Langmuir 1999, 15, 6395-6399
6395
Conformation of Human Carbonic Anhydrase II Variants Adsorbed to Silica Nanoparticles Peter Billsten,† Uno Carlsson,*,‡ Bengt Harald Jonsson,§ Gerd Olofsson,| Fredrik Ho¨o¨k,⊥ and Hans Elwing*,⊥ IFM-Laboratory of Applied Physics and IFM-Department of Chemistry, Linko¨ ping University, S-581 83 Linko¨ ping, Sweden, Department of Biochemistry, Umeå University, S-901 87 Umeå, Sweden , Department of Thermochemistry, University of Lund, S-221 00 Lund, Sweden, and Department of Cell and Molecular Biology, Interface Biophysics, Go¨ teborg University, S-413 90 Go¨ teborg, Sweden Received March 10, 1998. In Final Form: April 29, 1999 Conformational changes of human carbonic anhydrase II (HCAIIpwt) adsorbed on silica nanoparticles (with an average diameter of 9 nm) have been investigated using differential scanning calorimetry (DSC), and in some specific cases also using circular dichroism (CD) and intrinsic tryptophan fluorescence. To relate the observed conformational changes to the denaturation stability and/or chemical properties in solution, two N-terminally truncated variants and two mutants of HCAIIpwt containing specific single site mutations were also investigated. From the thermal transitions of HCAIIpwt adsorbed to the nanoparticles we found that this variant forms a state that was distinctly different from both the native and molten globule states in solution. No thermal transition at all was observed for any of the other variants adsorbed on nanoparticles. CD and intrinsic tryptophan fluorescence indicate that these variants attain a molten globule-like state at the surface.
Introduction There is a general tendency for proteins to accumulate even irreversibly at solid-liquid interfaces.1 In many practical situations this is made advantage of, as in, e.g., biosensor applications.2 In other situations, however, it is an unwanted process, as in, e.g., fouling processes during medical treatments.3 For these and related reasons a detailed understanding about the interactions are of great current interest, though still poorly understood. Due to the complex nature of protein molecules, spontaneous adsorption at solid-liquid interfaces is a phenomenon containing significantly more degrees of freedom than does adsorption of more uniform synthetic polymers or low-molecular-weight molecules. One question that has long been addressed to be of special importance is how the energy landscape (determining the native structure of proteins in solution) is changed at the interfacial region between a solid and a liquid. If the perturbation from the surface is large enough, the global free energy minimum for the protein might correspond to a structure very different from that of the native protein. Moreover, a protein in a denatured state might allow more attractive contact points to the surface and/or correspond to a state of higher entropy than would the native protein. Hence, surface-induced conformational changes might even drive the adsorption and be one important reason * Corresponding authors. † Laboratory of Applied Physics, Linko ¨ ping University. ‡ IFM-Department of Chemistry, Linko ¨ ping University. § Umeå University. | University of Lund. ⊥ Go ¨ teborg University. (1) Norde, W.; Haynes, C. A. Reversibility and Mechanism of Protein Adsorption In Proteins at Interfaces II; Horbett, T. J., Brash, J. L., Eds.; ACS Symposium Series 602, American Chemical Society: Washington, DC, 1995; pp 26-39. (2) Marco, M.; Barcelo, D. Meas. Sci. Technol. 1996, 7, 1547. (3) Proteins at Interfaces II; Horbett, T. A., Brash J. L., Eds.; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995.
