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Stability and binding properties of wild-type and c17s mutated human sterol carrier protein 2
Biochimica et Biophysica Acta 1432 (1999) 265^274 www.elsevier.com/locate/bba
Stability and binding properties of wild-type and c17s mutated human sterol carrier protein 2 Claudia Jatzke a , Hans-Ju«rgen Hinz
a;
*, Udo Seedorf b , Gerd Assmann
b;c
a
Institut fu«r Physikalische Chemie, Westfa«lische Wilhelms-Universita«t, SchloMplatz 4/7, 48149 Mu«nster, Germany Institut fu«r Arterioskleroseforschung, Westfa«lische Wilhelms-Universita«t, Domagkstr. 3, 48149 Mu«nster, Germany Institut fu«r Klinische Chemie und Laboratoriumsmedizin, Zentralklinikum der Westfa«lische Wilhelms-Universita«t, 48149 Mu«nster, Germany b
c
Received 23 April 1999; accepted 4 May 1999
Abstract The temperature- and solvent-induced denaturation of both the SCP2 wild-type and the mutated protein c71s were studied by CD measurements at 222 nm. The temperature-induced transition curves were deconvoluted according to a two-state mechanism resulting in a transition temperature of 70.5³C and 59.9³C for the wild-type and the c71s, respectively, with corresponding values of the van't Hoff enthalpies of 183 and 164 kJ/mol. Stability parameters characterizing the guanidine hydrochloride denaturation curves were also calculated on the basis of a two-state transition. The transition of the wild-type occurs at 0.82 M GdnHCl and that of the c71s mutant at 0.55 M GdnHCl. These differences in the half denaturation concentration of GdnHCl reflect already the significant stability differences between the two proteins. A quantitative measure are the Gibbs energies vG 0D (buffer) at 25³C of 15.5 kJ/mol for the wild-type and 8.0 kJ/mol for the mutant. We characterized also the alkyl chain binding properties of the two proteins by measuring the interaction parameters for the complex formation with 1-O-Decanyl-L-D-glucoside using isothermal titration microcalorimetry. The dissociation constants, Kd , for wild-type SCP2 are 335 WM at 25³C and 1.3 mM at 35³C. The corresponding binding enthalpies, vHb , are 321.5 kJ/ mol at 25³C and 72.2 kJ/mol at 35³C. The parameters for the c71s mutant at 25³C are Kd = 413 WM and vHb = 16.6 kJ/mol. These results suggest that both SCP2 wild-type and the c71s mutant bind the hydrophobic compound with moderate affinity. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Isothermal titration calorimetry; SCP2; Protein stability
1. Introduction Sterol carrier protein 2 (SCP2, also called non-speAbbreviations: SCP2, sterol carrier protein 2; 10-GLC, 1-O-Decanyl-L-D-glucoside; wt, wild-type; c71s, Cys-71CSer mutant form of sterol carrier protein 2; GdnHCl, guanidine hydrochloride; SDS-PAGE, sodiumdodecylsulfate polyarcylamide gel electrophoresis ; ITC, isothermal titration calorimetry; CD, circular dichroism; O0:1%;1cm 280nm , molar extinction coe¤cient of a 0.1% solution and a light path of 1 cm at 280 nm; vG 0D , molar standard change in Gibbs energy of unfolding; vg0 , speci¢c standard change in Gibbs energy; q, partition function ; K, equilibrium constant; Tm , transition temperature; vHb , binding enthalpy; Kd , dissociation constant; cmc, critical micellar concentration; L-FABP, liver fatty acid binding protein * Corresponding author. Fax: +49-251-832-9163; E-mail: [email protected] 0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 9 ) 0 0 1 1 4 - 4
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ci¢c lipid transfer protein) is a small, basic protein of molecular mass Mr = 13 380 Da, which binds fatty acids, long chain fatty acyl Coenzymes A, and sterols with a 1:1 molar stoichiometry and binding constants in the order of 0.23^0.4 WM for fatty acids [1], 4.5^2.8 nM for fatty acyl CoAs [2], 1 WM for sterols [3], and 0.3 WM for [3 H]cholesterol [4]. The protein occurs at high concentrations in peroxisomes, but signi¢cant concentrations are also found in the cytoplasm [5] and associated with other organelles, such as mitochondria [6,7] and the endoplasmatic reticulum [7]. In vitro, the protein activates the enzymatic conversion of 7-dehydrocholesterol to cholesterol by liver microsomes [8] and stimulates the acylCoA cholesterol acyltransferase-mediated esteri¢cation of intracellular cholesterol [9]. Furthermore, SCP2 promotes the exchange of various sterols [10,11] and phospholipids [12] between membranes in vitro. The latter activities led to the hypothesis that the protein participates in the intracellular transfer of phospholipids and sterols, especially cholesterol, also in vivo [1,12^16]. However, a recently characterized SCP2 knockout mouse model was not associated with a phenotype expected to result from defective cholesterol or phospholipid tra¤cking [17]. Instead, these mice showed a defect in the peroxisomal catabolism of the methyl-branched fatty acids 3,7,11,15-tetramethylhexadecanoic acid (phytanic acid) and 2,6,10,14-tetramethylpentadecanoic acid (pristanic acid) [17]. Because recombinant rat SCP2 protein exhibited high a¤nity binding of phytanoyl-CoA, it was presumed that the protein is involved in peroxisomal uptake and transport of this branched chain fatty acyl CoA. These ¢ndings suggest that the possible biological function of SCP2 is perhaps not predominantly associated with sterol transfer but involves a broader spectrum of activities relating to alkyl chain containing compounds. This interesting hypothesis prompted us to characterize quantitatively the interaction with 10-GLC of wild-type SCP2 protein and a mutated form in which cysteine 71 was replaced by serine. There was another reason for the particular choice of the c71s mutant. Activity studies with di¡erent deletion mutants had shown that helix A (residues 9^22), L-strand V (residues 100^102) and Asn-104
are essential elements for the lipid transfer activity of SCP2 [18]. Therefore, it had been assumed that the only cysteine, which is H-bonded to amino acid 102 plays an important role in the binding process. This hypothesis was obviously not correct, since it could be shown by lipid transfer assays that the mutant c71s had the same activity as the wild-type. However, the single point mutation has another intriguing e¡ect. We demonstrate in the present study that at 25³C wild-type and c71s mutant proteins differ signi¢cantly in stability, while the binding constants for 10-GLC are almost similar. A prerequisite for a role of SCP2 in the transport of alkyl chain containing compounds over the membrane in the form of a complex is the proof of the interaction between SCP2 and the corresponding ligand. Therefore, knowledge of the thermodynamics of complex formation is a necessary basis for the mechanistic understanding of the biological function. Since estimates of binding constants were published previously [1^4] we focused our e¡orts on the direct determination by ITC of the enthalpy changes associated with binding of 10-GLC as a model compound for hydrophobic alkyl chain containing ligands. 2. Materials and methods 2.1. Preparation of 10-GLC and SCP2 1-O-Decanyl-L-D-glucoside (10-GLC) was synthesized after a modi¢ed Ko«nigs^Knorr reaction from 1-K-bromo-2,3,4,6-tetra-O-acetylglucose with 1-decanol [19]. SCP2 and the c71s mutant were expressed and puri¢ed according to Seedorf et al. [18]. 2.2. Preparation of micellar solutions of 10-GLC 4.6 mM suspension of 10-GLC were prepared in bu¡er (bu¡er P: 43 mM KH2 PO4 /43 mM K2 HPO4 / 1.0 mM EDTA, pH 7.6). Before use the bu¡er was routinely ¢ltered through a 0.45 Wm ¢lter (Millipore, Badford, MA, USA) and degassed. The critical micellar concentration of 10-GLC was determined by measurements of the surface tension using the Wilhelmy method [20] (Fig. 4). The stock solution of
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4.6 mM 10-GLC was diluted with aliquots of bu¡er P to the various ¢nal concentrations between 0.1 and 1.5 mg/ml used in the present study. 2.3. CD measurements CD experiments were conducted using a CD6 Jobin-Yvon spectropolarimeter. Spectra were obtained from an average of ¢ve scans between 205 and 260 nm. Data were taken at 0.5 nm intervals with an integration time of 1 s. All spectra were corrected for the bu¡er signal (bu¡er B: 15 mM K2 HPO4 /15 mM KH2 PO4 /1 mM EDTA, pH 6.8). For all measurements the bu¡er was degassed and the sample compartment at the instrument was purged with a constant £ow of nitrogen (5 l/min). The light path was 1 cm at low concentrations of the protein or 0.01 cm at 1.6 mg/ml. The proteins were dialysed in bu¡er B. To avoid possible dimerization of the wild-type protein 5 Wl L-mercaptoethanol (obtained from Sigma) was added to the dialysis tubing and dialysed at RT (room temperature) for 1/2 h. Another dialysis overnight removed the L-mercaptoethanol from the protein solution prior to the measurements. SDS-PAGE (12.5%) was made under non-reducing conditions after the dialysis and veri¢ed the monomeric state of SCP2. Protein concentrations were determined by absorbance measurements at 280 nm using absorption coe¤cients of O0:1%;1cm 280nm A(wt) = 0.439 ml/(mg cm) and O0:1%;1cm (c71s) = 0.428 ml/(mg cm), respectively. The 280nm coe¤cients were calculated from the amino acid compositions following the procedure suggested by Gill and von Hippel [21]. Protein concentrations of the wild-type were 0.044 or 1.6 mg/ml, that of the mutant (c71s) was 0.055 mg/ml. Temperature scans were carried out at 222 nm using a programmable Haake F3 cryothermostat. The scan rate was 1 K/ min and the temperature ranges were 10³C to 95³C for the wild-type and 10³C to 85³C for c71s. The temperature was controlled and monitored using a 100 6 platinum resistance thermometer immersed in the protein solution. The van't Ho¡ enthalpy and the midpoint temperatures of the CD transition curves were determined by simulation of the transition curves according to a two-state mechanism using the following equations:
