Langmuir 2005, 21, 4299-4307
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Interactions of Humicola insolens Cutinase with an Anionic Surfactant Studied by Small-Angle Neutron Scattering and Isothermal Titration Calorimetry Anders D. Nielsen,†,‡,§ Lise Arleth,|,⊥ and Peter Westh*,† Department of Life Sciences and Chemistry, Roskilde University, 1 Universitetsvej, P.O. Box 260, DK-4000 Roskilde, Denmark, Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark, and Danish Polymer Centre, Risoe National Laboratory, DK-4000 Roskilde, Denmark Received November 3, 2004. In Final Form: January 12, 2005 The interaction of cutinase from Humicula insolens (HiC) and sodium dodecyl sulfate (SDS) has been investigated by small-angle neutron scattering (SANS) and isothermal titration calorimetry (ITC). The concerted interpretation of structural and thermodynamic information for identical systems proved valuable in attempts to elucidate the complex modes of protein-detergent interaction. Particularly so at the experimental temperature 22 °C, where the formation of SDS micelles is athermal (∆H ) 0), and the effects of protein-detergent interactions stand out clearly in the thermograms. It was found that the effect of SDS on cutinase depended strongly on the sample composition. Thus, addition of SDS corresponding to a molar ratio, nS ) nSDS/nHiC of about 10, was associated with the formation of HiC/SDS aggregates, which include more than one protein molecule. The SANS results suggested that on the average such adducts contained two HiC, and the ITC traces showed that they form and break down slowly. At slightly higher SDS concentrations (nS ) 10-25) these “dimers” dissociated, and the protein denatured. The denaturation showed the characteristic positive enthalpy change, but the SDS denatured state of HiC was unusually compact with a radius of gyration close to that of the native conformation. Further titration with SDS was associated with exothermic binding to the denatured protein until the saturation point at about nS ) 90. At this point, the free monomer concentration was 2.2 mM and the binding number was ∼40 SDS/HiC. Interestingly, this degree of SDS binding (∼0.5 g of SDS/g of HiC) is less than half the amount bound to typical water-soluble proteins.
1. Introduction Cutinases are extracellular enzymes excreted by various fungi. Their biological role is to hydrolyze ester bonds in cutin, a lipid-polyester, which is found in the cuticle of higher plants.1,2 The enzyme belongs to the serine R/βhydrolase family3,4 and is able to hydrolyze a range of water-soluble esters and long-chain triglycerides.2,5 These properties have made cutinases possible candidates for use in detergent applications with the purpose of facilitating the removal of fat-based stains. However, the use of cutinase in detergents is challenging due to the presence of anionic surfactants,6 which have been shown to diminish the catalytic activity and stability of the enzyme.2,7-9 Cutinases are also used as biocatalysts in organic synthesis † Department of Life Sciences and Chemistry, Roskilde University. ‡ Novozymes A/S. § Present address: Novo Nordisk A/S, Novo Alle ´ , DK-2880 Bagsvaerd, Denmark. | Danish Polymer Centre, Risoe National Laboratory. ⊥ Present address: Department of Natural Sciences, Royal Veterinary and Agricultural University, Bu¨lowsvej 17, DK-1870 Frederiksberg C, Denmark.
(1) Kolattukudy, P. E. Science 1980, 208, 990-1000. (2) Kolattukudy, P. E. In Lipases; Borgstro¨m, B., Brockman, H. L., Eds.; Elsevier: Amsterdam, 1984; pp 470-504. (3) Schrag, J. D.; Cygler, M. Methods Enzymol. 1997, 284, 85-107. (4) Martinez, C.; De Geus, P.; Lauwereys, M.; Matthyssens, G.; Cambillau, C. Nature 1992, 356, 615-618. (5) Carvalho, C. M. L.; Cabral, J. M. S.; Aires-Barros, M. R. Enzyme Microb. Technol. 1999, 24, 569-576. (6) Creveld, L. D.; Amadei, A.; Van Schaik, R. C.; Pepermans, H. A. M.; De Vlieg, J.; Berendsen, H. J. C. Proteins Struct. Funct. Genet. 1998, 33, 253-264. (7) Goncalves, A. M. D.; Aires-Barros, M. R.; Cabral, J. M. S. Enzyme Microb. Technol. 2003, 32, 868-879.
and in this context it is advantageous to encapsulate the cutinase in reversed micelles.5 The anionic surfactant bis(2-ethylhexyl) sodium sulfosuccinate (AOT) is frequently used because it readily forms reversed micelles. However, AOT has been reported to have destabilizing effects on cutinase.10,11 The mechanism of anionic surfactant induced inactivation has been suggested to be unfolding of the protein due to binding of anionic surfactants. Furthermore, kinetic data indicate that anionic surfactants, like sodium dodecyl sulfate (SDS), at low concentration interact with the active site in a somewhat complex way. It has been suggested that SDS leads to local conformational changes near the active site that result in inhibition, partial reversible unfolding, and ultimately inactivation.8 Thus, Kolattukudy,2 based on spectroscopic observations on Fusarium solani pisi cutinase (FsC), reported that monomeric SDS binds and causes local conformational changes around the active site. In addition, binding results with labeled SDS suggested that at low concentration two surfactant molecules bind to a cutinase molecule. Increasing the concentration resulted in a significant increase in the number of bound SDS molecules. Kinetic observations indicate that SDS is a competitive inhibitor at low concentration whereas a more complex mechanism is necessary to account for the interactions at higher (8) Pocalyko, D. J.; Tallman, M. Enzyme Microb. Technol. 1998, 22, 647-651. (9) Goncalves, A. M.; Serro, A. P.; Aires-Barros, M. R.; Cabral, J. M. S. Biochim. Biophys. Acta 2000, 1480, 92-106. (10) Sebastiao, M. J.; Cabral, J. M. S.; Aires-Barros, M. R. Biotechnol. Bioeng. 1993, 42, 326-332. (11) Melo, E. P.; Costa, S. M. B.; Cabral, J. M. S. Photochem. Photobiol. 1996, 63, 169-175.
