Eur. J. Biochem. 264, 479±487 (1999) q FEBS 1999

Stability, refolding and Ca2+ binding of pullulanase from thehyperthermophilic archaeon Pyrococcus woesei

Ruth M. Schwerdtfeger1, Roberta Chiaraluce2, Valerio Consalvi2, Roberto Scandurra2 and Garabed Antranikian1

1Technical University Hamburg-Harburg, Institute of Biotechnology, Department of Technical Microbiology, Hamburg, Germany;2Dipartimento di Scienze Biochimiche `A. Rossi Fanelli', UniversitaÁ `La Sapienza', Roma, Italy

The unfolding and refolding of the extremely heat-stable pullulanase from Pyrococcus woesei has been

investigated using guanidinium chloride as denaturant. The monomeric enzyme (90 kDa) was found to be very

resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was

determined to be 4.86 ^ 0.29 m for intrinsic fluorescence and 4.90 ^ 0.31 m for far-UV CD changes. The

unfolding process was reversible. Reactivation of the completely denatured enzyme (in 7.8 m guanidinium

chloride) was obtained upon removal of the denaturant by stepwise dilution; 100% reactivation was observed

when refolding was carried out via a guanidinium chloride concentration of 4 m in the first dilution step.

Particular attention has been paid to the role of Ca2+ which activates and stabilizes this archaeal pullulanase

against thermal inactivation. The enzyme binds two Ca2+ ions with a Kd of 0.080 ^ 0.010 mm and a Hill

coefficient H of 1.00 ^ 0.10. This cation enhances significantly the stability of the pullulanase against

guanidinium chloride-induced unfolding and the DGH2OD increased from 6.83 ^ 0.43 to 8.42 ^ 0.55 kcal´mol21.

The refolding of the pullulanase, on the other hand, was not affected by Ca2+.

Keywords: pullulanase; archaea; thermostable proteins; stability; calcium binding.

A number of thermophilic, extreme thermophilic andhyperthermophilic microorganisms belonging to bacteria andarchaea have been found to be capable of degrading starchand other biopolymers such as hemicellulose, cellulose andproteins [1,2]. In addition to the industrial relevance of heat-stable amylolytic enzymes, the existence of these enzymes inall kingdoms of life provides the possibility of comparativestudies on (thermo)stability and folding of proteins frommesophilic and thermophilic microorganisms.

Pyrococcus woesei, a hyperthermophilic archaeon of theorder Thermococcales grows optimally at 100±103 8C on acomplex medium under strictly anaerobic conditions [3]. Thisarchaeon is equipped with a polysaccharide-degrading enzymesystem which is composed of an a-amylase [4], a pullulanase[5] and an a-glucosidase [6].

The pullulanase from P. woesei belongs to pullulanase oftype II (also named amylopullulanase) and it hydrolyzes thea-1,6 glycosidic linkages in pullulan and branched oligosac-charides as well as a-1,4 glycosidic linkages in linear andbranched polysaccharides such as amylose and amylopectin.Many pullulanases of type II have been purified and character-ized from anaerobic thermophilic bacteria and archaea [7±14].On the other hand, type I pullulanases which attack specificallya-1,6 glycosidic linkages have been generally found in

mesophilic, aerobic microorganisms [1,2,15]. Only recently aheat-stable pullulanase of type I has been detected in the newlyisolated thermophilic anaerobic bacterium Fervidobacteriumpennavorans Ven5 [16].

The pullulanase from P. woesei is a monomeric enzyme witha molecular mass of 90 kDa. Its gene has been cloned andexpressed in Escherichia coli. The enzyme is optimally activeat 100 8C and pH 6.0 and is activated by the addition of Ca2+

which also increases its thermal stability. In the presence of1 mm Ca2+ the half-life of the pullulanase for thermalinactivation at 110 8C increased from 7 to 20 min [5]. Enzymeactivation and stabilization by the addition of Ca2+ was alsoreported for the pullulanases from Pyrococcus furiosus andThermococcus litoralis [7], whereas for a-amylases fromP. furiosus [17] and P. woesei [4] no effect of Ca2+ wasobserved.

In this paper the guanidinium chloride (GdmCl)-inducedreversible denaturation of pullulanase from P. woesei wasstudied by fluorescence and far-UV CD spectroscopy and byactivity measurements. Appropriate conditions for the reacti-vation of the heat-stable enzyme upon GdmCl-inducedunfolding have been found. The striking effect of Ca2+ onthermostability and activity led us to investigate the physico-chemical properties of Ca2+ binding by fluorescence quenchingstudies and its influence on the protein structure in solution.

M A T E R I A L S A N D M E T H O D S

Enzyme preparation and assay

E. coli PL2118 cells expressing recombinant P. woesei pull-ulanase were grown aerobically on tryptone and yeast extract aspreviously described [5]. The recombinant pullulanase waspurified as reported by RuÈdiger et al. [5]. The proteinconcentration was determined by the Folin phenol method

Correspondence to G. Antranikian, Technical University

Hamburg-Harburg, Institute of Biotechnology, Department of Technical

Microbiology, Denickestr. 15, 21071 Hamburg, Germany.