267
f T nb T ub T3nb TW q T31=q T 1 nb T a bW T3T m ub T c dW T3T m nb(T) is the temperature dependence of the native state baseline, ub(T) that of the unfolded state. Both are assumed to be linear. q T 1 K T is the partition function of a two-state transition involving the equilibrium constant K(T). The temperature dependence of the equilibrium constant depends on the standard Gibbs energy change vG0 for unfolding, which can be calculated from the experimental transition enthalpy vH0 and the transition temperature Tm according to: K T exp 3vG0 =RT and vG 0 vH 0 13T=T m
2.3.1. Stability studies In the isothermal denaturation studies using guanidine hydrochloride (GdnHCl) the protein concentrations were 0.05 mg/ml for the wild-type and 0.036 mg/ml for the mutant protein. CD measurements were made from 215 to 230 nm at 20³C with an integration time of 1 s and an increment of 0.5 nm. Data of ¢ve scans were averaged and corrected for the bu¡er signal. Denaturant-induced unfolding was monitored at 222 nm. The GdnHCl concentration was determined refractometrically according to Nozaki [22]. Evaluation of the isothermal unfolding curves was performed graphically as described below and numerically following the procedure of Santoro and Bolen [23] using the equations: 3222nm Y N mN GdnHCl Y D mD GdnHClWZ 1Z 2 Z exp3 vG 0D buffer mG GdnHCl= RT YN and YD are the intercepts, and mN and mD are
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the slopes of the pre- and post-unfolding baselines, respectively. [GdnHCl] is the concentration of the denaturant. vG0D (bu¡er) is the standard Gibbs energy change for the unfolding reaction in the absence of GdnHCl, and mG is the slope of the plot of vG 0app: versus GdnHCl concentration according to: vG 0app: vG0D buffer mG WGdnHCl The two-state equilibrium constants required to calculate vG 0app: were obtained from the plots of [3]222nm vs. GdnHCl concentration using linear extrapolated baselines as indicated by the dotted lines in Fig. 3. Independent determination of the baselines reduces the number of ¢t parameters in the Santoro and Bolen equation (Eq. 2) to two. The graphical analysis of the plot vG0app: vs. [GdnHCl] results in the standard Gibbs energy change, vG0D (bu¡er) by extrapolation to zero GdnHCl concentration as shown in the inserts of the corresponding ¢gure.
bu¡er, and the pure dialysis bu¡er was titrated with the solution of 10-GLC. The heat e¡ects observed in these controls were subtracted from the heat e¡ects associated with the titration of the protein with 10-GLC to obtain the net enthalpy of binding. The ligand (10-GLC) consists of a saturated alkyl chain of 10 carbon atoms linked to glucose in Lcon¢guration. The sugar is responsible for the good solubility in water. We determined the critical micellar concentration (cmc) of 10-GLC according to the Wilhelmy method [20]. The plot of surface tension vs. the concentration of 10-GLC provided a cmc value of 0.24 mg/ml (Fig. 4). This value rendered it possible to know the state of aggregation of 10GLC under all titration conditions. The values for the molar binding enthalpy, vHb , and the binding constant, Kb , were determined from non-linear least squares ¢ts of the data according to Eqs. 3 and 4. 1
vQ
2.4. Isothermal titration calorimetry (ITC) measurements The titration experiments were performed using an improved heat conduction twin microcalorimeter of Gill type, whose construction was based on the description by McKinnon et al. [24]. The calorimeter was calibrated by the dilution of propanol(1) into water [25] and, alternatively, by the protonation of the imidazole group of histidine [26] at 25³C and 35³C, respectively. The calorimeter was ¢lled with 800 Wl freshly prepared protein solution (50^73 WM). The calorimetrically active volume has been determined by calibration to be 410 Wl. Stable baselines were achieved after approximately 20 min after ¢lling. For the titration 5 Wl aliquots of a 4.6 mM 10GLC solution were injected automatically by means of a rotating stirrer syringe, which was controlled by a stepping motor. Each injection of 10-GLC (c = 1.48 mg/ml) produced a sharp exothermic signal which returned to the baseline within 2 min. An injection was made every 6 min to provide for su¤ciently long baselines before and after each measurement (Fig. 5). The reference cell contained water. To determine the heat contribution from dilution, two controls were made. The protein solution was titrated with dialysis
1 Pt nK b K b X t 3 1 Pt nK b K b X t 2 34Pt nK 2b X t 2 2K b VvH 0 3
The terms are de¢ned in the following manner [27]: vQ: corrected, cumulative heat (J); [P]t : total protein concentration (mol/l); n: number of binding sites ( = 1); Kb : intrinsic association constant (l/mol); [X]t : total concentration of ligand (mol/l); V: volume of the reaction cell ( = 410 Wl); vH0 : change of binding enthalpy per mol of ligand (J/mol). The alternative ¢t after Wiseman et al. [28] uses the following equation: 0 1 1 r Xr 13 3 1 dQ B C 2 2 A W vH W@0:5 p V dX tot X 2r 32X r 13r 1 r2 4
Binding is also assumed to exhibit 1:1 stoichiometry and the other terms are de¢ned as given in the following: Xr : ratio Xtot /Ptot ; Ptot : total protein concentration (mol/l); V: volume of the reaction cell ( = 410 Wl); Xtot : total ligand concentration in the cell after each injection; r: ratio 1/(Kb cPtot ), with Kb being the binding constant; vH0 : change of binding enthalpy per mol of ligand (J/mol).
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The vG0 and vS0 values at temperature T for binding were calculated from the standard thermodynamic relationship: 3RT ln K b vG0b vH 0b 3TvS 0b
5
3. Results 3.1. Stability of SCP2 and its c71s mutant 3.1.1. CD measurements of temperature-induced transitions Unfolding of SCP2 can be monitored conveniently by the disappearance of the CD absorption at 222 nm due to the loss of K-helical structure with the increase in temperature. This is illustrated in Fig. 1 which shows CD spectra of wild-type and mutant protein at di¡erent temperatures. The spectra of the native proteins have been determined at 10³C, those for the unfolded proteins at 96³C (SCP2 wt) and 85³C (c71s), respectively. Fig. 2 illustrates the corresponding transition curves of SCP2 wt (c = 0.044 mg/ ml) and c71s (c = 0.055 mg/ml) monitored at 222 nm. The symbols refer to the experimental points, the sigmoidal lines are the ¢tted curves using Eq. 1 and a non-linear least squares algorithm. The dotted lines indicate the CD absorption of the fully native and fully unfolded proteins as a function of temperature. It is evident that the transition curves can be described perfectly by the assumption of a two-state unfolding mechanism according to Eq. 1. The transition temperature Tm of the wild-type protein was found to be around 10 K higher than that of the mutant protein. This is indicative of the greater thermal stability of the wild-type protein. The thermodynamic parameters obtained from these measurements are summarized in Table 1. They are not dependent on protein concentration as shown for the wild-type protein by the fact that both the transition temperature and the transition enthalpy are identical for protein concentration di¡ering by a factor of about 40 (0.044 mg/ml and 1.6 mg/ml). Therefore, we can conclude that no oligomers were formed during the unfolding process. 3.1.2. Isothermal denaturant unfolding studies GdnHCl-induced denaturation curves of SCP2 wt
Fig. 1. CD spectra of SCP2 wild-type and c71s at di¡erent temperatures. The solid lines refer to the wild-type protein (c = 0.044 mg/ml) at 10 and 96³C. The dotted lines correspond to the c71s serine mutant (c = 0.055 mg/ml) at 10 and 85³C.
and c71s are shown in Fig. 3. The standard Gibbs energies of unfolding in the absence of the denaturant, vG 0D (bu¡er), were obtained either by plotting vG0app: vs. [GdnHCl] (inserts) and linearly extrapolating to zero GdnHCl concentration or by ¢tting the data to Eq. 2. The ¢ts are shown as solid lines in Fig. 3 [23]. These graphs show clearly the higher stability of the wild-type protein. Both the vG0 (bu¡er) values and the c50% concentrations are larger GdnHCl (c50% = 0.82 M for the wild-type and 0.55 M for GdnHCl the c71s mutant protein). All parameters are summarized in Table 2.