10.1021/la047299+ CCC: $30.25 © 2005 American Chemical Society Published on Web 04/02/2005
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surfactant concentrations.8 Recent spectroscopic studies indicate that submicellar concentrations of AOT initiate conformational changes in the tertiary structure due to the disruption of essential electrostatic interactions in the native structure.7 Earlier studies on FsC indicate that inactivation by SDS occurs at much lower concentrations than those needed for unfolding.2,8 Hence, a half-life of 12.5 min for FsC in 0.5 mM SDS has been reported.8 These observations imply, as recently suggested by Ternstro¨m,12 that low concentrations of anionic surfactant can cause local unfolding of the active site region and thereby cause a reduction in catalytic activity. Time-resolved fluorescence studies on FsC have revealed an increased correlation time with increasing concentrations of lithium dodecyl sulfate (LDS).13 The observations indicate that a complex four times the size of the monomeric cutinase is formed in the presence of ∼0.6 mM LDS. Increasing the surfactant concentration further apparently decreases the size of the complexes. All these data on cutinase-surfactant interrelationships is based on FsC, whereas similar information on other cutinases such as the one excreted by Humicola insolens remains sparse. However, Ternstro¨m12 recently reported that 50% of Humicola insolens cutinase (HiC) is unfolded at an AOT concentration of 0.3 mM. In this paper, we have studied the interactions of SDS with HiC at surfactant concentrations ranging from submicellar to high concentrations far beyond the critical micelle concentration (cmc). Complementary techniques have been used to elucidate the interactions. Calorimetric data obtained by isothermal titration calorimetry (ITC) provides direct, quantitative information about interaction and transition processes in the HiC/SDS system. Smallangle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) both provide structural information on a length scale of 10-1000 Å.14 This is the relevant length scale for investigating protein-surfactant interactions, and both techniques have previously been used to obtain detailed structural information about protein-surfactant interactions and protein unfolding.15-17 We have analyzed HiC/SDS with SANS and ITC, and the combined interpretation of the data obtained from these two experimental techniques has made it possible to rationalize molecular events in this system as a function of SDS/HiC molar ratio. 2. Experimental Section 2.1. Materials. Recombinant HiC was expressed in Aspergillus oryzae and purified to >95%, determined by SDS/PAGE, at Novozymes A/S, Bagsvaerd, Denmark. The enzyme was extensively dialyzed, at 5 °C, against 50 mM TRIS, 2 mM EDTA, pH 7.0 buffer. The following chemicals were used: TRIS (>99%, Merck, Darmstadt, Germany), ethylenediaminetetraacetic acid, EDTA (>99%, Merck, Darmstadt, Germany), sodium dodecyl sulfate, SDS (>99%, Fluka, Buchs, Switzerland), and D2O (>99.9%, Merck, Darmstadt, Germany). Samples for SANS. The following samples were prepared for the SANS experiments: pure protein, 0.1 mM HiC in 50 mM TRIS, 2 mM EDTA, pH 7 buffer; pure SDS, 7.5 mM SDS, 15 mM SDS, and 30 mM SDS in 50 mM TRIS, 2 mM EDTA, pH 7 buffer; mixed HiC-SDS, 0.1 mM HiC with, respectively, 0.45, 1.5, 5.1, (12) Ternstro¨m, T. Protein Folding Studied by Site Directed Mutagenesis. Lund University, Sweden, 2002. (13) Egmond, M. R.; van Bemmel, C. J. Methods Enzymol. 1997, 284, 119-129. (14) Guinier, A. and Fournet, G. Small-angle Scattering and X-rays; John Wiley & Sons: New York 1955. (15) Chen, S.-H. and Teixeira, J. Phys. Rev. Lett. 1986, 57, 25832586. (16) Ibel, K.; May, R. P.; Kirschner, K.; Szadkowski, H.; Mascher, E.; Lundahl, P. Eur. J. Biochem. 1990, 190, 311-318. (17) Santos, S. F.; Zanette, D.; Fischer, H.; Itri, R. J. Colloid Interface Sci. 2003, 262, 400-408.