Fax: + 49 40 42878 2909, Tel: + 49 40 42878 3117,

E-mail: Antranikian@tu-harburg.de

Abbreviations: ANS, 8-anilinonaphthalene-1-sulfonic acid ammonium salt;

GdmCl, guanidinium chloride; SVD, singular value decomposition.

Enzymes: pullulanase (EC 3.2.1.41).

(Received 29 April 1999; accepted 11 June 1999)

480 R. M. Schwerdtfeger et al. (Eur. J. Biochem. 264) q FEBS 1999

with bovine serum albumin as standard [18] and at 280 nmusing 1280 = 206 390 m21´cm21 calculated according to Gilland von Hippel [19]. Pullulanase activity was determined bymeasuring the amount of reducing sugars released uponenzyme incubation in the presence of pullulan according toBergmeyer and Grassl [20]. The enzyme sample (0.2±0.9 mg)was incubated in 100 mL of 25 mm Tris/HCl buffer, pH 7.4,containing 0.5% (w/v) pullulan at 908C for 10 min. One unit ofpullulanase catalyzes the formation of 1 mmol of reducingsugars per min under the above defined conditions; maltose wasused as a standard.

Spectroscopic techniques

Intrinsic fluorescence emission and light scattering measure-ments were carried out with a LS50B Perkin Elmer spectro-fluorimeter using a 1-cm pathlength quartz cuvette.Fluorescence emission spectra were recorded at 300±400 nm(1 nm sampling interval) at 208C with the excitation wave-length set at 290 nm. Light scattering was measured at 208Cwith both excitation and emission wavelengths set at 480 nm.

CD spectra were recorded on a Jasco J-720 spectro-polarimeter. Far-UV CD measurements (200±250 nm) wereperformed at 208C in a 0.5-cm pathlength quartz cuvette at anenzyme concentration of 35 mg´mL21. The results wereexpressed as the mean residue ellipticity ([Q]) assuming amean residue mass of 110 per amino acid residue.

Experiments with the fluorescent dye 8-anilinonaphthalene-1-sulfonic acid ammonium salt (ANS) were performed at 208Cby incubating the protein and ANS at a 1 : 180 molar ratio.After 5 min, fluorescence emission spectra were recorded at400±600 nm with the excitation wavelength set at 390 nm. Themaximum fluorescence emission wavelength and intensity ofthe hydrophobic probe (ANS) depend on the environmentalpolarity, e.g. on the hydrophobicity of the accessible surface ofthe protein [21].

Unfolding and refolding studies

For equilibrium transition studies the enzyme (final concen-tration 35 mg´mL21) was incubated at 208C at increasingconcentrations of GdmCl (0±8 m) in 25 mm Tris/HCl, pH 7.4,and 100 mm EGTA, with and without 120 mm CaCl2. After20 h (in the absence and the presence of calcium) and 48 h (inthe presence of calcium) intrinsic fluorescence emission andfar-UV CD spectra were recorded in parallel at 208C. Residualcatalytic activity was measured with the standard activity assaywhich resulted in a sixfold dilution of the unfolding mixture inthe activity assay solution. To test the reversibility of theunfolding, pullulanase was unfolded at 208C in 7.8 m GdmClat 0.4 mg´mL21 protein concentration in 25 mm Tris/HCl,pH 7.4, and 100 mm EGTA, with and without 120 mm CaCl2.After 20 h (in the absence of calcium) and 48 h (in the presenceof calcium), refolding was started by 12-fold dilution of theunfolding mixture at 208C into solutions of the same bufferused for unfolding containing decreasing GdmCl concen-trations. The final enzyme concentration was 33 mg´mL21.After 4 h and 24 h intrinsic fluorescence emission and far-UVCD spectra were recorded at 208C.

For unfolding kinetic studies the pullulanase was manuallymixed at 208C with 8 m GdmCl in 25 mm Tris/HCl, pH 7.4,and 100 mm EGTA, with and without 120 mm CaCl2. The finalGdmCl concentration was 7.8 m and the enzyme concentrationwas 20 mg´mL21. The decrease in intrinsic fluorescenceemission at 340 nm (excitation at 290 nm) was measured

every 2.5 s at 208C under continuous stirring. In identicalexperiments the unfolding process was followed by recordingemission spectra at 300±400 nm (excitation at 290 nm) at 208Cwith 1 min intervals.

Renaturation of the pullulanase was initiated by dilution ofthe enzyme unfolded in 7.8 m GdmCl, 25 mm Tris/HCl,pH 7.4, and 100 mm EGTA, with and without 120 mm CaCl2at 208C into the same buffer without the denaturant. Thereactivation of the pullulanase was achieved by dialysis usingCentricon-30 tubes (Amicon). Therefore the unfolding mixture(30±1000 mL) was diluted with 25 mm Tris/HCl, pH 7.4, and100 mm EGTA (^ 120 mm CaCl2) up to 2 mL directly in thetubes, concentrated to a volume of 0.1 mL by ultrafiltration at158C and again diluted up to 2 mL. This procedure (i.e. 20-folddilution followed by ultrafiltration) was repeated several timesuntil complete removal of GdmCl was achieved. The renatura-tion process during dialysis was followed by monitoringintrinsic fluorescence emission, far-UV CD and residualcatalytic activity 10 min after each dilution/concentration step.

