. The physiological significance of theinteraction with CaM is as yet undetermined; as forMARCKS, it might be a means of regulating intracellularCaM pools (Arbuzova et al., 2002). Other proteins, as yet

Fig. 6. Representative intrinsic Trp fluorescence emission spectra forrmJ37, at 2.8 �M concentration (averages of three replicates). Thebuffers consisted of 50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 1 mMCaCl2 (a); 50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 8 mM EDTA,2 mM EGTA (b); 50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 1 mMCaCl2, 6 M GdnHCl (c). Solid circles represent rmJ37 alone, and opencircles represent rmJ37 in the presence of an equimolar amount ofCaM.

Fig. 7. Stern-Volmer plots (averages of three replicates) for quenchingof the intrinsic Trp fluorescence emission at 340 nm of rmJ37 (2.8 �M)by acrylamide. The values of the Stern-Volmer coefficients were ob-tained by regression analysis using equation 1 (all r2 0.996). Circles:rmJ37 alone in 50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 1 mM CaCl2(Ksv � 5.92 � 0.35 M–1, V � 2.59 � 0.12 M–1). Solid triangles: rmJ37in 50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 1 mM CaCl2, and anequimolar amount of CaM (Ksv � 6.33 � 0.53 M–1, V � 1.52 �0.18 M–1). Open triangles: The soluble Trp analogue NATA, used asa reference (Ksv � 17.9 � 0.6 M–1, V � 1.5 � 0.1 M–1).

782 Kaur et al.

unidentified, appear to function in translocating CaM tothe nucleus (Mermelstein et al., 2001). Insofar as theGolli-MBPs are expressed in early oligodendrocyte devel-opment or differentiation, they would be expected tofunction in some signalling pathways. Here, the inabilityof rmJ37 to aggregate lipid vesicles suggests a functionalrole prior to the assembly of compact myelin. It is alsoclear from this study that J37 is a member of what isrecognized as a growing class of “natively unfolded” pro-teins (Dunker et al., 2002; Dyson and Wright, 2002),whose lack of intrinsic structure might be integral to theirfunction as a means of speeding molecular recognition viaa “fly-casting” mechanism (Shoemaker et al., 2000). Here,the demonstrated increase in regular secondary structure ofrmJ37 upon lipid binding suggests that an association witha phosphoinositide in particular, or a membrane in gen-eral, might be important in vivo. However, the weaker(albeit still specific) binding to CaM compared with 18.5kDa MBP suggests that this interaction might not beimportant. Nonetheless, further investigations of the in-teractions in vitro of J37, BG21, and TP8, and evaluationof posttranslational modifications in vivo, are warranted inorder to define more precisely the roles of the Golli-MBPs.

CONCLUSIONSA recombinant form of the murine Golli-MBP iso-

form J37 (rmJ37) has been purified to 95% purity via metalchelation and ion exchange chromatography, with a yieldof 0.4 mg per 1 liter of E. coli culture. The protein wasunmodified posttranslationally as determined by electro-spray ionisation mass spectrometry. In contrast to the 18.5kDa isoform of MBP, rmJ37 did not aggregate lipid ves-icles. Circular dichroic spectroscopy showed that rmJ37had a large proportion of random coil in aqueous solutionbut gained significant amounts of ordered secondary struc-ture in the presence of lipids, namely, monosialoganglio-side GM1 and PI(4)P, showing that it bound them. The-oretical analyses of the amino acid sequence of rmJ37predicted an N-terminal CaM-binding site. It has beenshown that rmJ37 and CaM interacted weakly when com-bined in an equimolar ratio in the presence of Ca2�, thephysiological significance (if any) of which is yet to bedetermined. Acrylamide quenching of the single trypto-phanyl residue of rmJ37 showed it to be moderately ex-posed to the environment and further sequestered whencomplexed with CaM. Knowledge of the properties ofrmJ37 determined here will be helpful for future structuraland functional studies of this protein.

ACKNOWLEDGMENTSThe authors are grateful to Dr. Lillian DeBruin and

Messrs. Jeffery Haines, Ian Bates, and Christopher Hill forassistance and advice with many experiments. This workwas supported by the Canadian Institutes of Health Re-search (G.H., M.A.M.), by the Natural Sciences and En-gineering Research Council of Canada (G.H.), by theMultiple Sclerosis Society of Canada (G.H., M.A.M.), and

by the National Multiple Sclerosis Society (grant RG2693to A.T.C.).

