Analytical Biochemistry 352 (2006) 299–301

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ANALYTICALBIOCHEMISTRY

Notes & Tips

A nondetergent sulfobetaine prevents protein aggregation in microcalorimetric studies

Tony Collins 1, Salvino D’Amico 1, Daphné Georlette, Jean Claude Marx, Adrienne L. Huston, Georges Feller ¤

Laboratory of Biochemistry, University of Liège, Institute of Chemistry B6a, B-4000 Liège-Sart Tilman, Belgium

Received 2 December 2005Available online 8 February 2006

DiVerential scanning calorimetry (DSC)2 is widely usedto study protein stability and has allowed an in-depth ther-modynamic analysis of heat-induced unfolding that has sig-niWcantly contributed to the elucidation of proteinenergetics [1–4]. However, such studies were generallyrestricted to small proteins that undergo two-state revers-ible unfolding, whereas most intracellular and extracellularproteins are large and frequently multidomain polypep-tides. The stability of large proteins diVers signiWcantly bythe level of enthalpic stabilization, the cooperativity ofunfolding, or the occurrence of multiple stability domains[5,6]. As a rule, these large proteins are not suitable formicrocalorimetric investigations as a result of their strongaggregation tendency that distorts the thermograms andprecludes any reliable calculations (Fig. 1). Having beeninvolved in stability studies of large proteins for many years[7–9], we have explored numerous experimental conditionsto avoid heat-induced aggregation in DSC. Here we reportthe use of 3-(1-pyridinio)-1-propanesulfonate (NDSB), anondetergent sulfobetaine, as an eYcient compound to pre-vent protein aggregation.

The following proteins were produced and puriWedessentially as described: �-amylase [10] and xylanases [7]from Pseudoalteromonas haloplanktis, cellulase from Clos-tridium thermocellum[7], DNA ligases from P. haloplanktis,Escherichia coli, and Thermus scotoductus[8], aminopepti-dase from Colwellia psychrerythraea[11], and phosphoglyc-erate kinase from Pseudomonas sp. [12]. The following

* Corresponding author. Fax: +32 4 366 33 64.E-mail address: gfeller@ulg.ac.be (G. Feller).

1 Both authors contributed equally to this work.2 Abbreviations used: NDSB, 3-(1-pyridinio)-1-propanesulfonate; DSC,

diVerential scanning calorimetry; Mops, 3-(N-morpholino)propanesulfon-ic acid.

0003-2697/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2006.01.035

proteins were kindly provided by collaborators: xylanaseXyl1 from Streptomyces sp. S38 (J. Georis, University ofLiège), beta-lactamase L1 from Stenotrophomonas malto-philia (N. Rhazi, University of Liège), cellulases from P.haloplanktis and Erwinia chrysanthemi (G. Sonan, Univer-sity of Liège), human leukotriene A4 hydrolase (J. Haegg-strom, Karolinska Institutet Stockholm), alcoholdehydrogenase from Moraxella sp. (J. Tsigos, University ofCrete), and glutamate dehydrogenases from Psychrobactersp. and from the iceWsh Chaenocephalus aceratus (M.A.Ciardiello, CNR Naples). Pig pancreatic �-amylase wasfrom Roche and Bacillus amyloliquefaciens �-amylase,bovine glutamate dehydrogenase, yeast alcohol dehydroge-nase, and phosphoglycerate kinase were from Sigma.Microcalorimetric measurements were performed usingeither a MicroCal MCS-DSC instrument or a MicroCalVP-DSC calorimeter as detailed [10]. Samples (3–6 mg/ml)were dialyzed overnight against 30 mM 3-(N-morpho-lino)propanesulfonic acid (Mops), pH 7.5. Stock solutionsof 2–3 M NDSB (Fluka) were prepared in the dialysisbuVer (after dialysis) and the pH was adjusted. NDSB wasadded to the protein samples at 0.5–1 M Wnal concentrationand a buVer sample was prepared under identical condi-tions. It was essential that the NDSB concentration in thereference cell matches that in the sample cell. To avoid sol-vent mismatch, the sample can be further dialyzed to equi-librium against a small volume of the NDSB-containingbuVer. Several baselines were recorded until a Xat thermo-gram was obtained and the sample cell was Wlled with thedegassed protein–NDSB solution. Thermograms were ana-lyzed according to a non-two-state model in which themelting point Tm, the calorimetric enthalpy �Hcal, and thevan’t HoV enthalpy �HvH of individual transitions areWtted independently using the MicroCal Origin softwareversion 2.9 (MCS-DSC) or version 7.0 (VP-DSC). Protein

300 Notes & Tips / Anal. Biochem. 352 (2006) 299–301

concentrations were determined by the BCA Protein Assay(Pierce) or by absorbance at 280 nm on aliquots withdrawnbefore NDSB addition.