Data analysis

The changes in intrinsic fluorescence emission spectra atincreasing GdmCl concentrations were quantified as thefluorescence intensity at 340 nm and as the intensity-averagedemission wavelength, l [22], calculated according to thefollowing equation

l �P�Iili�P�Ii� �1�

where l i and Ii are the emission wavelength and itscorresponding fluorescence intensity at that wavelength.

Far-UV CD spectra from GdmCl titration were analyzed bythe singular value decomposition algorithm (SVD) [23,24]using the software matlab (MathWorks, South Natick, MA,USA). SVD is useful to find the number of independentcomponents in a set of spectra and to remove the high-frequency noise and the low-frequency random error. CDspectra in the 210±250 nm region (0.2 nm sampling interval)were placed in a rectangular matrix A of n columns, onecolumn for each spectrum collected in the titration. The Amatrix is decomposed by SVD into the product of threematrices: A = U*S*VT where U and V are orthogonal matricesand S is a diagonal matrix. The columns of U matrix contain thebasis spectra and the columns of the V matrix contain thedenaturant dependence of each basis spectrum. Both U and Vcolumns are arranged in terms of their decreasing order of therelative weight of information, as indicated by the magnitude ofthe singular values in S. The diagonal S matrix contains thesingular values which quantify the relative importance of eachvector in U and V. An important feature of SVD analysis is thatthe signal-to-noise ratio is very high in the earliest columns ofU and V and the random noise is mainly accumulated in thelatest U and V columns. The wavelength-averaged spectralchanges induced by increasing denaturant concentrations arerepresented by the columns of matrix V. Therefore the plot ofthe columns of V vs the denaturant concentration providesinformation about the observed transition.

GdmCl-induced equilibrium unfolding was analyzed byfitting baseline and transition region data to a two-state linearextrapolation model [25] according to the following equation:

DGunfolding � DGH2O � mg�GdmCl� � ± RTlnKunfolding �2�where DGunfolding is the free energy change for unfolding for agiven denaturant concentration, DGH2O is the free energy

q FEBS 1999 P. woesei pullulanase: stability and Ca2+ binding (Eur. J. Biochem. 264) 481

change for unfolding in the absence of denaturant and mg is aslope term which quantitates the change in DGunfolding per unitconcentration of denaturant, R is the gas constant, T is thetemperature and Kunfolding is the equilibrium constant forunfolding. The model expresses the signal as a function ofdenaturant concentration:

yi �yN � mN�X�i � �yD � mD�X�i� � e��2DGH2O2mg�X�i�=RT�

1� e�2DGH2O2mg�X�i=RT� �3�

where yi is the observed signal, yN and yD are the native anddenatured baseline intercepts, mN and mD are the native anddenatured baseline slopes, [X]i is the denaturant concentrationafter the ith addition, DGH2O is the extrapolated free energy ofunfolding in the absence of denaturant, mg is the slope of aDGunfolding vs [X] plot, R is the gas constant and T is thetemperature. The [GdmCl]0.5 is the denaturant concentration atthe midpoint of the transition and, according to Eqn (2), iscalculated as:

�GdmCl�0:5 � DGH2O

mg

�4�

Calcium binding experiments

The affinity of the native pullulanase for calcium ions wasdetermined by following the changes in fluorescence emissionat 208C upon Ca2+ addition to the Ca2+-free enzyme solution.The enzyme was considered to be Ca2+-free when the additionof EGTA to an enzyme preparation that was extensivelydialysed against 25 mm Tris/HCl, pH 7.4, containing 100 mmEGTA, did not cause further changes in protein fluorescenceemission spectra. Aliquots (2.5±5.0 mm) of CaCl2 were addedunder continuous stirring to a fluorescence cuvette containingCa2+-free pullulanase in 25 mm Tris/HCl, pH 7.4, 100 mmEGTA. Pullulanase concentration was varied over the concen-tration range 20±100 nm. After each Ca2+ addition, fluores-cence emission spectra were recorded at 300±400 nm with theexcitation wavelength set at 290 nm. The excitation andemission slit were set at 5 nm. The concentration of unchelatedCa2+ in the solution was calculated by using the programmaxchelator (v 6.95) [26]. The quenching of the proteinintrinsic fluorescence emission intensity at 340 nm uponincreasing calcium concentration was expressed as FB = (F±F0)/(F1±F0), where F is the observed fluorescence signal at 340 nmat each titration step, F0 the fluorescence of the Ca2+-freeenzyme and F1 the fluorescence at the fully liganded state ofpullulanase. The concentration of free Ca2� was determinedaccording to: [free Ca2+] = [unchelated Ca2+] ± FB � [pullu-lanase] � 2, following the determination of a bindingstoichiometry of 2 mol of Ca2+ bound per mole ofpullulanase (see Results). Data pairs of FB vs free calciumconcentration were analyzed by nonlinear regression to thefollowing equation:

FB � FBmax��free Ca2���HKH

d � ��free Ca2���H �5�

where FB max is the maximal signal change, H is the Hillcoefficient and Kd is the ligand dissociation constant.

Acrylamide quenching

Fluorescence quenching was carried out by adding increasingamounts of acrylamide (0±160 mm) to a pullulanase solution(23 mg´mL21) in 25 mm Tris/HCl, pH 7.4, containing 100 mm

EGTA and 120 mm Ca2+ (if indicated). Emission spectra(300±400 nm) were recorded at 208C 10 min after eachacrylamide addition with the excitation wavelength set at290 nm. The effective quenching constants were obtained frommodified Stern±Vollmer plots by analyzing F0/DF vs 1/[acryl-amide] (20 data points) [27].