Table 1 Thermodynamic parameters determined from CD-monitored, thermal unfolding curves Tm (³C) vHvH (kJ/mol)
SCP2 wt
c71s
70.5 þ 0.5 183 þ 10
59.9 þ 0.9 164 þ 22
Two di¡erent experiments were performed with the wild-type protein at concentrations di¡ering by a factor of 40 (0.044 or 1.6 mg/ml). The di¡erence had no e¡ect on the transition temperature. The concentration of the c71s mutant was 0.055 mg/ml in both measurements. The parameters were evaluated on the basis of Eq. 1 using the two-state approximation.
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C. Jatzke et al. / Biochimica et Biophysica Acta 1432 (1999) 265^274 Table 2 Stability parameters from isothermal GdnHCl-induced unfolding studies c50% GdnHCl
(M) YN (deg cm2 /dmol) mN (deg cm2 l/(dmol mol)) YD (deg cm2 /dmol) mD (deg cm2 l/(dmol mol)) mG (kJ l/mol2 ) vG0D (bu¡er)Fit (kJ/mol) vG0D (bu¡er) (kJ/mol)
SCP2 wt
c71s
0.82 39226 0 33820 565 319.4 15.5 15.5
0.55 38100 0 33278 385 313.6 7.5 8.4
Two methods have been applied. The value vG0D (bu¡er)Fit has been obtained from ¢tting the data to Eq. 2. To avoid the errors involved in a six-parameter ¢t, the pre- and post-transitional baselines were determined independently by linear least squares ¢ts. This reduces the number of the ¢t parameters to two. vG0D (bu¡er) is the result of the linear extrapolation to zero GdnHCl concentration as shown in the inserts of Fig. 3. Protein concentrations were 0.05 mg/ml for the wild-type protein and 0.036 mg/ml for the c71s mutant. The temperature was 20³C.
Fig. 2. Thermal transition curve of SCP2 wt (upper panel) and c71s (lower panel). The transition was monitored by the change in mean residue ellipticity at 222 nm using a heating rate of 1 K/min. Upper panel: SCP2 wt concentration was 0.044 mg/ ml. Open squares refer to the experimental points. The solid line represents the simulated curve according to Eq. 1. The dotted lines re£ect the linear baselines. Lower panel: c71s concentration was 0.055 mg/ml. Open circles refer to the experimental points. The solid line represents the simulated curve according to Eq. 1. The dotted lines re£ect the linear baselines.
3.2. ITC measurements on the interaction of 10-GLC with SCP2 3.2.1. Determination of the critical micellar concentration (cmc) of 10-GLC The critical micellar concentration of 10-GLC was determined by measuring the surface tension of a 10GLC monolayer using the Wilhelmy method. The results are illustrated in Fig. 4. The intercept of the two linear-regression lines occurs at the critical micellar concentration. The value found at 20³C is of 0.24 mg/ml. Using this value one can calculate that throughout the titration 10-GLC occurs in the calorimetric vessel in the monomeric non-micellar state (14U5 Wl of the stock solution of 10-GLC 1.48 mg/
ml into an active volume of 410 Wl results in the ¢nal concentration of 0.22 mg/ml of 10-GLC). 3.2.2. Determination of the binding parameters Fig. 5A shows the uncorrected enthalpimetric titration data. Each peak represents the heat e¡ect resulting from injecting of 5 Wl of a 4.6 mM (1.48 mg/ml) solution of 10-GLC into a SCP2 solution (50^73 WM) in bu¡er P at 25³C. Fig. 5B illustrates the control experiment, i.e. the titration of 10-GLC into bu¡er. Dilution of 10-GLC is associated with a signi¢cant negative heat evolution. No heat e¡ect was observed for the dilution of SCP2 wt protein at 25³C in the corresponding control titration. But we observed an exothermic heat e¡ect of around 340 to 3160 WJ associated with protein dilution for SCP2 c71s at 25³C and for SCP2 wt at 35³C. The raw data were corrected for these heat e¡ects. Because these heats of dilution are in the order of the heats of ligand binding proper correction is very important. The binding data show that the binding enthalpy for the wild-type protein changes from 321.5 kJ/mol at 25³C to 72.2 kJ/mol at 35³C. The cumulative integral heats corrected for the controls are shown as a function of the total concentration of 10-GLC in Fig. 6A. The solid curve has been calculated using Eq. 3.
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Table 3 Thermodynamic parameters for the interaction of SCP2 with 10-GLC at 25³C and 35³C.
Kd /mM vG0binding /kJ/mol vg0binding /J/g vH0binding /kJ/mol -TvS0binding /kJ/mol
25³C SCP2 wt
25³C c71s
35³C SCP2 wt
0.3 þ 0.02 319.8 þ 0.1 31.48 321.5 þ 0.7 +1.7 þ 0.6
0.4 þ 0.001 319.3 þ 0.1 31.44 +16.6 þ 0.6 335.9 þ 0.7
1.3 þ 0.1 317.1 þ 0.2 31.28 +72.2 þ 3.4 389.2 þ 3.2
The data are the average values from two di¡erent titrations and the analysis procedures given in the references [27,28].
4. Discussion
Fig. 3. GdnHCl-induced CD denaturation curve of SCP2 wt (upper panel) and c71s (lower panel) at 20³C. Upper panel: The protein concentration was 0.05 mg/ml. The open squares show the measured values, the solid line refers to the ¢tted curve according to Eq. 2. The dotted lines correspond to the pre- and post-unfolding baselines. From the plot of vG0app: vs. [GdnHCl] the value of vG0D (bu¡er) = 15.5 kJ/mol is obtained (r = 0.976). Lower panel: The protein concentration was 0.036 mg/ml. The open circles refer to the experimental data, the solid line shows the ¢tted curve according to Eq. 2. The dotted lines correspond to the pre- and post-unfolding baselines. From the plot vG 0app: vs. [GdnHCl] the value of vG0D (bu¡er) = 8.4 kJ/mol is obtained (r = 0.965).
The binding enthalpy vHb and the binding constant Kb have been calculated on the basis of a stoichiometry of 1:1. This appears to be justi¢ed in view of the results of Schroeder et al. [3,29]. For comparison Fig. 6B shows the alternative ¢t according to Wiseman et al. [28] using Eq. 4. Both ¢ts provide identical results. The corresponding parameters are summarized in Table 3. The numbers given here are mean values based on two independent titration series and two di¡erent ¢tting procedures. The values agree within standard deviations. Because of the lack of mutant protein the titration of c71s could not be performed at 35³C.
The present study is concerned with the quantitative description of the energies involved in the binding of 10-GLC to SCP2 wt and c71s mutant protein. Since the sterol carrier protein has been reported to exhibit signi¢cant a¤nity for fatty acids and fatty acyl CoAs, 10-GLC is an excellent ligand to mimic short alkyl chain binding characteristics. On the basis of the cmc determination we can assume that the 10GLC concentration is always below the cmc during the titration experiment. This assumption is supported by the identical magnitude of the heat e¡ects associated with each injection of 5 Wl samples (see
Fig. 4. Determination of the critical micellar concentration of 10-GLC at 20³C. Bu¡er: 43 mM potassium phosphate, 1 mM EDTA, pH 7.6. The stock solution of 4.6 mM 10-GLC was diluted with bu¡er to the respective ¢nal concentrations used for the langmuir balance. The squares correspond to the experimental values and the dotted lines are linear ¢ts. The critical micellar concentration of 0.24 mg/ml results from the point of intersection.
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Fig. 5. Titration of SCP2 wt with 10-GLC (upper frame) and control (titration of bu¡er with 10-GLC, lower frame) at 25³C. Titrand: 800 Wl of 69 WM SCP2 wt solution in bu¡er P, pH 7.6 (15 mM potassium phosphate, 1 mM EDTA) (upper frame) or 800 Wl bu¡er P, pH 7.6 (lower frame). Addition: 14U5 Wl of 4.6 mM 10-GLC solution in bu¡er P, pH 7.6.