Preliminary trials have shown that the choice of thebuVer is important for DSC experiments. Indeed, lowerprotein aggregation occurs with Mops than with inorganic(e.g., phosphate), organic (e.g., citrate), or other Good’sbuVers. On the other hand, high concentrations of NDSBhave been shown to improve the solubility of various pro-teins [13,14] and this prompted us to check the eVect of thiscompound in DSC. As illustrated in Fig. 1, addition ofNDSB delays or prevents protein aggregation in the micro-calorimeter cell, allowing recording of a stable posttransi-tion baseline and analysis of the heat-absorption peak. Abaseline can then be calculated under the transition,thereby giving access to the calorimetric enthalpy �Hcal(area of the transition), the van’t HoV enthalpy �HvH (cal-culated from the slope of the transition), the melting point

Fig. 1. (Top) Raw thermograms illustrating the eVect of NDSB in a typicalDSC experiment. Bottom curve: xylanase from Pseudoalteromonas halo-planktis in Mops buVer unfolds around 54 °C and aggregates, as indicatedby the downward drift of the microcalorimetric signal. Top curves:increasing concentrations of NDSB delay aggregation to higher tempera-tures, hence allowing full analysis of the unfolding transition. Inset: analy-sis of the thermogram recorded with 1 M NDSB. (Bottom) Dissociationof protein aggregates by NDSB in DSC samples. Top thermogram: pigpancreatic �-amylase stored in ammonium sulfate, extensively dialyzedagainst the buVer and centrifuged still displays a slight transition (arrow)arising from heat-induced disruption of protein aggregates. Bottom ther-mogram: addition of 0.5 M NDSB dissociates these aggregates, allowingaccurate analysis of the DSC transition.

Tm (top of the transition), and a rough estimate of thediVerence in heat capacity �Cp (Fig. 1, inset; reliable deter-mination of �Cp requires additional experiments) that arethe basic parameters for further thermodynamic analysis. Itshould be noted that, if aggregation takes place, the endo-thermic unfolding and exothermic aggregation and precipi-tation occur simultaneously, therefore lowering theapparent melting point Tm (Fig. 1). Addition of NDSBdelays this eVect and provides a more accurate determina-tion of this parameter. This protective eVect of NDSB wasobserved with 21 diVerent proteins, including �-amylases(bacterial, porcine), xylanases (bacterial), cellulases (bacte-rial), DNA-ligases (bacterial), aminopeptidases (bacterial,human), beta-lactamase (bacterial), alcohol dehydrogen-ases (bacterial, yeast) glutamate dehydrogenases (bacterial,Wsh, bovine), and phosphoglycerate kinases (bacterial,yeast), and with mutants and isolated domains of some ofthese proteins (data not shown). This indicates that the pro-tective eVect of NDSB is independent of the protein size,fold, or origin. In addition, NDSB was found to be eVectiveover a wide range of temperatures accessible to high-sensi-tivity microcalorimeters as shown by the absence of aggre-gation of proteins with melting temperatures as low as35 °C or as high as 100 °C [8].

Commercial enzymes are generally sold as suspensionsof ammonium sulfate precipitates for storage purposes.However, even after prolonged dialysis and centrifugation,a small fraction of protein frequently remains in the formof light aggregates inducing a discrete but nonnegligibletransition centered around 45 °C (Fig. 1, bottom). We havefound with four diVerent commercial enzymes that additionof 0.5 M NDSB abolishes this transition, presumably bydissociation of these aggregates.