Ultracentrifugation analysis

Sedimentation velocity experiments were carried out in aBeckman Optima XL-A analytical ultracentrifuge at 100 000 gand 208C. The movement of the protein toward the bottom ofthe cell was determined by absorption scans along thecentrifugation radius at a wavelength of 280 nm. Proteinconcentration was 0.1 mg´mL21. Sedimentation coefficientswere calculated with the software provided by Beckman.

Chemicals and buffers

GdmCl, ANS and EGTA were from Fluka. Pullulan, 3 0,5 0-dinitrosalicylic acid (DNS) and CaCl2 (0.02 mol´L21 solution)were purchased from Sigma. Buffer solutions were filtered(0.22 mm) and carefully degassed. All buffers and solutionswere prepared with ultra-high quality water (ELGA UHQ, UK).

R E S U L T S

Unfolding and refolding

The equilibrium unfolding of the pullulanase from P. woeseiwas monitored by following changes in the intrinsic fluores-cence emission and far-UV CD. In the native state pullulanaseexhibits a fluorescence emission spectrum with a maximum at340 nm when excited at 290 nm (data not shown). The far-UVCD spectrum shows a local minimum at 222 nm, typical for aprotein with a significant content of helical structure (data notshown).

Incubation of pullulanase at increasing GdmCl concen-trations (0±8 m) in 25 mm Tris/HCl, pH 7.4, containing100 mm EGTA for 20 h at 208C resulted in a progressivedecrease in intrinsic fluorescence emission intensity (Fig. 1A).At GdmCl concentrations higher than 6.5 m, only 50% of theintrinsic fluorescence emission intensity of the native enzymewas measured and the maximum fluorescence emissionwavelength was shifted to 355 nm. The red-shift of the intrinsicfluorescence emission was determined by calculation of theintensity-averaged emission wavelength, l, according toEqn (1). This quantity is an integral measurement, negligiblyinfluenced by the noise, which reflects changes in the shape andposition of the emission spectrum. The plots of the fluorescencechanges as a function of the GdmCl concentration showed asigmoidal denaturant dependence, with transition midpoints at4.86 ^ 0.31 m for the fluorescence emission intensity at340 nm and at 4.86 ^ 0.29 m for l, (Fig. 1A). Far-UV CDspectra changed at the increase of GdmCl concentration, andabove 6.5 m GdmCl less than 2% of the CD signal of the nativeenzyme was measurable, indicating a complete loss of thesecondary structure. The GdmCl-induced changes of the CDspectra were analyzed by SVD which indicated that only twospectral components contribute to the CD spectra. The mostsignificant singular values were 395.7, 101.3 and 42.2. All theother singular values were below 10% of the largest singularvalue and progressively decreased approaching to zero. A plotof the first column of the V matrix (V1) as a function of GdmClconcentration shows a sigmoidal profile, with a transition

482 R. M. Schwerdtfeger et al. (Eur. J. Biochem. 264) q FEBS 1999

midpoint at 4.90 ^ 0.31 m (Fig. 1B). The same plot of thesecond column of V (V2) approximately showed a sigmoidaldependence on GdmCl, shifted to a lower denaturant concen-tration (data not shown). However, a quantitative fitting of theV2 data has not been possible to perform due to the scattereddata in the transition region and to the noisy post-transitionbaseline. The third column of V (V3) randomly changed inmagnitude and sign as a function of the GdmCl concentration(data not shown). The sigmoidal denaturation curves shown inFig. 1B indicate that the loss of structure of the first spectralcomponent (V1) follows a two-state mechanism from the nativeto the unfolded state without any detectable intermediates.

Table 1. Analysis of GdmCl-induced unfolding equilibria of pull-

ulanase in the absence and presence of Ca2+. Data are reported ^ curve

fitting standard error. Far-UV CD data refer to the transition of the molar

ellipticity at 222 nm from the raw spectra (Q222) or to the molar ellipticity

at the same wavelength reconstructed using the first SVD component (SVD

Q222) or to the transition of the first column of V matrix (V1), as described

in the text. FI340 is the intrinsic fluorescence emission intensity at 340 nm.

l is the intensity-averaged emission wavelength calculated according to

Eqn (1). DGH2OD and mg values were obtained by nonlinear regression

analysis to Eqn (3). [GdmCl]0.5 is the transition midpoint corresponding to

the GdmCl concentration at which 50% of the protein is unfolded.