Fig. 5B). These peaks re£ect the heat e¡ects involved in the transition of 10-GLC going from the micellar into a monomeric fully hydrated state. Therefore, the thermodynamic parameters for the binding reaction refer to complex formation with monomeric 10GLC. The ITC experiments show clearly, that SCP2 binds 10-GLC, though with rather low a¤nity as indicated by the dissociation constant of 0.3 mM. The lower a¤nity of SCP2 for 10-GLC compared with that for long chain fatty acids, fatty acyl CoA or dehydroergosterol is likely the result from the shorter carbon chain length. This interpretation is in line with the results of Frolov et al. [30], who observed an optimal interaction of SCP2 with fatty acids containing 14^22 CH2 groups. In previous studies by Rolf et al. [31], two binding constants were reported for the binding of oleic acid to L-FABP. The dissociation constant for the ¢rst
Fig. 6. Determination of the binding parameters according to [27]. A: Integrated and dilution corrected data (open squares) are shown as function of concentration of 10-GLC after each injection at 25³C. The best-¢t curve (line) is shown calculated according to Eq. 3 with the assumption of one binding site (n = 1). The ¢t parameters are Kd = 0.3 mM and vH0 = 321.5 kJ/mol. Titrand: 800 Wl of 73 WM SCP2 wt in bu¡er P, pH 7.6. Addition: 14U5 Wl of a 4.6 mM solution of 10-GLC in bu¡er P, pH 7.6. B: Determination of the binding parameters according to [28]. Changes of heat per injection of 10-GLC (open circles) are shown as function of the ratio of total ligand to total macromolecule concentration at 25³C and pH 7.6. The solid line refers to the theoretical curve calculated according to Eq. 4. Titrand: 800 Wl of 73 WM SCP2 wt in bu¡er P, pH 7.6. Addition: 14U5 Wl of a 4.6 mM solution of 10-GLC in bu¡er P, pH 7.6.
binding site was 0.26 WM that for the second 4.9 WM. The corresponding binding enthalpy values were 316 kJ/mol and +22 kJ/mol, respectively. It is worth noting that the enthalpy value observed for 10-GLC binding to SCP2 wt at 25³C is of comparable magnitude (322 kJ/mol) as that observed by Rolf et al. for the ¢rst binding site.
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From spectroscopic studies involving time-resolved tryptophan £uorescence an ellipsoidal shape of the SCP2 had been deduced with the lipid binding site perpendicular to the long symmetry axis of the protein, in analogy to the shape of the phosphatidylcholine transfer protein [32]. This result has been con¢rmed by recent NMR studies [33]. These NMR data [33] have shown that SCP2 consists of three K-helices (residues 9^22, 25^30, 78^84) and a ¢ve-stranded L-sheet that comprises the residues 33^41, 47^54, 60^62, 71^76 and 100^102. The Lstructure is supposedly a central feature of the molecular architecture of the protein. Depending on the relative orientation of the residues 77^99 (around helix C) and the C-terminus to the L-structure the formation of a hydrophobic pocket is possible. Observations based on site-directed mutagenesis of SCP2 [18] favor the existence of a speci¢c lipid binding site involving the residues (77^99 and the carboxy-terminus). The replacement of Cys in position 71 with Ser results in only a small change in Kd from 0.3 mM to 0.4 mM which corresponds to a minor change in the standard Gibbs energy from 319.8 kJ/ mol to 319.3 kJ/mol at 25³C. These results are consistent with the ¢nding of identical lipid transfer activities for wild-type and mutated protein and they suggest that the position 71 is not important for the binding properties [18]. Interpretation of the binding constants shows that the temperature dependence of the binding enthalpy is large. The dissociation constant of SCP2 wt increases from 0.3 mM at 25³C to 1.3 mM at 35³C. The very similar binding properties at 25³C contrast markedly with the large di¡erences in stability that have been observed for the two proteins, both with regard to temperature- and GdnHCl-induced unfolding. The c50% GdnHCl value of the wild-type protein as well as the Gibbs energy is approximately twice as large as the corresponding value for the serine mutant. The conservation of the binding a¤nity is accomplished with vastly di¡erent energy and entropy parameters. While the binding of 10-GLC to the wildtype protein is associated with a favorable enthalpy change of 321.5 kJ/mol and a slightly unfavorable entropy term of 3TvS0 = +1.7 kJ/mol, the complex formation with the mutant is characterized by a positive enthalpy change of +16.6 kJ/mol but an over-
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compensating favorable entropy contribution to vG0binding of 3TvS0 = 335.9 kJ/mol. It is the latter term that apparently drives the reaction of the mutant protein at 25³C. The signs of the binding enthalpies and entropies for the mutant protein suggest that the binding of 10-GLC induces energy consuming restructuring processes which are associated with favorable entropy changes. A possible mechanism would be the release of surface bound water molecules into the bulk phase on binding of the hydrophobic ligand. Since the mutant protein is signi¢cantly less stable than the wild-type protein water release could also result from restructuring of parts of the protein other than the direct hydrophobic binding pocket. Actually this has to be assumed, because the number of water molecules replaced in the binding pocket can be expected to be roughly comparable for both proteins. It is an intriguing idea to envisage the ligand as creating its own optimal binding pocket. Acknowledgements H.-J.H. gratefully acknowledges support by the DFG (Deutsche Forschungsgemeinschaft) and the Fonds der Chemischen Industrie.
References [1] F. Schroeder, S.C. Myers-Payne, J.T. Billheimer, W.G. Wood, Probing the lipid binding sites of fatty acid and sterol carrier proteins: E¡ect of ethanol, Biochemistry 34 (1995) 11919^11927. [2] A. Frolov, T.H. Cho, J.T. Billheimer, F. Schroeder, Sterol carrier protein 2, a new fatty acyl Coenzyme A-binding protein, J. Biol. Chem. 271 (1996) 31878^31884. [3] F. Schroeder, P. Butko, G. Nemecz, T.J. Scallen, Interaction of £uorescent D5;7;9 11;22 -Ergostatetraen-3b-ol with sterol carrier protein 2, J. Biol. Chem. 265 (1990) 151^157. [4] S.M. Colles, J.K. Woodford, D. Moncecchi, S.C. MyersPayne, L.R. McLean, J.T. Billheimer, F. Schroeder, Cholesterol interaction with recombinant human sterol carrier protein-2, Lipids 30 (1995) 795^803. [5] B.C. McNamara, C.R. Jefcoate, The role of sterol carrier protein 2 in stimulation of steroidgenesis in rat adrenal mitochondria by adrenal cytosol, Arch. Biochem. Biophys. 275 (1989) 53^62. [6] S.M. Mendis-Handagama, R.F. Aten, P.A. Watkins, T.J. Scallen, H.R. Berhman, Peroxisomes and sterol carrier pro-
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C. Jatzke et al. / Biochimica et Biophysica Acta 1432 (1999) 265^274 tein-2 in luteal cell steroidogenesis: a possible role in cholesterol transport from lipid droplets to mitochondria, Tissue Cell 27 (1995) 483^490. M.P. Reinhart, S.J. Avart, T.O. Dobson, T.A. Foglia, The presence and subcellular distribution of sterol carrier protein 2 in embryonic-chick tissues, Biochem. J. 295 (1993) 787^ 792. B.J. Noland, R.E. Arebalo, E. Hansbury, T.J. Scallen, Puri¢cation and properties of sterol carrier protein 2, J. Biol. Chem. 255 (1980) 4282^4289. K.L. Gavey, B.J. Noland, T.J. Scallen, The participation of sterol carrier protein 2 in the conversion of cholesterol to cholesterol ester by rat liver microsomes, J. Biol. Chem. 256 (1981) 2993^2999. K.W.A. Wirtz, T.W.J. Gadella Jr., Properties and modes of action of speci¢c and non-speci¢c phospholipid transfer proteins, Experientia 46 (1990) 592^599. F. Schroeder, J.R. Je¡erson, A.B. Kier, T.J. Scallen, Membrane cholesterol dynamics: cholesterol domains and kinetic pools, Proc. Soc. Exp. Biol. Med. 196 (1991) 235^252. B. Bloj, D.B. Zilversmit, Rat liver proteins capable of transferring phosphatidyl-ethanolamine, J. Biol. Chem. 252 (1977) 1613^1619. B. Bloj, D.B. Zilversmit, Accelerated transfer of neutral glycosphingolipids and ganglioside GM1 by a puri¢ed lipid transfer protein, J. Biol. Chem. 256 (1981) 5988^5991. R.C. Crain, D.B. Zilversmit, Two non-speci¢c phospholipid exchange proteins from beef liver. 1. Puri¢cation and characterization, Biochemistry 19 (1980) 1433^1439. T.J. Billheimer, J.L. Gaylor, E¡ect of lipid composition on transfer of sterol mediated non-speci¢c lipid transfer protein (sterol carrier protein 2), Biochim. Biophys. Acta 1046 (1990) 136^143. A. van Amerongen, R.A. Demel, J. Westermann, K.W.A. Wirtz, Transfer of cholesterol and oxysterol derivatives by non-speci¢c transfer-protein (sterol carrier protein 2): A study on its mode of action, Biochim. Biophys. Acta 1004 (1989) 36^43. U. Seedorf, M. Raabe, P. Ellinghaus, F. Kannenberg, M. Fobker, T. Engel, S. Denis, F. Wouters, K.W.A. Wirtz, R.J.A. Wanders, N. Maeda, G. Assmann, Defective peroxisomal catabolism of branched fatty acyl coenzyme A in mice lacking the sterol carrier protein-2/sterol carrier protein-x gene function, Genes Dev. 12 (1998) 1189^1201. U. Seedorf, S. Scheek, T. Engel, C. Steif, H.J. Hinz, G. Assmann, Structure-activity studies of human sterol carrier protein 2, J. Biol. Chem. 269 (1994) 2613^2618. ë ber einige Derivate des TraubenW. Ko«nigs, E. Knorr, U zuckers und der Galactose, Ber. Dtsch. Chem. Ges. 34 (1901) 957.