proteins tend to bind irreversibly at the solid-liquid interface.1 As a model system to investigate surface-induced conformational changes, we have focused on human carbonic anhydrase II (HCAII) adsorbed to silica particles with an average diameter of 9 ( 0.3 nm. The wild-type protein has a molar mass of 29 300 Da and consists of 259 amino acid residues. The crystal structure has been determined at a resolution of 1.54 Å,4 and the structure and folding properties of the protein have been extensively characterized,5-9 which makes HACII a good model protein in the present case. Folding experiments have revealed the existence of an equilibrium folding intermediate, referred to as a molten globule,10 which has been observed also at solid surfaces for HCAII11 and for human growth hormone.12 In addition, site-specific mutations of HCAII (Figure 1) can be used to study variations in the adsorption behavior with respect to conformational changes using small, precise variations of the structure. Pioneering work using site-directed mutants in adsorption studies was performed by McGuire et al.13,14 In this investigation we focused on the adsorption of five variants of HCAII. In the pseudo wild type (HCAIIpwt) (4) Håkansson, K.; Carlsson, M.; Svensson, L. A.; Liljas, A. J. Mol. Biol. 1992, 227, 1192. (5) Andersson, D.; Freskga˚rd, P.-O.; Jonsson, B.-H.; Carlsson, U. Biochemistry 1997, 36, 4623. (6) Aronsson, G.; Ma˚rtensson, L.-G.; Carlsson, U.; Jonsson, B.-H. Biochemistry 1995, 34, 2153. (7) Carlsson, U.; Jonsson, B.-H. Curr. Opin. Struct. Biol. 1995, 5, 482. (8) Freskga˚rd, P.-O.; Carlsson, U.; Ma˚rtensson, L.-G.; Jonsson, B.H. FEBS Lett. 1991, 289, 117. (9) Ma˚rtensson, L.-G.; Jonsson, B.-H.; Freskga˚rd, P.-O.; Kihlgren, A.; Svensson, M.; Carlsson, U. Biochemistry 1993, 3, 224. (10) Kuwajima, K. Proteins: Struct., Funct., Genet. 1989, 6, 87. (11) Billsten, P.; Freskga˚rd, P.-O.; Carlsson, U.; Jonsson, B.-H.; Elwing, H. FEBS Lett. 1997, 402, 67. (12) Buijs, J.; Hlady, V. J. Colloid Interface Sci. 1997, 190, 171. (13) McGuire, J.; Wahlgren, M.; Arnebrant, T. J. Colloid Interface Sci. 1995, 170, 182. (14) McGuire, J.; Wahlgren, M.; Arnebrant, T. J. Colloid Interface Sci. 1995, 170, 193.
10.1021/la980288u CCC: $18.00 © 1999 American Chemical Society Published on Web 07/07/1999
6396 Langmuir, Vol. 15, No. 19, 1999
Billsten et al.
which the heat capacity reaches its maximum (Tm) and the area of the transition peak both relates to the enthalpy change (∆H) and denotes therefore the stability of the protein. The width of the peak is related to the cooperativity of the process. In addition, DSC has also been used to study conformational changes of proteins adsorbed to solid surfaces.19-21 The solid surface used in this investigation was a silica nanoparticle with an average diameter of 9 nm. The small size makes them useful for spectroscopic studies due to the low-light scattering properties. Furthermore, the large effective surface area of the nanoparticles enables them to be used in methods which requires a high protein concentration such as circular dichroism (CD) spectroscopy11,22 or as in the present study DSC. Experimentals Section Figure 1. Structure of HCAII different mutation sites marked by arrows. The figure was produced using the program Molscript,26 and the coordinates were kindly provided by Dr. Kjell Håkansson.
the only naturally occurring cysteine residue in position 206 was replaced by a serine. The stability and activity of HCAIIpwt were apparently identical to that of the wildtype enzyme.8 All other variants used in this investigation were derived from HCAIIpwt. In trunc 5 and trunc 17 the 4 and 16 N-terminal amino acid residues have been removed, respectively. Such truncations result in structural destabilization of the protein, as measured by guanidine hydrochloride (GuHCl) denaturation. Interestingly, the native state is for both of these modifications destabilized relative to the molten globule state, whereas the molten globule state relative the denatured state is not significantly changed, compared to HCAIIpwt.6 In the mutant W16C (Trp-16 f Cys) and W97C (Trp-97 f Cys) the large amino acid tryptophan has been replaced by the smaller amino acid cysteine. Trp-16 is included in a small hydrophobic cluster, together with Trp-5, Tyr-7, and Phe-20. Trp-97 is located in a β-strand and is a part of a large hydrophobic cluster involving approximately 30 amino acids, including the aromatic amino acid residues Phe-66, -70, -93, -95, -176, -179, -226 and Trp-97. The Trp mutations destabilize the native state relative to the molten globule state, whereas the stability of the molten globule state relative to the unfolded state is only marginally affected for both W16C15 and W97C mutant16 compared to HCAIIpwt. In a previous study, CD and fluorescence methods were used to study the conformation of three carbonic anhydrase variants (HCAIIpwt, trunc 5, and trunc17) adsorbed to silica nanoparticles.11 However, an alternative method was required for further characterization of the conformation that accompanies the adsorption. In this investigation we therefore applied differential scanning calorimetry (DSC). The unfolding of proteins is generally an endothermic process that can be studied by DSC. For example, valid information on protein stability in solution can be obtained from a thermogram of unfolding.17,18 The temperature at (15) Lindgren, M.; Svensson, M.; Freskga˚rd, P.-O.; Carlsson, U.; Jonsson, B.-H.; Ma˚rtensson, L.-G.; Jonasson, P. J. Chem. Soc., Perkin Trans. 2 1993, 2003. (16) Svensson, M.; Jonasson, P.; Freskga˚rd, P.-O.; Jonsson, B.-H.; Lindgren, M.; Ma˚rtensson, L.-G.; Gentile, M.; Boren, K.; Carlsson, U. Biochemistry 1995, 34, 8606. (17) Sturtevant, J. M. Annu. Rev. Phys. Chem. 1987, 38, 463. (18) Privalov, P. L.; Gill, S. J. Adv. Protein Chem. 1988, 39, 194.