[Ca 2+]

(mm)

DGH2OD

(kcal´mol21)

mg

(kcal´mol21´m21)

[GdmCl]0.5

(m)

Q222 0 6.045 �^ 0.651 1.238 �^ 0.133 4.88 �^ 0.53

SVD Q222 0 6.309 �^ 0.339 1.300 �^ 0.070 4.85 �^ 0.26

V1 0 6.834 �^ 0.432 1.393 �^ 0.088 4.90 �^ 0.31

FI340 0 6.286 �^ 0.401 1.294 �^ 0.083 4.86 �^ 0.31

l 0 7.051 �^ 0.421 1.450 �^ 0.086 4.86 �^ 0.29

Q222 120 8.457 �^ 1.099 1.684 �^ 0.219 5.02 �^ 0.65

SVD Q222 120 8.950 �^ 0.537 1.775 �^ 0.107 5.04 �^ 0.30

V1 120 8.420 �^ 0.551 1.670 �^ 0.109 5.04 �^ 0.33

FI340 120 8.583 �^ 0.553 1.695 �^ 0.105 5.06 �^ 0.31

l 120 8.687 �^ 0.545 1.705 �^ 0.107 5.10 �^ 0.32

Fig. 1. Effect of Ca2+ on GdmCl-induced unfolding/refolding equilibria

of P. woesei pullulanase. Fluorescence and far-UV CD spectra were

recorded at the indicated GdmCl concentrations at 35 mg´mL21 enzyme

concentration and 208C in the absence (solid symbols) and in the presence

(open symbols) of 120 mm Ca2+. (A) Fluorescence changes are reported as

fluorescence intensity at 340 nm (X, W, O, K, solid lines) and intensity-

averaged emission wavelength (l) (X, W, O, K, dashed lines) in the

presence (open symbols) and in the absence (solid symbols) of Ca2+. l was

obtained by Eqn (1). Solid and dashed lines result from nonlinear regression

to Eqn (3) (see Materials and methods). Reversibility points (O, K) were

not included in the nonlinear regression analysis. (B) Far-UV CD changes

in the presence (A) and in the absence (B) of Ca2+ are reported as the first

column of the V matrix (V1). V1 was obtained by SVD of the far-UV CD

spectral data as described in the text. Solid lines result from nonlinear

regression to Eqn (3) (see Materials and methods). (C) Molar ellipticity

changes at 222 nm in the presence (open symbols, left axis) and absence

(solid symbols, right axis) of Ca2+ are reported from the raw far-UV CD

spectra (V, S, dashed lines) and from the spectra reconstructed using the

principal SVD component (B, A, solid lines). Solid and dashed lines result

from nonlinear regression to Eqn (3) (see Materials and methods).

Reversibility points are reported as the molar ellipticity changes at

222 nm from the raw far-UV CD spectra (P, L) and were not included

in the nonlinear regression analysis.

q FEBS 1999 P. woesei pullulanase: stability and Ca2+ binding (Eur. J. Biochem. 264) 483

Incubation of the pullulanase at increasing concentrations ofGdmCl in the presence of 120 mm Ca2+ caused a progressivechange of the fluorescence properties and of the dichroicactivity with the same amplitudes observed for the unfolding inthe absence of the cation (Fig. 1A±C). Above 6.5 m GdmCl, ared-shift of the maximum fluorescence emission wavelength to355 nm was also observed. In the presence of the cation, theunfolding transition midpoint shifted to 5.06 ^ 0.31 m and5.10 ^ 0.32 m GdmCl for the changes in the fluorescenceintensity at 340 nm and in the intensity-averaged emissionwavelength l, respectively (Fig. 1A). The changes of the far-UV CD spectra upon the increase of the GdmCl concentrationin the presence of Ca2+ were analyzed by SVD. The threelargest singular values were 454.59, 98.09 and 63.91. Two mainspectral components contributed significantly. Figure 1B showsthe sigmoidal dependence of the first column of V (V1) onGdmCl concentration in the presence of Ca2+, yielding atransition midpoint at 5.04 ^ 0.33 m. The dependence of thesecond column of V (V2) on the denaturant was apparentlyshifted to lower GdmCl concentrations. However, a quantitativefitting was impossible due to the noisy pre-transition and post-transition baselines (data not shown). Random changes of V3

were observed at the increase of denaturant concentration. Thechanges in molar ellipticity at 222 nm at increasing denaturantconcentration were analysed on the raw far-UV CD spectra andafter reconstruction of the spectra corresponding to the firstspectral component (Fig. 1C). The unfolding process wasreversible, independently of the presence of Ca2+, as indicatedby the comparable ellipticity at 222 nm and by the identity ofthe changes in the fluorescence intensity at 340 nm and in theintensity-averaged emission wavelength, l, monitored duringthe unfolding and refolding transitions (Fig. 1A,C).

Table 1 reports the GdmCl-induced unfolding parameters ofpullulanase calculated by nonlinear regression fitting of thedata reported in Fig. 1 according to Eqns (3) and (4). TheDGH2O

D values are within the range of those expected for afolded globular protein, i.e. 5±15 kcal´mol21 [28]. Theunfolding parameters obtained from fluorescence and far-UVCD changes are comparable and the standard error of fittingafter SVD analysis of CD spectra is significantly decreased.The presence of Ca2+ influences all the parameters byincreasing their values, independently of the spectral probeused for monitoring the unfolding.

The fluorescent probe ANS (180 m excess) was added toeach unfolding mixture at different GdmCl concentrations.Fluorescence emission spectra at 400±600 nm were recorded at208C after 5 min with the excitation wavelength set at 390 nm.The maximum fluorescence emission wavelength was at515 nm in the presence of the native enzyme and was notchanged in the presence of the enzyme incubated at variousconcentrations of GdmCl. The fluorescence emission intensityat 515 nm increased progressively with increasing GdmCl

Fig. 2. Effect of Ca2+ on the unfolding kinetics of P. woesei pullulanase.