To check the possible interference of high NDSB con-centrations on the microcalorimetric parameters, a bacte-rial �-amylase [10] that unfolds reversibly (and therefore isnot prone to aggregation) was assayed in the absence andpresence of 0.5 M NDSB. It was found that neither themelting point Tm, the calorimetric enthalpy (�Hcal), thevan’t HoV enthalpy (�HvH), nor the reversibility wereaVected by NDSB. Furthermore, a bacterial cellulaseunfolding irreversibly in Mops buVer was found to fullyrefold upon cooling in the presence of 0.5 M NDSB asshown by an identical second up-scan (data not shown).

The possible interference of high NDSB concentrationswith ligand binding was also investigated. This was checkedusing a bacterial phosphoglycerate kinase that adopts anopen conformation in the unbound state and a closed con-formation in the presence of 5 mM 3-phosphoglycerate andMg+2-ADP as ligands, resulting in drastically distinct DSCthermograms [12]. These experiments have been repeated inthe presence of 0.5 M NDSB without alteration of the typi-cal microcalorimetric proWles. In addition, the isolated C-terminal domain produced by gene truncation and contain-ing the nucleotide-binding site was found to bind 5 mMMg+2-ADP with or without 0.5 M NDSB but was prone tostrong aggregation before completion of the unfolding

Notes & Tips / Anal. Biochem. 352 (2006) 299–301 301

transition in the absence of the sulfobetaine (data notshown).

Finally, the recorded thermodynamic parameters haveto be normalized in molar units and therefore an accuratedetermination of the protein concentration is required.NDSB possesses a high absorbance at 280 nm (�» 2.7 M¡1

cm¡1) and this precludes reliable protein determination bythe UV method. By contrast, no interference was observedwith methods based on Coomassie blue or bicinchoninicacid. Nevertheless, protein concentrations were routinelydetermined in this work on aliquots withdrawn beforeNDSB addition.

NDSB at concentrations between 0.5 and 1 M was foundto protect several large proteins against heat-inducedaggregation without altering the unfolding pattern, thethermodynamic parameters, or the ligand binding proper-ties. Aggregation is either abolished or, at least, delayed tohigher temperatures, allowing recording of the requiredposttransition baseline. These Wndings open the prospect ofanalyzing proteins that were previously considered unsuit-able for DSC experiments and considerably increase thenumber of proteins amenable to microcalorimetric studiesfor thermodynamic (reversible unfolding) or kinetic (irre-versible unfolding) approaches. Comparison of the struc-tures of Mops and NDSB (Fig. 2) reveals strikingsimilarities that can help to elucidate their protective eVectagainst protein aggregation. It is commonly believed thatprotein aggregation arises from the solvent exposure ofburied hydrophobic groups in the unfolded state followedby the entropy-driven, nonspeciWc association of thesegroups, mainly at elevated temperatures that favor thehydrophobic eVect [15]. In this respect, the propyl aliphaticchain and the 4- (Mops) or 5- (NDSB) carbon rings canshield the nonspeciWc interactions between protein hydro-phobic groups, especially at the high NDSB concentrationsused. Interestingly, it has been suggested that NDSB canadopt a cyclic conformation by formation of an ion pairbetween the two opposite charges [13]. This will result in anadditional six-atom ring that can further improve thehydrophobic shield. On the other hand, one cannot neglectthe high charge density provided by NDSB that can inducea charge-screening eVect between the polar groups in theunfolded polypeptides. One can therefore propose that the

Fig. 2. Structures of the nondetergent sulfobetaine 3-(1-pyridinio)-1-pro-panesulfonate (NDSB) and of the buVer 3-(N-morpholino)propanesul-fonic acid (Mops).

occurrence of chemical functions responsible for bothproperties (hydrophobic shield and charge-screeningeVects) within a single molecule is the main determinant ofthe NDSB eYciency in preventing heat-induced proteinaggregation.

Acknowledgments

We thank M.A. Ciardiello, J. Tsigos, J. Georis, J. Haegg-strom, N. Rhazi, and G. Sonan for providing protein sam-ples. We also thank N. Gerardin-Otthiers, R. Marchand,and A. Dernier for their skillful technical assistance. Thiswork was supported by the Fonds National de la Recher-che ScientiWque, Belgium (Grants FRFC 2.4515.00 and2.4536.04). S.D’A. is a FNRS postdoctoral researcher.

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