REFERENCESArbuzova A, Schmitz AA, Vergeres G. 2002. Cross-talk unfolded:

MARCKS proteins. Biochem J 362:1–12.Bates IR, Matharu P, Ishiyama N, Rochon D, Wood DD, Polverini E,

Moscarello MA, Viner NJ, Harauz G. 2000. Characterization of a recom-binant murine 18.5-kDa myelin basic protein. Protein Expr Purif 20:285–299.

Bates IR, Libich DS, Wood DD, Moscarello MA, Harauz G. 2002. AnArg/Lys3Gln mutant of recombinant murine myelin basic protein as amimic of the deiminated form implicated in multiple sclerosis. ProteinExpr Purif 25:330–341.

Baumann N, Pham-Dinh D. 2001. Biology of oligodendrocyte and myelinin the mammalian central nervous system. Physiol Rev 81:871–927.

Beniac DR, Luckevich MD, Czarnota GJ, Tompkins TA, Ridsdale RA,Ottensmeyer FP, Moscarello MA, Harauz G. 1997. Three-dimensionalstructure of myelin basic protein. I. Reconstruction via angular reconsti-tution of randomly oriented single particles. J Biol Chem 272:4261–4268.

Beniac DR, Wood DD, Palaniyar N, Ottensmeyer FP, Moscarello MA,Harauz G. 2000. Cryoelectron microscopy of protein-lipid complexes ofhuman myelin basic protein charge isomers differing in degree of citrul-lination. J Struct Biol 129:80–95.

Boggs JM, Wood DD, Moscarello MA. 1981. Hydrophobic and electro-static interactions of myelin basic proteins with lipid. Participation ofN-terminal and C-terminal portions. Biochemistry 20:1065–1073.

Boggs JM, Yip PM, Rangaraj G, Jo E. 1997. Effect of posttranslationalmodifications to myelin basic protein on its ability to aggregate acidic lipidvesicles. Biochemistry 36:5065–5071.

Boggs JM, Rangaraj G, Koshy KM. 1999. Analysis of the membrane-interacting domains of myelin basic protein by hydrophobic photolabel-ing. Biochim Biophys Acta 1417:254–266.

Boggs JM, Rangaraj G, Koshy KM, Mueller JP. 2000. Adhesion of acidiclipid vesicles by 21.5 kDa (recombinant) and 18.5 kDa isoforms of myelinbasic protein. Biochim Biophys Acta 1463:81–87.

Campagnoni AT, Skoff RP. 2001. The pathobiology of myelin mutantsreveal novel biological functions of the MBP and PLP genes. Brain Pathol11:74–91.

Campagnoni AT, Pribyl TM, Campagnoni CW, Kampf K, Amur-UmarjeeS, Landry CF, Handley VW, Newman SL, Garbay B, Kitamura K. 1993.Structure and developmental regulation of Golli-mbp, a 105-kilobasegene that encompasses the myelin basic protein gene and is expressed incells in the oligodendrocyte lineage in the brain. J Biol Chem 268:4930–4938.

Dunker AK, Brown CJ, Lawson JD, Iakoucheva LM, Obradovic Z. 2002.Intrinsic disorder and protein function. Biochemistry 41:6573–6582.

Dyson HJ, Wright PE. 2002. Coupling of folding and binding for unstruc-tured proteins. Curr Opin Struct Biol 12:54–60.

Givogri MI, Bongarzone ER, Campagnoni AT. 2000. New insights on thebiology of myelin basic protein gene: the neural-immune connection.J Neurosci Res 59:153–159.

Givogri MI, Costa RM, Schonmann V, Silva AJ, Campagnoni AT, Bon-garzone ER. 2002. Central nervous system myelination in mice withdeficient expression of Notch1 receptor. J Neurosci Res 67:309–320.

Hill C, Bates I, White G, Hallett FR, Harauz G. 2002. Effects of theosmolyte trimethylamine-N-oxide on conformation, self-association, andtwo-dimensional crystallization of myelin basic protein. J Struct Biol139:13–26.