The unfolding was followed upon manual mixing of native pullulanase to

7.8 m GdmCl in the absence and presence of 120 mm Ca2+ by recording the

decrease of intrinsic fluorescence emission at 340 nm (excitation at

290 nm) every 2.5 s (A), and the shift of the intensity-averaged emission

wavelength, l, at the indicated time intervals (B). Enzyme concentration

was 20 mg´mL21. The lower panels show the residuals (difference between

the data and the fitted function).

484 R. M. Schwerdtfeger et al. (Eur. J. Biochem. 264) q FEBS 1999

concentrations. There was no evidence for intermediate(s)between the native and the GdmCl-unfolded state (data notshown).

In addition to the structural parameters the residual catalyticactivity after incubation at various GdmCl concentrations wasdetermined after sixfold dilution of the unfolding mixture in theactivity assay. The activity was affected by much lower GdmClconcentrations than the conformational changes; at $ 0.7 mGdmCl no activity was detectable in the absence of Ca2+ whilein the presence of 120 mm Ca2+ 15% of residual activity werestill measurable at 1.4 m GdmCl.

The kinetics of pullulanase unfolding were studied bymonitoring the decrease in intrinsic fluorescence emission at340 nm at 208C as a function of time upon manual mixing ofthe native pullulanase into 8 m GdmCl. The decrease in thefluorescence emission intensity can be described as the sum oftwo first-order decay processes with half-times of 93 s and781 s, respectively. The amplitude of the first fast unfoldingprocess accounts for 60% of the overall changes (Fig. 2A). Theoverall kinetic amplitude observed in the unfolding reactionwas identical to the static amplitude measured at equilibriumbetween the native (N) and the unfolded state (D). A series ofemission spectra was also recorded in one-minute intervalsunder the same conditions. The red-shift of the intensity-averaged emission wavelength, l, can be described as a sum oftwo first-order decay processes with half-times of 140 s and960 s, respectively (Fig. 2B). These values are within a timescale similar to that of the phases of the unfolding processmonitored continuously at a single wavelength, but the first fastphase accounted for only 35% of the total change. In thepresence of 120 mm Ca2+ the unfolding kinetics were best fittedas the sum of two first-order decay processes with half-times of2574 s and 21815 s (Fig. 2A). The first fast process corres-ponded to 75% of the total amplitude. Control experiments inthe absence of GdmCl indicated that less than 10% of thesecond phase changes were due to photobleaching. Record-ing a series of emission spectra in one-minute intervalsrevealed a first-order process for the red-shift of theintensity-averaged emission wavelength, l, with a half-timeof 2738 s (Fig. 2B).

To study the regaining of enzymatic activity, refolding of thepullulanase was induced by 20-fold dilution of the denaturant(from 7.8 m to 0.39 m GdmCl) with 25 mm Tris/HCl, pH 7.4,containing 100 mm EGTA. The native-like fluorescence emis-sion intensity at 340 nm and the ellipticity at 222 nm wererecovered within the manual mixing dead time (3 s), but lessthan 30% of activity were measurable. To improve thereactivation yield the removal of GdmCl was carried out bydialysis in Centricon tubes till the denaturant concentration was, 1 mm. The protein concentration after the first dilution of theunfolding mixture was always 15 mg´mL21. Reactivation yieldsof 35±100% were observed depending on the first dilution stepof the unfolding mixture before dialysis. In particular, 67-folddilution (i.e. from 7.8 m GdmCl to 0.1 m residual GdmCl afterthe first dilution step) and subsequently thorough dialysisresulted in a reactivation yield of 35%, whereas 20-fold dilution(0.4 m residual GdmCl) enhanced the extent of reactivation to70% when dialysis was finished. Upon twofold dilution (4.0 mGdmCl) of the unfolding mixture, 100% reactivation wasachieved after removal of the denaturant by dialysis. Thereactivation yield was not changed upon incubation of therefolding mixtures at 60 and 908C after removal of GdmCl bydialysis. In any case, native-like spectral properties of theenzyme were observed and no aggregation to particles,detectable by right-angle light scattering at 480 nm, occurred

Fig. 3. Fluorescence quenching of P. woesei pullulanase with Ca2+.

Calcium was added in 2.5±5 mm-aliquots to 80 nm (W) and 30 nm (A)

pullulanase in 25 mm Tris buffer, pH 7.4, containing 100 mm EGTA. The

unchelated Ca2+ concentrations were calculated according to [26].

Fluorescence emission spectra were recorded at 208C (290 nm excitation

wavelength) after each addition of Ca2+. The quenching of the protein

intrinsic fluorescence emission intensity at 340 nm was expressed as

FB = (F ± F0)/(F1 ± F0) where F is the observed fluorescence signal at

340 nm at each titration step, F0 the fluorescence of the Ca2+-free enzyme

and F1 the fluorescence at the fully liganded state of pullulanase. (A) The

two limiting slopes intersecting at the arrow were calculated by nonlinear

regression analysis to the fluorescence quenching data at 80 nm pullulanase.

The intersection point corresponds to [Ca2+ unchelated]/[pullulanase] = 2.