Nielsen et al. 7.5, 9.9, 15, 22.5, and 30 mM SDS in 50 mM TRIS, 2 mM EDTA, pH 7 buffer. In this way SDS/HiC molar mixing ratios of 4.5, 15, 51, 75, 99, 150, 225, and 300 were obtained. The samples for the SANS experiments were prepared using D2O for the buffer solutions. This was done in order to optimize the neutron scattering contrast of the aggregates. The pure (i.e., protein-free samples) SDS samples were prepared with 100% D2O, while the pure HiC samples and the mixed HiC/SDS samples were prepared with 90% D2O and 10% H2O. The samples were measured in 1 mm Hellma quartz cells. 2.2. Small-Angle Neutron Scattering (SANS) Experiments. Small-angle neutron scattering experiments were performed at the SANS-II instrument at the Swiss Spallation Source at the Paul Scherrer Institute in Switzerland. Using two different combinations of neutron wavelengths and sample-to-detector distances, a q-range from 0.01 to 0.3 Å-1 was covered. The SANS data were azimuthally averaged and background subtracted by the standard approach used at the facility and normalized to absolute units of differential scattering cross section per unit volume (cm2/cm3) by division by the scattering spectrum of H2O.18 The wavelength spread ∆λ/λ was 10.5%. The resolution effects arising from wavelength spread and finite collimation were taken into account in the data analysis by convolution with the appropriate resolution function at each setting.19 This is done automatically in the programs used for the data analysis. 2.3. Isothermal Titration Calorimetric (ITC) Experiments. The calorimetric measurements were conducted on a MCS-ITC (MicroCal Inc., Northampton, MA) isothermal titration calorimetry equipment.20 The reference cell was filled with water. In a typical experiment, the sample cell was loaded with a solution of 0.1 mM HiC. The cell solution was titrated with 100-104 5 µL aliquots of 30 mM SDS. This was accomplished by making approximately 50 injections in the initial experimental trial. Upon refill of the syringe, another 50 injections were made into the resulting cell solution obtained in the first trial. The two data files were merged into a single file using the ITC-merge software written by Dr. Bent W. Sigurskjold, University of Copenhagen. The critical micelle concentration (cmc) and micellization enthalpy change were obtained at different temperatures by injection of concentrated SDS solutions (30 mM) into a buffer solution as described previously.21 All solutions were degassed by stirring under vacuum before use. The heat signals from the ITC were integrated using the Origin software supplied by MircoCal Inc. This software was also used to calculate the concentrations of surfactant and protein throughout the titrations. These calculations are based on two assumptions. First, it is assumed that the liquid displaced from the (full) calorimetric cell during an injection has the same composition as the cell contents prior to the injection. Second, once displaced from the cell, liquid in the access shaft of the calorimetric cell is presumed not mix with the contents of the cell. For the current protocols, the protein concentration was reduced by 25-30% at the end of the experiment. 2.4. SANS Theory and Methods. 2.4.A. The Small-Angle Scattering Intensity and the Forward Scattering, I(0). For an isotropic suspension of monodisperse particles the elastic small-angle scattering intensity as a function of the scattering vector q can be expressed by (see, e.g., ref 22)
I(q) ) φV(∆F)2P(q)S(q)
(1)
where φ is the volume fraction of the particles, V is the volume of a single particle, and ∆F is the excess scattering length density of the particles as defined by F - F0, where F is the integrated particle scattering length per unit volume and F0 is the integrated solvent scattering length per unit volume. P(q) is the scattering form factor and describes the q-dependence of the scattering (18) Jacrot, B. Rep. Prog. Phys. 1976, 39, 911. (19) Pedersen, J. S.; Posselt, D.; Mortensen, K. J. Appl. Crystallogr. 1990, 23, 321. (20) Wiseman, T.; Wiliston, S.; Brandts, J. F.; Lin, L.-N. Anal. Biochem. 1989, 179, 131-137. (21) Nielsen, A. D.; Borch, K.; Westh, P. Biochim. Biophys. Acta 2000, 1479, 321-331. (22) Porod, G. In Small-angle X-ray scattering; Glatter, O., Kratky, O., Eds.; Academic Press: New York, 1982; pp 17-51.
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from the single particle. P(q) is normalized such that P(0) ) 1. S(q) is the effective structure factor and describes the interparticle interactions. S(q) is normalized such that S(q) f 1 as φ f 0. At low concentrations (sufficient for S(0) ≈ 1) and q ) 0, eq 1 reduces to
I(0) ) φV(∆F)2 For a dilute suspension of n different types of particles, the total forward scattering is simply the sum of the scattering from the single types of components n
I(0) )
∑ φ V (∆F )
2
i
i
i
become non-negligible and the corresponding p(r) will have an oscillatory behavior at high r values. By omission of the lowest q values in the IFT analysis, the structure factor effects can to a good approximation be filtered out of the p(r) and the information corresponding to the pure form factor obtained.26 This approach has been used in the case of some of the most concentrated samples. A Fortran program developed by Pedersen is used for the IFT.27 The program uses a smoothness constraint that is a modification of the constraint used in Glatter’s original procedure.25 The program also provides an estimate of the incoherent background and thus allows for separating the experimental scattering intensity into a contribution from the small-angle scattering and a contribution from incoherent background.