Ishiyama N, Bates IR, Hill CM, Wood DD, Matharu P, Viner NJ,Moscarello MA, Harauz G. 2001. The effects of deimination of myelinbasic protein on structures formed by its interaction withphosphoinositide-containing lipid monolayers. J Struct Biol 136:30–45.

Properties of Recombinant Murine J37 783

Ishiyama N, Hill CM, Bates IR, Harauz G. 2002. The formation of helicaltubular vesicles by binary monolayers containing a nickel-chelating lipidand phosphoinositides in the presence of basic polypeptides. Chem PhysLipids 114:103–111.

Jo E, Boggs JM. 1995. Aggregation of acidic lipid vesicles by myelin basicprotein: dependence on potassium concentration. Biochemistry 34:13705–13716.

Lakowicz JR. 1999. Principles of fluorescence spectroscopy, 2 ed. NewYork: Kluwer Academic. 698 p.

Libich DS, Harauz G. 2002a. Interactions of the 18.5 kDa isoform ofmyelin basic protein with Ca2�-calmodulin: in vitro studies using fluo-rescence microscopy and spectroscopy. Biochem Cell Biol 80:395–406.

Libich DS, Harauz G. 2002b. Interactions of the 18.5 kDa isoform ofmyelin basic protein with Ca2�-calmodulin: in vitro studies using gel shiftassays. Mol Cell Biochem 241:45–52.

Lintner RN, Dyer CA. 2000. Redistribution of cholesterol in oligoden-drocyte membrane sheets after activation of distinct signal transductionpathways. J Neurosci Res 60:437–449.

Mermelstein PG, Deisseroth K, Dasgupta N, Isaksen AL, Tsien RW. 2001.Calmodulin priming: nuclear translocation of a calmodulin complex andthe memory of prior neuronal activity. Proc Natl Acad Sci USA 98:15342–15347.

Nowak MW, Berman HA. 1991. Fluorescence studies on the interactionsof myelin basic protein in electrolyte solutions. Biochemistry 30:7642–7651.

Pedraza L, Fidler L, Staugaitis SM, Colman DR. 1997. The active transportof myelin basic protein into the nucleus suggests a regulatory role inmyelination. Neuron 18:579–589.

Polverini E, Fasano A, Zito F, Riccio P, Cavatorta P. 1999. Conformationof bovine myelin basic protein purified with bound lipids. Eur Biophys J28:351–355.

Pribyl TM, Campagnoni CW, Kampf K, Kashima T, Handley VW, Mc-Mahon J, Campagnoni AT. 1993. The human myelin basic protein geneis included within a 179-kilobase transcription unit: expression in theimmune and central nervous systems. Proc Natl Acad Sci USA 90:10695–10699.

Reyes SD, Campagnoni AT. 2002. Two separate domains in the Gollimyelin basic proteins are responsible for nuclear targeting and processextension in transfected cells. J Neurosci Res 69:587–596.

Schleiff E, Schmitz A, McIlhinney RA, Manenti S, Vergeres G.1996. Myristoylation does not modulate the properties of MARCKS-related protein (MRP) in solution. J Biol Chem 271:26794–26802.

Shoemaker BA, Portman JJ, Wolynes PG. 2000. Speeding molecular rec-ognition by using the folding funnel: the fly-casting mechanism. ProcNatl Acad Sci USA 97:8868–8873.

Staugaitis SM, Colman DR, Pedraza L. 1996. Membrane adhesion andother functions for the myelin basic proteins. Bioessays 18:13–18.

Walker AG, Rumsby MG. 1985. The induction of liposome aggregation bymyelin basic protein. Neurochem Int 7:441–447.

Wood DD, Moscarello MA. 1997. Molecular biology of the glia: compo-nents of myelin-myelin basic protein—the implication of posttranslationalchanges for demyelinating disease. In: Russell WC, editor. Molecularbiology of multiple sclerosis. Chichester: John Wiley & Sons. p 37–54.

Yap KL, Kim J, Truong K, Sherman M, Yuan T, Ikura M. 2000. Cal-modulin target database. J Struct Funct Genom 1:8–14.

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