The dashed lines represent the fitting of the data to a hyperbola. (B) Data at

30 nm pullulanase concentration were analyzed by nonlinear regression to

Eqn (5). [Free Ca2+] was calculated as [free Ca2+] = [unchelated Ca2+] ±

FB * [pullulanase] * 2. The fit gives a Kd of 0.080 ^ 0.010 mm, and a Hill

coefficient (H) of 1.00 ^ 0.10. The insets to A and B show the residuals

(difference between the data and the fitted function).

q FEBS 1999 P. woesei pullulanase: stability and Ca2+ binding (Eur. J. Biochem. 264) 485

at any denaturant concentration since no changes in 908scattering signals were observed.

Ca2+ does not seem to affect the refolding processsignificantly. The regain of structure upon 20-fold dilution ofthe unfolded pullulanase was not altered by the presence of120 mm Ca2+. Upon twofold dilution followed by dialysis100% reactivation was observed in the absence andpresence of Ca2+ while upon 20-fold and 67-fold dilutionin the presence of Ca2+ the reactivation yield was decreased by5±10%.

Calcium binding studies

Titration of native pullulanase with increasing amounts of Ca2+

(1.0 nm to 5.7 mm unchelated Ca2+, calculated according toBers et al. [26]) resulted in a progressive decrease influorescence intensity at 340 nm. The fluorescence intensityof the Ca2+-loaded state of pullulanase (i.e. in the presence of$ 1 mm unchelated Ca2+) was about 74% of the value of thenative, Ca2+-free state of the enzyme. No change in themaximum fluorescence emission wavelength was observed.Typical quenching curves resulting from the titration of 30 and80 nm pullulanase with Ca2+ are reported in Fig. 3A. The dataobtained at the highest protein concentration were analyzed bynonlinear regression to define two limiting slopes intersectingat a value which suggested that essentially 2 mol of Ca2+ arebound per mole of pullulanase. The hyperbolic shape of thepullulanase intrinsic fluorescence changes upon addition ofcalcium (Fig. 3A) indicates that the two binding sites displaythe same decremental signal change. The nonlinear fitting ofthe data to Eqn (5) (Fig. 3B) yielded a Hill coefficientH = 1.00 ^ 0.10, which suggests a lack of interaction betweenthe two binding sites, and a Kd value of 0.080 ^ 0.010 mMover a protein concentration range from 20 to 100 nm. Thevalues of binding stoichiometry, Kd and H did not vary morethan 10% using different enzyme preparations. Ca2+-bindingstudies in the presence of 150 mm NaCl showed a decrease inthe fluorescence emission intensity to 80% of that of the native,Ca2+-free state of the enzyme. The Kd was slightly enhanced inthe presence of salt (data not shown). The binding of Ca2+ didnot induce any change in the enzyme sedimentation coefficientas shown by analytical ultracentrifugation.

Acrylamide quenching

The uncharged fluorescence quencher acrylamide was used toprobe the accessibility of the hydrophobic core and the dynamicproperties of the pullulanase in the absence and presence ofCa2+. Effective acrylamide quenching constants obtained fromthe modified Stern±Vollmer plots were 12.1 m21 for theCa2+-free and 9.1 m21 for the Ca2+-loaded state (120 mm Ca2+)of the enzyme. A quantitative analysis of the data is notpossible because pullulanase is a heterogeneously emittingsystem, but the data suggest a decreased accessibility of proteinfluorophores to the quencher in the presence of Ca2+.

D I S C U S S I O N

Ca2+ has been reported to activate and/or stabilize a number ofthermostable pullulanases, e.g. from Thermoanaerobium Tok 6B1 [13], Thermococcus litoralis [7], Thermus spp. [29]. Brownand Kelly [7] showed that the pullulytic activity and the thermalstability of P. furiosus amylopullulanase are increased in thepresence of Ca2+. A similar effect was reported for P. woeseipullulanase [5].

Fluorescence quenching studies on the pullulanase fromP. woesei revealed that Ca2+ binds with high affinity to theprotein with a 2 : 1 stoichiometry. The hyperbolic dependenceof the fluorescence changes on the ligand concentration suggestthat the two binding sites are indistinguishable and noninter-acting [30], as confirmed by the Hill coefficient value. TheKd of 0.080 ^ 0.010 mM is comparable to that found fortypical Ca2+-binding proteins, e.g. calbindin D9k [31], vitaminD-dependent Ca2+-binding protein [32], bovine a-lactalbumin[33], and whiting parvalbumin [34]. The binding of Ca2+ toP. woesei pullulanase entails a conformational shift as indicatedby the decrease in intrinsic fluorescence emission intensity. Thelack of change in the maximal fluorescence emissionwavelength over a wide range of Ca2+ concentrations suggeststhat the local environment of the tryptophanyl residues is notperturbed. Parallel studies with the fluorescence quencheracrylamide indicate that Ca2+ binding to the protein induces aconformational change to a more compact form, without anychange in the monomeric state of the enzyme association, asshown by the sedimentation velocity analysis in the presence ofsaturating Ca2+ concentration.