Chemicals. A 10 mM sodium-/potassium-phosphate buffer (pH 7.5) was used in all experiments. Water was distilled, passed through an ion exchanger and an active charcoal filter (Millipore Corp., Bedford, MA), and then distilled twice on a glass distiller. Guanidine hydrochloride (GuHCl) was of sequence grade, and its concentration was determined refractometrically.23 The colloidal silica nanoparticles (kindly provided by EKA-Nobel, Stenungsund, Sweden) have a negative charge at the pH of the experiment. The stock solution contained 5.1 × 1017particles /mL. All proteins were filtered prior to use (Millipore; pore size, 0.45 µm), and the concentration was at 280 nm using a Beckman DU-50 spectrophotometer. The proteins were mixed with particles to obtain a 1:1 molar ratio of proteins to particles and allowed to interact for 24 h with the particles prior to measurements. In some cases W16C and W97C were unfolded using 1.5 M GuHCl for 24 h to induce the molten globule state. DSC Measurements. Temperature scanning differential calorimetry was performed using a Microcal MC-2 high-sensitivity differential calorimeter (MicroCal, Northampton, MA). It is a differential instrument using twin, 1.2 mL total-fill cells. The Origin (version 2.9) software for data collection and analysis, supplied by the manufacturer, was used for instrument control, data acquisition, and analysis. The sample cell was filled with protein solution, and the reference cell was filled with an equal amount of water. Samples and water were degassed and transferred to the cells with a syringe. The instrument measures the power required to keep the temperature of the sample and reference cell equal while the temperature is raised at a constant rate. The results recorded in this way are called sample traces. The reference traces were recorded with the sample cell filled with a solution without the protein but otherwise identical to the sample solution. The excess heat capacity Cp,ex of the sample solution was calculated as a function of temperature from the difference between the sample and the reference traces. The ∆H for the transition was calculated from the area under the peak. The progress base-line option, which takes into account the heat capacity change accompanying the transition, was used to determine the base line. The protein concentration was 34 mM, and the scan rate was 60 °C/h. CD Measurements. The CD spectra were recorded using a spectrodichrograph CD6 (Jobin-Yvon Instruments SA, Longjumeau, France) under constant N2 flushing. Each CD spectrum presented is the average of three scans obtained by collecting data at 0.5 nm intervals with an integration time of 2 s. The CD (19) Yan, G.; Li, J.-C.; Huang, S.-H.; Caldwell, K. D. In Proteins at Interfaces: Fundamentals and Applications; Horbett, T. A., Ed.; American Chemical Society: San Diego, 1995; Vol. 602, p 256. (20) Zoungrana, T.; Findenegg, G. H.; Norde, W. J. Colloid Interface Sci. 1997, 190, 437. (21) Steadman, B. L.; Thompson, K. C.; Middaugh, R. C.; Matsuno, K.; Vrona, S.; Lawson, E. Q.; Lewis, R. V. Biotechnol. Bioeng. 1992, 40, 8. (22) Kondo, A.; Oku, S.; Higashitani, K. J. Colloid Interface Sci. 1991, 143, 214. (23) Nozaki, Y. Methods Enzymol. 1972, 26, 43.
Particle-Adsorbed Carbonic Anhydrases
Langmuir, Vol. 15, No. 19, 1999 6397
Figure 2. Differential scanning calorimetry thermograms of the different HCAII variants in the absence of nanoparticles (solid line) and after adsorption to nanoparticles (broken line): (a, top left) HCAIIpwt, (b, top right) trunc 5, (c, middle left) trunc 17, (d, middle right) W16C, and (e, bottom) W97C. Protein (68 µM) was resuspended in 10 mM phosphate buffer (pH 7.5) was used. spectra of the proteins were corrected by subtracting scans of a reference solution without the protein but otherwise identical. Measurements were carried out in two wavelength regions, farUV (190-260 nm) and near-UV (240-320 nm). A 0.5 mm quartz cell was used in the far-UV measurements and a 5 mm cell in the near-UV range. In the near-UV region a protein concentration of 17 µM was used, and in the far-UV region the concentration was 8.5 µM. Fluorescence Measurements. The fluorescence measurements were carried out using a Hitachi F-4500 spectofluorometer. The intrinsic tryptophan fluorescence was selectively collected by exciting the protein at 295 nm. Excitation and emission bandwidths were 2.5 and 10 nm, respectively, and the cuvette path length was 10 mm. The fluorescence emission was recorded between 300 and 500 nm. The protein concentration was 3.4 µM.