i)1
In the case of a suspension of particles of similar excess scattering length density (e.g., polydisperse particles) I(0) can be expressed as
I(0) ) φV(∆F)2 n φi is the total volume fraction of the particles and where φ ) ∑i)1 V h is the volume-fraction weighted mean particle volume defined n n φiVi)/∑i)1 φi. In a suspension of particles of different by V h ) (∑i)1 composition and size, the excess scattering length density will generally vary from particle to particle and I(0) becomes
I(0) ) φV(∆F)2 where
∑
∑
n i)1
V(∆F)2 ) (
φiVi(∆Fi)2)/
n i)1
φi
In all cases I(0) gives information on the average size and composition of particles in solution. Furthermore, the fact that I(0) is independent of the detailed structure and conformation of the particles (assuming S(0) ) 1), makes it relatively easy to perform model calculations that can be used for interpretation of the forward scattering. 2.4.B. The Pair-Distance Distribution Function. The pairdistance distribution function p(r) gives the distribution of distances between pairs of scattering centers in a sample, where the distances are weighted by the excess scattering length of each scattering center. p(r) ) r2γ(r) where γ(r) is the correlation function and the Fourier transform of the scattering intensity, I(q). For an isotropic solution p(r) and I(q) are therefore related via
I(q) ) 4π
∫
∞
0
p(r)
sin qr dr qr
For samples sufficiently dilute that interaction effects can be neglected, the p(r) only contains information about the distribution of distances within the single aggregates. This means that p(r) equals zero for distances larger than the maximal distance, Dmax, within a single aggregate. The overall size and shape of the aggregates can in many cases be determined from a visual inspection of p(r).23,24 Furthermore, the radius of gyration, RG, and the forward scattering, I(0), of the aggregates can be calculated by integration over p(r)25
∫ ) 2∫
Dmax
2
RG
0
r2p(r) dr
Dmax
0
p(r) dr
and
I(0) )
∫
Dmax
0
p(r) dr
We determined the p(r), RG, and I(0) from the experimental I(q) by means of the method of indirect Fourier transform (IFT).23-25 For slightly concentrated samples, the structure factor effects
3. Results 3.1. ITC Results. 3.1.1. Thermodynamics of Demicellization. Previous calorimetric experiments using concentrated SDS solutions have revealed that addition of SDS to a solution free of the surfactant will result in heat signals proportional to the demicellization enthalpy change (∆Hdemic) (e.g., ref 21). Therefore, we determined the temperature dependence of ∆Hdemic to find a temperature where the demicellization heat is zero (Figure 1). The obtained ∆Hdemic and cmc values at temperatures between 22 and 45 °C are listed in Table 1. It is evident that ∆Hdemic is approximately zero at 22 °C and therefore all calorimetric experiments with HiC were conducted at this temperature to minimize the heat contribution from the demicellization process. A linear correlation between ∆Hdemic and temperature is observed which corresponds to a change in heat capacity of -470 ( 11 J mol-1 K-1. From Table 1 it is also evident that cmc increases from 2.2 mM at 22 °C to 2.8 mM at 45 °C. 3.1.2. Enthalpogram of HiC/SDS. Titration of HiC with the anionic surfactant SDS results in characteristic enthalpograms as demonstrated in Figure 2. To cover a large SDS concentration range, and at the same time maintain a sufficient resolution of the enthalpogram, it has been necessary to combine two consecutive titration experiments. The rather complex course of the titration trials was found to be highly reproducible. At low SDS concentration, corresponding to low SDS: HiC molar ratios (nS ) nSDS/nHiC), an endothermic heat flow is observed with maximum at 0.36 mM SDS (nS ) 3.7). In the SDS concentration range from 0.36 to 2.2 mM (nS ) 3.7-25) a significant shift in heat flow from endothermic to exothermic is observed. A closer look at the injection peaks in this concentration range reveals that more time is needed to reach the baseline after injection, which suggests that a slow process takes place (see inset in Figure 2). The exothermic heat flow continues up to approximately 5 mM SDS (nS ∼ 60). Finally, the injection peaks gradually become zero and the inflection point is about 5.9 mM (nS ∼ 75). The onset of the inflection indicates that a fraction of the injected SDS solution will remain in a micellar form. Therefore, the inflection point is the apparent cmc value of SDS in the presence of HiC and corresponds to an increase in cmc of ∼3.7 mM (23) Glatter, O. In Small-angle X-ray scattering; Glatter, O., Kratky, O., Eds.; Academic Press:New York, 1982; pp 167-196. (24) Glatter, O. In Neutron, X-ray and light scattering: Introduction to an investigative tool for colloidal and polymeric systems; Lindner, P., Zemb, T., Eds.; Elsevier Science Publishers B.V.: Amsterdam, 1991; pp 33-82, (25) Glatter, O. J. Appl. Crystallogr. 1977, 10, 415-421. (26) Mu¨ller, K. and Glatter, O. Macromol. Chem. 1982, 183, 465479. (27) Pedersen, J. S.; Hansen, S.; Bauer, R. Eur. Biophys. J. 1994, 22, 379-389.