[20] L. Wilhelmy, Ann. Physik 119 (1963) 177. [21] S.C. Gill, P.H. von Hippel, Calculation of protein extinction coe¤cients from amino acid sequence data, Anal. Biochem. 182 (1989) 319^326. [22] Y. Nozaki, The preparation of guanidine hydrochloride, Methods Enzymol. 26 (1972) 43^50. [23] M.M. Santoro, D.W. Bolen, Unfolding free energy changes determined by linear extrapolation method. 1. Unfolding of Phenylmethanesulfonyl alpha-Chymotrypsin using di¡erent denaturants, Biochemistry 27 (1988) 8063^8068. [24] I.R. McKinnon, L. Fall, A. Parody, S.J. Gill, A twin titration microcalorimeter for study of biochemical reactions, Anal. Biochem. 139 (1984) 134^139. [25] L.E. Briggner, I. Wadso«, Test and calibration processes for microcalorimeters, with special reference to heat conduction instruments used with aqueous systems, J. Biochem. Biophys. Methods 22 (1991) 101^118. [26] J. Hermans Jr., G. Rialdi, Heat of ionisation and denaturation of sperm-whale myoglobin determined with a microcalorimeter, Biochemistry 4 (1965) 1277^1281. [27] E. Freire, O.L. Mayorga, M. Straume, Isothermal titration, Anal. Chem. 62 (1990) 1^11. [28] T. Wiseman, S. Williston, J.F. Brands, L.N. Lin, Rapid measurements of binding constants and heats of binding using a new titration calorimeter, Anal. Biochem. 179 (1989) 131^137. [29] F. Schroeder, P. Butko, I. Hapala, T.J. Scallen, Intermembrane cholesterol transfer: Role of sterol carrier protein 2 and phosphatidylserine, Lipids 25 (1990) 669^674. [30] A. Frolov, K. Miller, J.T. Billheimer, T.H. Cho, F. Schroeder, Lipid speci¢city of the sterol carrier protein 2 fatty acid-binding site: A £uorescence displacement and energy transfer study, Lipids 32 (1997) 1201^1209. [31] B. Rolf, E. Oudenampsen-Kru«ger, T. Bo«rchers, N.J. F×rgeman, J. Knudsen, A. Lezius, F. Spener, Analysis of the ligand binding properties of bovine liver-type fatty acid binding protein, Biochim. Biophys. Acta 1259 (1995) 245^ 253. [32] T.W.J. Gadella Jr., P.I.H. Bastiaens, J.W.G. Visser, K.W.A. Wirtz, Shape and lipid-binding site of non-speci¢c lipidtransfer protein (sterol carrier protein 2): A steady-state and time-resolved £uorescence study, Biochemistry 30 (1991) 5555^5564. [33] T. Szyperski, S. Scheek, J. Johansson, G. Assmann, U. Seedorf, K. Wu«thrich, NMR determination of the secondary structure and the three-dimensional polypeptide backbone fold of human sterol carrier protein 2, FEBS Lett. 335 (1993) 18^25.
BBAPRO 35939 1-7-99
Stability and binding properties of wild-type and c17s mutated human sterol carrier protein 2 Claudia Jatzke a , Hans-Ju«rgen Hinz
a;
*, Udo Seedorf b , Gerd Assmann
b;c
a
Institut fu«r Physikalische Chemie, Westfa«lische Wilhelms-Universita«t, SchloMplatz 4/7, 48149 Mu«nster, Germany Institut fu«r Arterioskleroseforschung, Westfa«lische Wilhelms-Universita«t, Domagkstr. 3, 48149 Mu«nster, Germany Institut fu«r Klinische Chemie und Laboratoriumsmedizin, Zentralklinikum der Westfa«lische Wilhelms-Universita«t, 48149 Mu«nster, Germany b
c
Received 23 April 1999; accepted 4 May 1999
Abstract The temperature- and solvent-induced denaturation of both the SCP2 wild-type and the mutated protein c71s were studied by CD measurements at 222 nm. The temperature-induced transition curves were deconvoluted according to a two-state mechanism resulting in a transition temperature of 70.5³C and 59.9³C for the wild-type and the c71s, respectively, with corresponding values of the van't Hoff enthalpies of 183 and 164 kJ/mol. Stability parameters characterizing the guanidine hydrochloride denaturation curves were also calculated on the basis of a two-state transition. The transition of the wild-type occurs at 0.82 M GdnHCl and that of the c71s mutant at 0.55 M GdnHCl. These differences in the half denaturation concentration of GdnHCl reflect already the significant stability differences between the two proteins. A quantitative measure are the Gibbs energies vG 0D (buffer) at 25³C of 15.5 kJ/mol for the wild-type and 8.0 kJ/mol for the mutant. We characterized also the alkyl chain binding properties of the two proteins by measuring the interaction parameters for the complex formation with 1-O-Decanyl-L-D-glucoside using isothermal titration microcalorimetry. The dissociation constants, Kd , for wild-type SCP2 are 335 WM at 25³C and 1.3 mM at 35³C. The corresponding binding enthalpies, vHb , are 321.5 kJ/ mol at 25³C and 72.2 kJ/mol at 35³C. The parameters for the c71s mutant at 25³C are Kd = 413 WM and vHb = 16.6 kJ/mol. These results suggest that both SCP2 wild-type and the c71s mutant bind the hydrophobic compound with moderate affinity. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Isothermal titration calorimetry; SCP2; Protein stability
1. Introduction Sterol carrier protein 2 (SCP2, also called non-speAbbreviations: SCP2, sterol carrier protein 2; 10-GLC, 1-O-Decanyl-L-D-glucoside; wt, wild-type; c71s, Cys-71CSer mutant form of sterol carrier protein 2; GdnHCl, guanidine hydrochloride; SDS-PAGE, sodiumdodecylsulfate polyarcylamide gel electrophoresis ; ITC, isothermal titration calorimetry; CD, circular dichroism; O0:1%;1cm 280nm , molar extinction coe¤cient of a 0.1% solution and a light path of 1 cm at 280 nm; vG 0D , molar standard change in Gibbs energy of unfolding; vg0 , speci¢c standard change in Gibbs energy; q, partition function ; K, equilibrium constant; Tm , transition temperature; vHb , binding enthalpy; Kd , dissociation constant; cmc, critical micellar concentration; L-FABP, liver fatty acid binding protein * Corresponding author. Fax: +49-251-832-9163; E-mail: [email protected] 0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 9 ) 0 0 1 1 4 - 4
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ci¢c lipid transfer protein) is a small, basic protein of molecular mass Mr = 13 380 Da, which binds fatty acids, long chain fatty acyl Coenzymes A, and sterols with a 1:1 molar stoichiometry and binding constants in the order of 0.23^0.4 WM for fatty acids [1], 4.5^2.8 nM for fatty acyl CoAs [2], 1 WM for sterols [3], and 0.3 WM for [3 H]cholesterol [4]. The protein occurs at high concentrations in peroxisomes, but signi¢cant concentrations are also found in the cytoplasm [5] and associated with other organelles, such as mitochondria [6,7] and the endoplasmatic reticulum [7]. In vitro, the protein activates the enzymatic conversion of 7-dehydrocholesterol to cholesterol by liver microsomes [8] and stimulates the acylCoA cholesterol acyltransferase-mediated esteri¢cation of intracellular cholesterol [9]. Furthermore, SCP2 promotes the exchange of various sterols [10,11] and phospholipids [12] between membranes in vitro. The latter activities led to the hypothesis that the protein participates in the intracellular transfer of phospholipids and sterols, especially cholesterol, also in vivo [1,12^16]. However, a recently characterized SCP2 knockout mouse model was not associated with a phenotype expected to result from defective cholesterol or phospholipid tra¤cking [17]. Instead, these mice showed a defect in the peroxisomal catabolism of the methyl-branched fatty acids 3,7,11,15-tetramethylhexadecanoic acid (phytanic acid) and 2,6,10,14-tetramethylpentadecanoic acid (pristanic acid) [17]. Because recombinant rat SCP2 protein exhibited high a¤nity binding of phytanoyl-CoA, it was presumed that the protein is involved in peroxisomal uptake and transport of this branched chain fatty acyl CoA. These ¢ndings suggest that the possible biological function of SCP2 is perhaps not predominantly associated with sterol transfer but involves a broader spectrum of activities relating to alkyl chain containing compounds. This interesting hypothesis prompted us to characterize quantitatively the interaction with 10-GLC of wild-type SCP2 protein and a mutated form in which cysteine 71 was replaced by serine. There was another reason for the particular choice of the c71s mutant. Activity studies with di¡erent deletion mutants had shown that helix A (residues 9^22), L-strand V (residues 100^102) and Asn-104