In the absence of Ca2+, however, pullulanase from thehyperthermophile P. woesei shows a remarkable resistance tothermal denaturation. Incubation of the enzyme at 90 8C for 4 hdid not cause any loss in catalytic activity [5]. Moreover,the equilibrium transition midpoint and the kinetics for theGdmCl-induced unfolding of the pullulanase indicate that theenzyme is not only highly thermostable but also veryresistant to chemical denaturation. The sigmoidal equilibriumdenaturation profiles of both probes tested (fluorescence andCD) indicate an apparent two-state mechanism for theunfolding process from the native state (N) to the unfoldedstate (D) [25]. The integrated measures of the equilibriumunfolding over the entire spectrum represented by the intensity-averaged emission wavelength and by the columns of V matrixallows a reliable quantitative evaluation of the transitions lessprone to instrumental noise or vagaries in sample preparation.The analysis of equilibrium data indicates that the unfoldingparameters DGH2O

D , mg and [GdmCl]0.5 are coincident, indepen-dently of the spectral probe used to monitor the process, thussuggesting a two-state denaturation. Despite the high transitionmidpoint, the free energy of unfolding is close to the lower limitof that observed for many globular proteins from mesophiles.This fact indicates that the protein can tolerate high concen-trations of denaturant without showing a remarkable confor-mational stability at 20 8C. Therefore, pullulanase shouldbe considered as a protein resistant to GdmCl-induceddenaturation rather than a stable protein [35]. The inactivationof P. woesei pullulanase at low denaturant concentrations maybe due to subtle changes at the active site, which precede thegross structural changes. This observation has also beenreported for other proteins, e.g. glyceraldehyde-3-phosphatedehydrogenase from Thermotoga maritima [36] and bovineuridine diphosphoglucose dehydrogenase [37]. Inactivationprior to denaturation may be due to the fact that active sitesof enzymes are often situated in regions that are moresensitive to the denaturant than the protein as a whole [38].Similarly, the lack of complete reactivation upon 20-folddilution despite the absence of measurable aggregation andthe native spectra might therefore be due to the presence ofinactive enzyme with the same spectral properties as thenative pullulanase.

The unfolding kinetics of pullulanase followed by monitoringthe decrease in fluorescence emission intensity are composedof two first-order processes. This might be attributed to the

486 R. M. Schwerdtfeger et al. (Eur. J. Biochem. 264) q FEBS 1999

different structural stability of parts of the protein which unfoldwith different rate constants [39]. The first fast unfolding phasemay account for most of the dynamic quenching since a smallerrelative amplitude of this phase is observed when the unfoldingis monitored by the shift of the intensity-averaged emissionwavelength. The stabilizing effect of Ca2+ on the GdmCl-induced unfolding is evident from an analysis of the kineticparameters for fluorescence changes. The presence of Ca2+ ledto a 27-fold increase in the half-time of the two unfoldingphases. Moreover, the increase in the relative amplitude of thefirst unfolding phase and the simultaneous occurrence of thered-shift of the intrinsic maximum emission wavelengthsuggest a role of the cation in inducing tertiary structurechanges. The positive effect of Ca2+ on the conformationalstability of the enzyme is also evident by the analysis ofequilibrium unfolding parameters which are all increased in thepresence of the cation (Table 1). In particular the increase ofthe m-value, which is a function of the amount of proteinsurface exposed to solvent upon unfolding [35], indicates anincreased exposure of accessible surface area upon transitionfrom the native to the unfolded state in the presence of Ca2+.This observation may be explained as an increase in thedifference between the accessible surface area of the native andthe unfolded state possibly due to an increased compactness ofthe native state, as indicated by the fluorescence quenchingwith acrylamide. A stabilizing effect of ligands, including ions,was often observed [40,41]. In lysozyme a metal binding sitewas especially introduced to enhance the stability of thisenzyme [42].

In spite of its stabilizing effects on pullulanase Ca2+ does notseem to affect the refolding of the enzyme. The rapid regain ofnative-like structure upon dilution of the unfolding mixture to aGdmCl concentration well below the transition region in theabsence or presence of Ca2+ was not sufficient to completelyregain the active conformation. The reactivation yield ofpullulanase was strictly dependent on the GdmCl concentrationafter the first dilution of the unfolding mixture beforeultrafiltration. The optimal denaturant concentration is in thevicinity of the transition region. Under these conditions GdmClmay allow a partial internalization of hydrophobic residues andprevent the formation of marginally soluble refolding inter-mediates. The role of the denaturant in favoring proteinrenaturation and reactivation during refolding procedures hasbeen reported also for urokinase [43] and bovine growthhormone [44].

The native state of pullulanase binds Ca2+ with high affinity.The lower stability of P. woesei pullulanase in the absence ofCa2+ could be due to the prevalence of the repulsive forces ofcharged residues at the Ca2+-binding site [45]. The decreasedconformational stability of the ligand-free pullulanase may alsofind an explanation in the conformational flexibility requiredfor the structural changes necessary to accommodate the ligand.The elucidation of the Ca2+-binding site by sequence analysisand X-ray studies which are in progress will give furtherinsights about the effect of the cation on pullulanase stabilityand activity.

A C K N O W L E D G E M E N T S

We are grateful to Dr Alberto Boffi for the help in SVD analysis and for the

helpful discussion and to Dr Laura Giangiacomo for analytical ultracentri-

fugation analysis. This work was supported by the European Commission

(Biotech Generic Project Extremophiles as Cell Factories), by the Deutsche

Forschungsgemeinschaft (Graduiertenkolleg Biotechnologie and Projekt

AN 154/2), by the CNR Target Project Biotechnology and by MURST.

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