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Figure 1. Titration of buffer with 10 µL aliquots of 30 mM SDS solution at temperatures between 22 and 45 °C (see legend). The heat signals signify the breakdown (demicellization) of micelles upon dilution. cmc corresponds to the SDS concentration at inflection point as previously described by Paula et al. (Paula, S.; Su¨s, W.; Tuchtenhagen, J.; Blume, A. J. Phys. Chem. 1995, 99, 11742-11751.) Table 1. Demicellization Enthalpies and cmc Values of SDS in 50 mM TRIS, 2 mM EDTA, pH 7 buffer at 22-45 °C temp, °C
∆Hdemic, kJ mol-1
cmc, mM
22 30 38 45
0.1 3.9 7.9 10.8
2.2 2.3 2.4 2.8
compared to SDS in pure buffer. At SDS concentrations higher than 8.3 mM (nS > 120) no heat of interaction is observed. All HiC/SDS SANS experiments were carried out in buffer prepared with 90% D2O and 10% H2O, whereas the above ITC experiments have been conducted in H2O buffer. To evaluate the possible effect of D2O on the SDS/HiC enthalpogram, we also conducted ITC experiments in D2O buffer. Figure 3 shows that the overall enthalpogram obtained in D2O buffer is similar to that of the H2O buffer. Hence, the shifts between endothermic and exothermic heat flow are found at similar surfactant concentrations. Therefore we find it reasonable to compare the SANS results with the ITC enthalpograms. However, the peaks are slightly more exothermic in the D2O buffer, as expected for binding processes including the formation of hydrogen bonds.28 3.2. SANS Results. 3.2.1. Pure HiC and Pure SDS. Samples containing, respectively, pure protein and pure SDS were measured first. The scattering data and the corresponding pair distance distribution functions are plotted in Figure 4. The almost bell-shaped p(r) obtained for the pure SDS is in agreement with the formation of nearly globular micelles. The p(r) for HiC is bell shaped at low r values and has a small tail at high r values. This shows either that HiC is slightly elongated or that a small population of either dimers or molecules larger than the HiC is present in the sample. Both micelles and proteins are rather small. Consequently the absolute scattering intensities are low and the error bars of the single I(q) points, as well as the obtained values for I(0) and RG, significant. As seen from Figure 4 this is mainly the case for the measurement on pure HiC. The results for I(0), (28) Chervenak, M. C.; Toone, E. J. J. Am. Chem. Soc. 1994, 116, 10533-10539.
Figure 2. Calorimetric data from the titration of a 98 µM HiC solution with 30 mM SDS in 50 mM TRIS, 2 mM EDTA, pH 7 at 22 °C. (Top) Raw heat signal from 101 injections of 5 µL SDS aliquots (except injection 1 and 51 which are only 1 µL). The inset shows the heat flow of first 10 injections, where a significant broadening of the peaks can be observed from the fourth injection. (Bottom) Each peak has been integrated to obtain the enthalpy change per mole of SDS added.
Figure 3. Comparison of calorimetric data obtained in 100% H2O buffer and 90% D2O + 10% H2O buffer.
RG, and Dmax as obtained by the IFT analysis are listed in Table 2. As seen from these values, HiC monomers and SDS micelles are of comparable size. The forward scattering from the pure protein can be expressed by
(
IP(0) ) NPνP2
)
BP - F0 νP
2
where NP denotes the protein concentration (number density), νP is the partial specific molecular volume of the
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Figure 4. SANS data from HiC (A) and SDS (C) in aqueous buffer. The fitted lines in (A) and (C) are obtained by indirect Fourier transformation and the corresponding pair-distance distribution functions are plotted in (B) HiC and (D) SDS. Notice the different intensity scales.
where NS denotes the number density of SDS molecules,
Ncmc the critical micelle concentration (number density) of SDS in the buffer solution, nagg the mean micellar aggregation number (number of surfactant molecules per micelle), νS the partial specific molecular volume of SDS in the buffer solution, and BS the total neutron scattering length of a SDS molecule. If we use the calorimetry result for the cmc, i.e., 2.2 mM or Ncmc ) 2.2 × 10-6 mol/cm3 and use νS ) 4.09 × 10-22 cm3, BS ) 53.4 fm, F0 ) 6.38 × 1010 cm-2 (100% D2O) and furthermore average over the three I(0) values from the measurements of pure SDS in buffer (see Figure 6A), we obtain a mean micellar aggregation number, nagg, of 80. This is in good agreement with the previously determined weight-averaged value of 84 as determined in pure D2O.30 3.2.2. Mixed SDS/HiC. Examples of scattering data and pair-distance distribution functions from the mixed SDS/HiC system are shown in Figure 5. At the three lowest mixing ratios, nS ) 4.5, 15, and 51, the p(r) function is bell shaped for r < 60 Å and has a tail for r > 60 Å. The largest aggregates are observed in the nS ) 15 sample. The I(q) and corresponding p(r) for this sample are shown in parts A and B of Figure 5, respectively. We find a Dmax of 120 Å and a radius of gyration of 32 Å. This shows that the aggregates formed at this molar ratio are significantly larger than both pure SDS micelles and pure HiC monomers. The obtained curve shape of the p(r) suggests either that slightly elongated particles (axis ratio 2:1) are formed or that the sample is heterogeneous and consists of a mixture of small and larger particles, e.g. monomers and higher-mers. At nS ) 51 (Figure 5C,D) most of the tail characteristics of nS ) 15 has vanished again and at the nS corresponding to the inflection point of the ITC curve, nS ) 75 (Figure 5E,F),
(29) Harpaz, Y.; Gerstein, M.; Chotia, C. Structure 1994, 2, 641649.
(30) Cabane, B.; Duplessix, R.; Zemb, T. J. Phys. 1985, 46, 21612178.