are essential elements for the lipid transfer activity of SCP2 [18]. Therefore, it had been assumed that the only cysteine, which is H-bonded to amino acid 102 plays an important role in the binding process. This hypothesis was obviously not correct, since it could be shown by lipid transfer assays that the mutant c71s had the same activity as the wild-type. However, the single point mutation has another intriguing e¡ect. We demonstrate in the present study that at 25³C wild-type and c71s mutant proteins differ signi¢cantly in stability, while the binding constants for 10-GLC are almost similar. A prerequisite for a role of SCP2 in the transport of alkyl chain containing compounds over the membrane in the form of a complex is the proof of the interaction between SCP2 and the corresponding ligand. Therefore, knowledge of the thermodynamics of complex formation is a necessary basis for the mechanistic understanding of the biological function. Since estimates of binding constants were published previously [1^4] we focused our e¡orts on the direct determination by ITC of the enthalpy changes associated with binding of 10-GLC as a model compound for hydrophobic alkyl chain containing ligands. 2. Materials and methods 2.1. Preparation of 10-GLC and SCP2 1-O-Decanyl-L-D-glucoside (10-GLC) was synthesized after a modi¢ed Ko«nigs^Knorr reaction from 1-K-bromo-2,3,4,6-tetra-O-acetylglucose with 1-decanol [19]. SCP2 and the c71s mutant were expressed and puri¢ed according to Seedorf et al. [18]. 2.2. Preparation of micellar solutions of 10-GLC 4.6 mM suspension of 10-GLC were prepared in bu¡er (bu¡er P: 43 mM KH2 PO4 /43 mM K2 HPO4 / 1.0 mM EDTA, pH 7.6). Before use the bu¡er was routinely ¢ltered through a 0.45 Wm ¢lter (Millipore, Badford, MA, USA) and degassed. The critical micellar concentration of 10-GLC was determined by measurements of the surface tension using the Wilhelmy method [20] (Fig. 4). The stock solution of
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4.6 mM 10-GLC was diluted with aliquots of bu¡er P to the various ¢nal concentrations between 0.1 and 1.5 mg/ml used in the present study. 2.3. CD measurements CD experiments were conducted using a CD6 Jobin-Yvon spectropolarimeter. Spectra were obtained from an average of ¢ve scans between 205 and 260 nm. Data were taken at 0.5 nm intervals with an integration time of 1 s. All spectra were corrected for the bu¡er signal (bu¡er B: 15 mM K2 HPO4 /15 mM KH2 PO4 /1 mM EDTA, pH 6.8). For all measurements the bu¡er was degassed and the sample compartment at the instrument was purged with a constant £ow of nitrogen (5 l/min). The light path was 1 cm at low concentrations of the protein or 0.01 cm at 1.6 mg/ml. The proteins were dialysed in bu¡er B. To avoid possible dimerization of the wild-type protein 5 Wl L-mercaptoethanol (obtained from Sigma) was added to the dialysis tubing and dialysed at RT (room temperature) for 1/2 h. Another dialysis overnight removed the L-mercaptoethanol from the protein solution prior to the measurements. SDS-PAGE (12.5%) was made under non-reducing conditions after the dialysis and veri¢ed the monomeric state of SCP2. Protein concentrations were determined by absorbance measurements at 280 nm using absorption coe¤cients of O0:1%;1cm 280nm A(wt) = 0.439 ml/(mg cm) and O0:1%;1cm (c71s) = 0.428 ml/(mg cm), respectively. The 280nm coe¤cients were calculated from the amino acid compositions following the procedure suggested by Gill and von Hippel [21]. Protein concentrations of the wild-type were 0.044 or 1.6 mg/ml, that of the mutant (c71s) was 0.055 mg/ml. Temperature scans were carried out at 222 nm using a programmable Haake F3 cryothermostat. The scan rate was 1 K/ min and the temperature ranges were 10³C to 95³C for the wild-type and 10³C to 85³C for c71s. The temperature was controlled and monitored using a 100 6 platinum resistance thermometer immersed in the protein solution. The van't Ho¡ enthalpy and the midpoint temperatures of the CD transition curves were determined by simulation of the transition curves according to a two-state mechanism using the following equations:
267
f T nb T ub T3nb TW q T31=q T 1 nb T a bW T3T m ub T c dW T3T m nb(T) is the temperature dependence of the native state baseline, ub(T) that of the unfolded state. Both are assumed to be linear. q T 1 K T is the partition function of a two-state transition involving the equilibrium constant K(T). The temperature dependence of the equilibrium constant depends on the standard Gibbs energy change vG0 for unfolding, which can be calculated from the experimental transition enthalpy vH0 and the transition temperature Tm according to: K T exp 3vG0 =RT and vG 0 vH 0 13T=T m
2.3.1. Stability studies In the isothermal denaturation studies using guanidine hydrochloride (GdnHCl) the protein concentrations were 0.05 mg/ml for the wild-type and 0.036 mg/ml for the mutant protein. CD measurements were made from 215 to 230 nm at 20³C with an integration time of 1 s and an increment of 0.5 nm. Data of ¢ve scans were averaged and corrected for the bu¡er signal. Denaturant-induced unfolding was monitored at 222 nm. The GdnHCl concentration was determined refractometrically according to Nozaki [22]. Evaluation of the isothermal unfolding curves was performed graphically as described below and numerically following the procedure of Santoro and Bolen [23] using the equations: 3222nm Y N mN GdnHCl Y D mD GdnHClWZ 1Z 2 Z exp3 vG 0D buffer mG GdnHCl= RT YN and YD are the intercepts, and mN and mD are
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the slopes of the pre- and post-unfolding baselines, respectively. [GdnHCl] is the concentration of the denaturant. vG0D (bu¡er) is the standard Gibbs energy change for the unfolding reaction in the absence of GdnHCl, and mG is the slope of the plot of vG 0app: versus GdnHCl concentration according to: vG 0app: vG0D buffer mG WGdnHCl The two-state equilibrium constants required to calculate vG 0app: were obtained from the plots of [3]222nm vs. GdnHCl concentration using linear extrapolated baselines as indicated by the dotted lines in Fig. 3. Independent determination of the baselines reduces the number of ¢t parameters in the Santoro and Bolen equation (Eq. 2) to two. The graphical analysis of the plot vG0app: vs. [GdnHCl] results in the standard Gibbs energy change, vG0D (bu¡er) by extrapolation to zero GdnHCl concentration as shown in the inserts of the corresponding ¢gure.
bu¡er, and the pure dialysis bu¡er was titrated with the solution of 10-GLC. The heat e¡ects observed in these controls were subtracted from the heat e¡ects associated with the titration of the protein with 10-GLC to obtain the net enthalpy of binding. The ligand (10-GLC) consists of a saturated alkyl chain of 10 carbon atoms linked to glucose in Lcon¢guration. The sugar is responsible for the good solubility in water. We determined the critical micellar concentration (cmc) of 10-GLC according to the Wilhelmy method [20]. The plot of surface tension vs. the concentration of 10-GLC provided a cmc value of 0.24 mg/ml (Fig. 4). This value rendered it possible to know the state of aggregation of 10GLC under all titration conditions. The values for the molar binding enthalpy, vHb , and the binding constant, Kb , were determined from non-linear least squares ¢ts of the data according to Eqs. 3 and 4. 1
vQ
2.4. Isothermal titration calorimetry (ITC) measurements The titration experiments were performed using an improved heat conduction twin microcalorimeter of Gill type, whose construction was based on the description by McKinnon et al. [24]. The calorimeter was calibrated by the dilution of propanol(1) into water [25] and, alternatively, by the protonation of the imidazole group of histidine [26] at 25³C and 35³C, respectively. The calorimeter was ¢lled with 800 Wl freshly prepared protein solution (50^73 WM). The calorimetrically active volume has been determined by calibration to be 410 Wl. Stable baselines were achieved after approximately 20 min after ¢lling. For the titration 5 Wl aliquots of a 4.6 mM 10GLC solution were injected automatically by means of a rotating stirrer syringe, which was controlled by a stepping motor. Each injection of 10-GLC (c = 1.48 mg/ml) produced a sharp exothermic signal which returned to the baseline within 2 min. An injection was made every 6 min to provide for su¤ciently long baselines before and after each measurement (Fig. 5). The reference cell contained water. To determine the heat contribution from dilution, two controls were made. The protein solution was titrated with dialysis
1 Pt nK b K b X t 3 1 Pt nK b K b X t 2 34Pt nK 2b X t 2 2K b VvH 0 3
The terms are de¢ned in the following manner [27]: vQ: corrected, cumulative heat (J); [P]t : total protein concentration (mol/l); n: number of binding sites ( = 1); Kb : intrinsic association constant (l/mol); [X]t : total concentration of ligand (mol/l); V: volume of the reaction cell ( = 410 Wl); vH0 : change of binding enthalpy per mol of ligand (J/mol). The alternative ¢t after Wiseman et al. [28] uses the following equation: 0 1 1 r Xr 13 3 1 dQ B C 2 2 A W vH W@0:5 p V dX tot X 2r 32X r 13r 1 r2 4
Binding is also assumed to exhibit 1:1 stoichiometry and the other terms are de¢ned as given in the following: Xr : ratio Xtot /Ptot ; Ptot : total protein concentration (mol/l); V: volume of the reaction cell ( = 410 Wl); Xtot : total ligand concentration in the cell after each injection; r: ratio 1/(Kb cPtot ), with Kb being the binding constant; vH0 : change of binding enthalpy per mol of ligand (J/mol).
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The vG0 and vS0 values at temperature T for binding were calculated from the standard thermodynamic relationship: 3RT ln K b vG0b vH 0b 3TvS 0b
5
3. Results 3.1. Stability of SCP2 and its c71s mutant 3.1.1. CD measurements of temperature-induced transitions Unfolding of SCP2 can be monitored conveniently by the disappearance of the CD absorption at 222 nm due to the loss of K-helical structure with the increase in temperature. This is illustrated in Fig. 1 which shows CD spectra of wild-type and mutant protein at di¡erent temperatures. The spectra of the native proteins have been determined at 10³C, those for the unfolded proteins at 96³C (SCP2 wt) and 85³C (c71s), respectively. Fig. 2 illustrates the corresponding transition curves of SCP2 wt (c = 0.044 mg/ ml) and c71s (c = 0.055 mg/ml) monitored at 222 nm. The symbols refer to the experimental points, the sigmoidal lines are the ¢tted curves using Eq. 1 and a non-linear least squares algorithm. The dotted lines indicate the CD absorption of the fully native and fully unfolded proteins as a function of temperature. It is evident that the transition curves can be described perfectly by the assumption of a two-state unfolding mechanism according to Eq. 1. The transition temperature Tm of the wild-type protein was found to be around 10 K higher than that of the mutant protein. This is indicative of the greater thermal stability of the wild-type protein. The thermodynamic parameters obtained from these measurements are summarized in Table 1. They are not dependent on protein concentration as shown for the wild-type protein by the fact that both the transition temperature and the transition enthalpy are identical for protein concentration di¡ering by a factor of about 40 (0.044 mg/ml and 1.6 mg/ml). Therefore, we can conclude that no oligomers were formed during the unfolding process. 3.1.2. Isothermal denaturant unfolding studies GdnHCl-induced denaturation curves of SCP2 wt
Fig. 1. CD spectra of SCP2 wild-type and c71s at di¡erent temperatures. The solid lines refer to the wild-type protein (c = 0.044 mg/ml) at 10 and 96³C. The dotted lines correspond to the c71s serine mutant (c = 0.055 mg/ml) at 10 and 85³C.
and c71s are shown in Fig. 3. The standard Gibbs energies of unfolding in the absence of the denaturant, vG 0D (bu¡er), were obtained either by plotting vG0app: vs. [GdnHCl] (inserts) and linearly extrapolating to zero GdnHCl concentration or by ¢tting the data to Eq. 2. The ¢ts are shown as solid lines in Fig. 3 [23]. These graphs show clearly the higher stability of the wild-type protein. Both the vG0 (bu¡er) values and the c50% concentrations are larger GdnHCl (c50% = 0.82 M for the wild-type and 0.55 M for GdnHCl the c71s mutant protein). All parameters are summarized in Table 2.
Table 1 Thermodynamic parameters determined from CD-monitored, thermal unfolding curves Tm (³C) vHvH (kJ/mol)
SCP2 wt
c71s
70.5 þ 0.5 183 þ 10
59.9 þ 0.9 164 þ 22
Two di¡erent experiments were performed with the wild-type protein at concentrations di¡ering by a factor of 40 (0.044 or 1.6 mg/ml). The di¡erence had no e¡ect on the transition temperature. The concentration of the c71s mutant was 0.055 mg/ml in both measurements. The parameters were evaluated on the basis of Eq. 1 using the two-state approximation.
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(M) YN (deg cm2 /dmol) mN (deg cm2 l/(dmol mol)) YD (deg cm2 /dmol) mD (deg cm2 l/(dmol mol)) mG (kJ l/mol2 ) vG0D (bu¡er)Fit (kJ/mol) vG0D (bu¡er) (kJ/mol)
SCP2 wt
c71s
0.82 39226 0 33820 565 319.4 15.5 15.5
0.55 38100 0 33278 385 313.6 7.5 8.4
Two methods have been applied. The value vG0D (bu¡er)Fit has been obtained from ¢tting the data to Eq. 2. To avoid the errors involved in a six-parameter ¢t, the pre- and post-transitional baselines were determined independently by linear least squares ¢ts. This reduces the number of the ¢t parameters to two. vG0D (bu¡er) is the result of the linear extrapolation to zero GdnHCl concentration as shown in the inserts of Fig. 3. Protein concentrations were 0.05 mg/ml for the wild-type protein and 0.036 mg/ml for the c71s mutant. The temperature was 20³C.
Fig. 2. Thermal transition curve of SCP2 wt (upper panel) and c71s (lower panel). The transition was monitored by the change in mean residue ellipticity at 222 nm using a heating rate of 1 K/min. Upper panel: SCP2 wt concentration was 0.044 mg/ ml. Open squares refer to the experimental points. The solid line represents the simulated curve according to Eq. 1. The dotted lines re£ect the linear baselines. Lower panel: c71s concentration was 0.055 mg/ml. Open circles refer to the experimental points. The solid line represents the simulated curve according to Eq. 1. The dotted lines re£ect the linear baselines.
3.2. ITC measurements on the interaction of 10-GLC with SCP2 3.2.1. Determination of the critical micellar concentration (cmc) of 10-GLC The critical micellar concentration of 10-GLC was determined by measuring the surface tension of a 10GLC monolayer using the Wilhelmy method. The results are illustrated in Fig. 4. The intercept of the two linear-regression lines occurs at the critical micellar concentration. The value found at 20³C is of 0.24 mg/ml. Using this value one can calculate that throughout the titration 10-GLC occurs in the calorimetric vessel in the monomeric non-micellar state (14U5 Wl of the stock solution of 10-GLC 1.48 mg/
ml into an active volume of 410 Wl results in the ¢nal concentration of 0.22 mg/ml of 10-GLC). 3.2.2. Determination of the binding parameters Fig. 5A shows the uncorrected enthalpimetric titration data. Each peak represents the heat e¡ect resulting from injecting of 5 Wl of a 4.6 mM (1.48 mg/ml) solution of 10-GLC into a SCP2 solution (50^73 WM) in bu¡er P at 25³C. Fig. 5B illustrates the control experiment, i.e. the titration of 10-GLC into bu¡er. Dilution of 10-GLC is associated with a signi¢cant negative heat evolution. No heat e¡ect was observed for the dilution of SCP2 wt protein at 25³C in the corresponding control titration. But we observed an exothermic heat e¡ect of around 340 to 3160 WJ associated with protein dilution for SCP2 c71s at 25³C and for SCP2 wt at 35³C. The raw data were corrected for these heat e¡ects. Because these heats of dilution are in the order of the heats of ligand binding proper correction is very important. The binding data show that the binding enthalpy for the wild-type protein changes from 321.5 kJ/mol at 25³C to 72.2 kJ/mol at 35³C. The cumulative integral heats corrected for the controls are shown as a function of the total concentration of 10-GLC in Fig. 6A. The solid curve has been calculated using Eq. 3.
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Table 3 Thermodynamic parameters for the interaction of SCP2 with 10-GLC at 25³C and 35³C.