Table 2. Results for I(0), RG, and Dmax in HiC and SDS Solutions As Determined from the Indirect Fourier Transforma sample
I(0), 1/cm
R G, Å
Dmax, Å
qmin, 1/Å
HiC, 0.1 mM SDS, 7.5 mM SDS, 15 mM SDS, 30 mM
0.018 ( 0.002 0.155 ( 0.001 0.360 ( 0.002 0.819 ( 0.004
16.7 ( 1.6 15.6 ( 0.1 15.6 ( 0.1 15.8 ( 0.1
55 45 45 45
0.01 0.01 0.04 0.05
a The q min column contains the minimal q values used for the indirect Fourier transform.
proteins, BP is the total scattering length of the proteins, and F0 is the scattering length density of the buffer solution. IP(0) is experimentally available and F0 can be calculated from the buffer composition. (See www.ncnr.nist.gov/ resources/sldcalc.html for further details.) The partial specific density of proteins only changes slightly from protein to protein,29 and in the present analysis we have used a partial specific density of 1.37 g/cm3 leading to νP ) 2.67 × 10-20 cm3 for the 22 kDa HiC. From these values, an experimental value for BP that includes the H-D exchange that takes place in the D2O containing buffer can be determined. Using NP ) 0.1 × 10-6 mol/cm3, νP ) 2.67 × 10-20 cm3, and F0 ) 5.69 × 1010 cm-2 (90% D2O and 10% H2O), we obtain BP ) 9.69 × 104 fm. The forward scattering from the pure surfactant micelles can be expressed by
(
IMic(0) ) (NS - Ncmc)naggνS2
)
BS - F0 νS
2
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Figure 5. SANS data from mixed SDS/HiC in aqueous buffer: (A) SDS:HiC molar ratio of 15; (C) SDS:HiC molar ratio of 51; (E) SDS:HiC molar ratio of 75; (G) SDS:HiC molar ratio of 150. The fitted curves in (A), (C), (E), and (G) are obtained by indirect Fourier transformation, and the corresponding pair-distance distribution functions are plotted in (B), (D), (F), and (H), respectively.
our SANS data show that the size of the aggregates in the mixed HiC/SDS sample has decreased a little further and is now only slightly larger than the HiC monomers and the SDS micelles. The same type of behavior is observed at the higher molar ratios. As an example we have plotted the data from the nS ) 150 sample in parts G and H of Figure 5. The p(r) function is bell shaped and resembles that of the pure HiC monomers or SDS micelles, and no reminiscence of the large aggregates observed at the lowest mixing ratios is found. This behavior differs from the small-angle scattering and dynamic light scattering observations that have been reported for other SDS-protein systems.16,31,32 The general observation is that the SDS-induced unfolding leads to the formation of rather large SDS-protein complexes that (31) Valstar, A.; Almgren, M.; Brown, W.; Vasilescu, M. Langmuir 2000, 16, 922-927. (32) Arleth, L.; Kirchhoff, C. F.; Onyuksel, H.; Hjelm, R. P. Submitted for publication in Macromolecules.
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Figure 6. (A): 0, experimental forward scattering of pure SDS micelles as a function of concentration. The cmc of SDS (2.2 mM) is indicated in the plot by the vertical full line. The forward scattering of pure HiC at 0.1 mM is indicated in the plot by the horizontal full line. Sloped full line, forward scattering as a function of concentration of SDS micelles with an aggregation number of 80 and cmc ) 2.2 mM. (B): ∆, experimental forward scattering of mixed SDS/HiC samples as a function of mixing ratio. cmc of SDS is indicated by the vertical full line. Sloped full line, I(0) versus nS assuming coexistence of HiC monomers and SDS micelles (eq 1). Long-dashed line, I(0) versus nS assuming the formation of SDS/HiC complexes, and SDS/HiC complexes with excess micelles above the saturation level of nS ) 107 (eq 2). Dotted line, I(0) versus nS as predicted from the ITC measurements (see further details in the Discussion). For these calculations a binding stoichiometry of the complexes of 47 SDS per HiC and a free monomer concentration of 2.2 mM is used. The error bars on the experimental points fall within the symbols used for the plot.
are clearly visible by both small-angle scattering and light scattering. Furthermore the complexes remain large and visible above the saturation level of the SDS. 3.3. Analysis of I(0). The results for the forward scattering, I(0), as a function of mixing ratio/SDS concentration are shown in Figure 6B. The following two model calculations are also plotted: No Interaction. In the “no interaction” model, plain HiC proteins and plain SDS micelles coexist and the total forward scattering is the sum of the forward scattering from HiC and SDS micelles at their respective concentrations
Cutinase and Surfactant Interactions
I1(0) ) IP(0) + IMic(0)
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(1)
Reynolds-Tanford (RT) Model. In the next step it is assumed that a simple type of protein-surfactant complexes is formed: The SDS is evenly distributed on the HiC proteins and the total volume fraction of the complexes is φC ) φHiC + φSDS. The volume of the single complexes is VC ) νHiC + nSνSDS, while their excess scattering length density is ∆FC ) (BHiC + nSBSDS)/(νHiC + nSνSDS) - F0. The corresponding forward scattering is then
I2(0) ) φCVC(∆FC)2 In the “RT model” it is furthermore assumed that there is an upper level, Ns, for how much SDS can be bound in the surfactant-protein complexes. This is in agreement with the observations by Reynolds and Tanford33 who have found that on average 1.4 g of SDS can bind to 1 g of “common water soluble” protein. For HiC this amount corresponds to a saturation level of Ns ) 107 SDS molecules per HiC. Above this saturation level, excess SDS will form micelles of the same size as in the pure SDS system. The volume fraction of the excess micelles is φMic ) φSDS φSDSnS)Ns, where φSDSnS)Ns signifies the volume fraction of SDS at the saturation level. The forward scattering becomes
I3(0) ) I2(0), for nS e Ns
{
I2ns)Ns(0) + φMicNaggνS(∆FSDS)2, for nS > Ns
(2)
In the first part of the “RT model”, I3(0) describes the scattering from the growing complexes, and in the second part, I3(0) describes the scattering from the saturated complexes and the growing number of excess micelles. In Figure 6B the model is plotted for a saturation level of 107 SDS molecules per HiC. The “RT model” might be developed further by including a free SDS monomer concentration in the calculations. On comparison of model calculations and scattering data (Figure 6B), it is seen that the scattering data are in reasonable agreement with the “no interaction” model at high molar ratios, but not at low molar ratios (nS < 100). We can therefore rule out this model. The “RT prediction” with a saturation level of Ns ) 107 SDS molecules per HiC as predicted by Reynolds and Tanford33 does also not agree with our experimentally determined I(0) points. We have tried including a cmc effect with a cmcSDS of 2.2 mM in the calculations (data not shown). Still, model and data disagree and we can thus also rule out the Reynolds-Tanford prediction of SDS-protein interaction and in particular the SDS saturation level for HiC. To summarize, the analysis of the p(r) functions determined from the SANS data shows that relatively large aggregates are formed at low SDS/HiC molar ratios (nS ) 4.5 and nS ) 15). This shows that SDS and HiC interacts. Apparently, the size of the aggregates decreases as the molar ratio increases, and at high molar ratios, nS g 100, the average size of the aggregates converges toward that of pure SDS micelles. The analysis of the absolute intensity of the forward scattering supports the analysis of the p(r) functions. It shows that there is interaction between SDS and HiC and that SDS/HiC complexes are (33) Reynolds, J. A.; Tanford, C. J. Biol. Chem. 1970, 245, 51615165.
formed. However, both the type of interaction and the saturation level of SDS in the complexes differ significantly from what has previously been found for a number of proteins.16,31-34 4. Discussion 4.1. SDS/HiC Complex Binding Stoichiometry at the Saturation Level. The ITC measurements showed that the cmc of SDS in a solution which was initially 0.10 mM HiC is 5.9 mM. Taking the dilution of HiC during the titration into account (see Experimental Section) this corresponds to a SDS:HiC molar ratio, nS ) 75. Assuming that the free monomer concentration of SDS is similar to that of SDS in pure buffer solutions, i.e., 2.2 mM, and assuming that the remaining SDS is bound in HiC/SDS complexes, we obtain an average binding ratio of 75(5.9 mM - 2.2 mM)/5.9 mM ) 47 SDS molecules per HiC. If we, for simplicity, assume that, as the SDS/HiC molar ratio increases in a 0.1 mM HiC solution, the first 47 SDS molecules (per HiC) bind to the HiC, the following 22 molecules remain as monomers, and additional SDS remain as pure SDS micelles, we obtain a modified version of the RT model. This model is plotted in Figure 6B. As seen from the plot, the agreement between model and experimental data is better for this model than for the “No interaction” and “RT prediction” models. However, an even better agreement is obtained below the saturation level if we allow the saturation level to vary slightly from the value determined from the ITC data and decrease the binding ratio to 36 SDS molecules per HiC while keeping the cmc of 2.2 mM (see Figure 7(top)). However, in either case the saturation level for SDS bound in the SDS/HiC complexes is significantly smaller than the values found in previously described protein systems.32-34 For the remaining discussion we have chosen to partition the enthalpogram and SANS forward scattering plots into four regions (A, B, C, and D) of characteristic thermal and scattering behavior (see Figure 7). 4.2. Region A. For the two lowest binding ratios (nS ) 4.5 and nS ) 15), the experimental values for I(0) still exceed the theoretical values (see Figure 7(top)). This shows that the average size of the aggregates is larger than that assumed in the model. The observation is supported by the analysis of the pair distribution functions which show that rather large aggregates are formed at low mixing ratios while smaller aggregates dominate at higher mixing ratios. Similar observations are described in the literature13 for the LDS/FsC system. A larger average complex size could be obtained if SDS/HiC complexes with two or more protein molecules are formed. If it is assumed that the complexes, on average, are dimers at the lowest nS, we obtain an I(0) that agrees with the observed scattering at nS ) 4.5 and 15 but not at higher nS. To explain the observed scattering at higher nS, these “dimerized” SDS/HiC complexes must break up into monomeric SDS/HiC complexes at higher binding ratios. The tendency to form dimers or larger aggregates at low molar ratios may be explained qualitatively by the combination of decreased charge due to the bound (anionic) SDS and increased hydrophobicity of the SDS/HiC complexes due to exposed alkyl chains of bound SDS. Whereas, the tendency for the dimers to break up into monomeric HiC/SDS complexes again at higher molar ratios may be due to an increasing total (negative) charge of the complexes in combination with an increasing surface hydrophilicity as more and more SDS molecules are hydrophobically associated and screen the hydrocarbon (34) Tanford, C. The hydrofobic effect; Wiley: New York, 1980.