Kd /mM vG0binding /kJ/mol vg0binding /J/g vH0binding /kJ/mol -TvS0binding /kJ/mol
25³C SCP2 wt
25³C c71s
35³C SCP2 wt
0.3 þ 0.02 319.8 þ 0.1 31.48 321.5 þ 0.7 +1.7 þ 0.6
0.4 þ 0.001 319.3 þ 0.1 31.44 +16.6 þ 0.6 335.9 þ 0.7
1.3 þ 0.1 317.1 þ 0.2 31.28 +72.2 þ 3.4 389.2 þ 3.2
The data are the average values from two di¡erent titrations and the analysis procedures given in the references [27,28].
4. Discussion
Fig. 3. GdnHCl-induced CD denaturation curve of SCP2 wt (upper panel) and c71s (lower panel) at 20³C. Upper panel: The protein concentration was 0.05 mg/ml. The open squares show the measured values, the solid line refers to the ¢tted curve according to Eq. 2. The dotted lines correspond to the pre- and post-unfolding baselines. From the plot of vG0app: vs. [GdnHCl] the value of vG0D (bu¡er) = 15.5 kJ/mol is obtained (r = 0.976). Lower panel: The protein concentration was 0.036 mg/ml. The open circles refer to the experimental data, the solid line shows the ¢tted curve according to Eq. 2. The dotted lines correspond to the pre- and post-unfolding baselines. From the plot vG 0app: vs. [GdnHCl] the value of vG0D (bu¡er) = 8.4 kJ/mol is obtained (r = 0.965).
The binding enthalpy vHb and the binding constant Kb have been calculated on the basis of a stoichiometry of 1:1. This appears to be justi¢ed in view of the results of Schroeder et al. [3,29]. For comparison Fig. 6B shows the alternative ¢t according to Wiseman et al. [28] using Eq. 4. Both ¢ts provide identical results. The corresponding parameters are summarized in Table 3. The numbers given here are mean values based on two independent titration series and two di¡erent ¢tting procedures. The values agree within standard deviations. Because of the lack of mutant protein the titration of c71s could not be performed at 35³C.
The present study is concerned with the quantitative description of the energies involved in the binding of 10-GLC to SCP2 wt and c71s mutant protein. Since the sterol carrier protein has been reported to exhibit signi¢cant a¤nity for fatty acids and fatty acyl CoAs, 10-GLC is an excellent ligand to mimic short alkyl chain binding characteristics. On the basis of the cmc determination we can assume that the 10GLC concentration is always below the cmc during the titration experiment. This assumption is supported by the identical magnitude of the heat e¡ects associated with each injection of 5 Wl samples (see
Fig. 4. Determination of the critical micellar concentration of 10-GLC at 20³C. Bu¡er: 43 mM potassium phosphate, 1 mM EDTA, pH 7.6. The stock solution of 4.6 mM 10-GLC was diluted with bu¡er to the respective ¢nal concentrations used for the langmuir balance. The squares correspond to the experimental values and the dotted lines are linear ¢ts. The critical micellar concentration of 0.24 mg/ml results from the point of intersection.
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Fig. 5. Titration of SCP2 wt with 10-GLC (upper frame) and control (titration of bu¡er with 10-GLC, lower frame) at 25³C. Titrand: 800 Wl of 69 WM SCP2 wt solution in bu¡er P, pH 7.6 (15 mM potassium phosphate, 1 mM EDTA) (upper frame) or 800 Wl bu¡er P, pH 7.6 (lower frame). Addition: 14U5 Wl of 4.6 mM 10-GLC solution in bu¡er P, pH 7.6.
Fig. 5B). These peaks re£ect the heat e¡ects involved in the transition of 10-GLC going from the micellar into a monomeric fully hydrated state. Therefore, the thermodynamic parameters for the binding reaction refer to complex formation with monomeric 10GLC. The ITC experiments show clearly, that SCP2 binds 10-GLC, though with rather low a¤nity as indicated by the dissociation constant of 0.3 mM. The lower a¤nity of SCP2 for 10-GLC compared with that for long chain fatty acids, fatty acyl CoA or dehydroergosterol is likely the result from the shorter carbon chain length. This interpretation is in line with the results of Frolov et al. [30], who observed an optimal interaction of SCP2 with fatty acids containing 14^22 CH2 groups. In previous studies by Rolf et al. [31], two binding constants were reported for the binding of oleic acid to L-FABP. The dissociation constant for the ¢rst
Fig. 6. Determination of the binding parameters according to [27]. A: Integrated and dilution corrected data (open squares) are shown as function of concentration of 10-GLC after each injection at 25³C. The best-¢t curve (line) is shown calculated according to Eq. 3 with the assumption of one binding site (n = 1). The ¢t parameters are Kd = 0.3 mM and vH0 = 321.5 kJ/mol. Titrand: 800 Wl of 73 WM SCP2 wt in bu¡er P, pH 7.6. Addition: 14U5 Wl of a 4.6 mM solution of 10-GLC in bu¡er P, pH 7.6. B: Determination of the binding parameters according to [28]. Changes of heat per injection of 10-GLC (open circles) are shown as function of the ratio of total ligand to total macromolecule concentration at 25³C and pH 7.6. The solid line refers to the theoretical curve calculated according to Eq. 4. Titrand: 800 Wl of 73 WM SCP2 wt in bu¡er P, pH 7.6. Addition: 14U5 Wl of a 4.6 mM solution of 10-GLC in bu¡er P, pH 7.6.
binding site was 0.26 WM that for the second 4.9 WM. The corresponding binding enthalpy values were 316 kJ/mol and +22 kJ/mol, respectively. It is worth noting that the enthalpy value observed for 10-GLC binding to SCP2 wt at 25³C is of comparable magnitude (322 kJ/mol) as that observed by Rolf et al. for the ¢rst binding site.
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From spectroscopic studies involving time-resolved tryptophan £uorescence an ellipsoidal shape of the SCP2 had been deduced with the lipid binding site perpendicular to the long symmetry axis of the protein, in analogy to the shape of the phosphatidylcholine transfer protein [32]. This result has been con¢rmed by recent NMR studies [33]. These NMR data [33] have shown that SCP2 consists of three K-helices (residues 9^22, 25^30, 78^84) and a ¢ve-stranded L-sheet that comprises the residues 33^41, 47^54, 60^62, 71^76 and 100^102. The Lstructure is supposedly a central feature of the molecular architecture of the protein. Depending on the relative orientation of the residues 77^99 (around helix C) and the C-terminus to the L-structure the formation of a hydrophobic pocket is possible. Observations based on site-directed mutagenesis of SCP2 [18] favor the existence of a speci¢c lipid binding site involving the residues (77^99 and the carboxy-terminus). The replacement of Cys in position 71 with Ser results in only a small change in Kd from 0.3 mM to 0.4 mM which corresponds to a minor change in the standard Gibbs energy from 319.8 kJ/ mol to 319.3 kJ/mol at 25³C. These results are consistent with the ¢nding of identical lipid transfer activities for wild-type and mutated protein and they suggest that the position 71 is not important for the binding properties [18]. Interpretation of the binding constants shows that the temperature dependence of the binding enthalpy is large. The dissociation constant of SCP2 wt increases from 0.3 mM at 25³C to 1.3 mM at 35³C. The very similar binding properties at 25³C contrast markedly with the large di¡erences in stability that have been observed for the two proteins, both with regard to temperature- and GdnHCl-induced unfolding. The c50% GdnHCl value of the wild-type protein as well as the Gibbs energy is approximately twice as large as the corresponding value for the serine mutant. The conservation of the binding a¤nity is accomplished with vastly di¡erent energy and entropy parameters. While the binding of 10-GLC to the wildtype protein is associated with a favorable enthalpy change of 321.5 kJ/mol and a slightly unfavorable entropy term of 3TvS0 = +1.7 kJ/mol, the complex formation with the mutant is characterized by a positive enthalpy change of +16.6 kJ/mol but an over-
273
compensating favorable entropy contribution to vG0binding of 3TvS0 = 335.9 kJ/mol. It is the latter term that apparently drives the reaction of the mutant protein at 25³C. The signs of the binding enthalpies and entropies for the mutant protein suggest that the binding of 10-GLC induces energy consuming restructuring processes which are associated with favorable entropy changes. A possible mechanism would be the release of surface bound water molecules into the bulk phase on binding of the hydrophobic ligand. Since the mutant protein is signi¢cantly less stable than the wild-type protein water release could also result from restructuring of parts of the protein other than the direct hydrophobic binding pocket. Actually this has to be assumed, because the number of water molecules replaced in the binding pocket can be expected to be roughly comparable for both proteins. It is an intriguing idea to envisage the ligand as creating its own optimal binding pocket. Acknowledgements H.-J.H. gratefully acknowledges support by the DFG (Deutsche Forschungsgemeinschaft) and the Fonds der Chemischen Industrie.
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