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Nielsen et al. Table 3. Results for I(0), RG, and Dmax in Mixed SDS/HiC Solutions As Determined from the Indirect Fourier Transforma SDS:HiC molar ratio
I(0), 1/cm
R G, Å
Dmax, Å
qmin, 1/Å
4.5 15 51 75 99 150 225 300
0.041 ( 0.003 0.086 ( 0.005 0.105 ( 0.002 0.106 ( 0.003 0.178 ( 0.003 0.301 ( 0.003 0.496 ( 0.003 0.651 ( 0.005
27.7 ( 1.1 34.6 ( 1.6 17.9 ( 0.3 17.3 ( 0.5 16.8 ( 0.2 16.1 ( 0.2 16.1 ( 0.1 16.1 ( 0.1
90. 120. 60. 55. 50. 50. 50. 50.
0.01 0.01 0.01 0.01 0.01 0.03 0.04 0.05
a HiC concentration is 0.1 mM in all samples. The q min column contains the minimal q values used for the indirect Fourier transform.
Figure 7. Forward scattering (top) and enthalpogram (bottom) plots: ∆, experimental forward scattering of mixed SDS/HiC samples as a function of mixing ratio. The error bars on the experimental points fall within the symbols used for the plot. Full line, I(0) versus nS as predicted from the ITC measurements, but using a slightly smaller binding number of 36 SDS per HiC at the saturation point (see discussion). Dotted line, I(0) versus nS assuming the formation of dimers of SDS/HiC complexes (see discussion). The letters A, B, C, and D in both plots refer to regions identified based on scattering or thermal behavior. A detailed description of the molecular events in each region is presented in the text.
chains of the initial electrostatically bound SDS molecules. A similar behavior has been described in the lysozyme/ SDS system35,36 where, upon titration with SDS, the system first aggregates and precipitates at low nS and then redissolves at larger nS. The observation is mainly explained by charge neutralization of the lysozyme due to electrostatically bound SDS. In the nS range from ∼4 to 15 the enthalpogram of HiC/SDS (Figure 3) shows a shift from endothermic to exothermic heat flow. This shift may include exothermic contributions from the formation of dimers or higher-mers. In addition a peak broadening is observed in this nS range (see inset Figure 2), which suggest that a slow process, possibly the formation of, e.g., dimers, takes place. We find this interpretation plausible as it is in accord with the observed behavior with large aggregates and large (35) Moren, A. K.; Khan, A. Langmuir 1995, 11, 3636-3643. (36) Lad, M. D.; Ledger, V. M.; Briggs, B.; Green, R. J.; Frazier, R. A. Langmuir 2003, 19, 5098-5103.
I(0) at low nS, followed by smaller aggregates and relatively smaller I(0) values at higher nS. 4.3. Region B. In region B there is a constant negative heat signal in the ITC curve showing that there is interaction between proteins and SDS. In the same region no or only weak growth of SANS I(0) is observed (see Figure 7(top)). (Unfortunately we only have one SANS data point within this region, so mixing ratios 15, 51, and 75 are compared.) Such a behavior of the I(0) could be obtained if SDS added in this nS range dissociated into monomers. However, as the demicellization heat is zero at the used temperature (22 °C), this would not explain the observed heat signal in the ITC. A combined interpretation of SANS and ITC results could be that the dimers (and highermers) formed in region A are breaking up while, at the same time, the SDS/HiC binding stoichiometry increases. However, we do not have sufficient data to quantify this process in more detail. The p(r) functions and the radii of gyration determined from the SANS data (Figure 5 and Table 3) show that the SDS/HiC complexes are only slightly larger than the pure HiC and the pure SDS micelles and of nearly the same shape. This indicates that the change in the tertiary structure of the HiC upon the SDS induced unfolding is unusually small as compared to previous observations on other protein systems. 4.4. Region C. In the range of nS ) 65 to nS ) 100 the heat signal in the ITC measurement gradually vanishes and no heat signal is observed above nS ) 100. In the same range of molar ratios, the increment of the SANS forward scattering as a function of molar ratio changes to the value expected if pure SDS micelles are formed. The two I(0) points in this region fall slightly below the model (full curve in Figure 7(top)). In the model we have, for simplicity, assumed that from the beginning of region C all added SDS form micelles. However, the ITC data clearly show that the transition is gradual and takes place over the full region C so that the model for I(0) may overestimate the scattering slightly. 4.5. Region D. Above nS ) 100 there is no heat signal in the ITC and the increment of the SANS forward scattering as a function of molar ratio equals the value for pure SDS micelles. Furthermore the radius of gyration, RG, of the aggregates as determined from the SANS data gradually converges toward that of the SDS micelles (see Table 3). Thus both techniques suggest that added SDS remain as free micelles. 5. Conclusions This work has shown that Humicola insolens cutinase interacts strongly with SDS at low concentration (