Results DSC Analysis. Figure 2 shows thermograms of unfolding for all variants of HCAII without particles present in the buffer solution. All variants of HCAII displayed significant transitions upon heating, as illustrated in the DSC thermograms (Figure 2). The thermal unfolding of all variants was accompanied by visible, irreversible aggregation of the protein. Irrespective of the aggregation, HCAIIpwt was more stable than the other variants, since its Tm and ∆H values were significantly larger (Table 1). This interpretation is also in consensus with previous measurements using spectroscopic methods.6,15,16 The Tm and ∆H values of unfolding of the proteins were inde-
6398 Langmuir, Vol. 15, No. 19, 1999
Billsten et al.
Table 1. Temperature at Which the Heat Capacity Reaches Its Maximum (Tm) and the Area of the Transition Peak, ∆H, Denoting the Stability of the Protein ∆H (kcal/mol)
Tm (°C) HCAII variant
without nanoparticles
adsorbed to nanoparticles
without nanoparticles
adsorbed to nanoparticles
HCAIIpwt Trunc 5 Trunc 17 W16C W97C
59 52 53 51 50
52 no transition no transition no transition no transition
180 130 130 140 140
80 0 0 0 0
pendent of the protein concentrations (34-136 µM) used in this study, and different scan rates in the range 30-90 °C/h did not affect the calorimetric results (not shown). The introduction of particles completely repressed the thermal transitions for all variants except for HCAIIpwt. However, Tm and ∆H values displayed were significantly lower, compared to experiments without particles (cf., Figure 2 and Table 1). In addition, no thermal aggregation was observed for any of the variants in the presence of nanoparticles. It is likely that the aggregation in solution is due to hydrophobic attraction between hydrophobic patches on individual molecules that become exposed upon unfolding. Hence, one possible explanation to the prevention of aggregation in the presence of nanoparticles might be that these hydrophobic patches are shielded when the protein is adsorbed in an unfolded state on the nanoparticles or, for that reason, thermally unfolded during the DCS measurement. An alternative explanation might be that the binding of proteins (with or without changed conformation) does not reduce the electrostatic repulsion between the silica particles enough to allow them to aggregate. Reheating of HCAIIpwt did not display any thermal transition, which indicates that the thermal denaturation was irreversible, despite the lack of aggregation upon heating. CD Analysis. Since DSC thermograms cannot generally differentiate between the molten globule and the fully unfolded state, CD analysis was performed. This is because the unfolding of the molten globule state is often accompanied by only a small ∆H change.10 CD spectra of the W16C and W97C mutants were recorded in the far- and near-UV wavelength region in the absence of particles (Figures 3 and 4). There were some differences between the variants in both near- and far-UV spectra due to spectral contributions of the replaced tryptophans.24 However, addition of particles resulted in a striking similarity of the CD spectra independent of the mutants. Both near-UV and far-UV CD spectra of adsorbed W16C and W97C mutants agree well with previously reported CD spectra of trunc 5 and trunc 17 mutant adsorbed to nanoparticles.11 The latter CD spectra indicated that the contents of secondary structure were almost intact, whereas the interactions characteristic for the tertiary structure were lost. These spectral properties are characteristic for the molten globule state. Thus, W16C, W97C, trunc 5, and trunc 17 all attain a molten globule-like conformation upon adsorption to the nanoparticles. Intrinsic Trp Fluorescence. The intrinsic Trp fluorescence of W16C and W97C was measured when the variants were adsorbed on nanoparticles or were in a 1.5 M GuHCl (Table 2). The variants displayed fluorescence emission maxima when adsorbed to the particles that were similar to that of the molten globule state induced by 1.5 M GuHCl. (24) Freskga˚rd, P.-O.; Ma˚rtensson, L.-G.; Jonasson, P.; Jonsson, B.H.; Carlsson, U. Biochemistry 1994, 33, 14281.
Figure 3. Far-UV circular dichroism of HCAII variants in the absence of particles (solid line) and after adsorption to nanoparticles (broken line) (where the rotation “2 103”, for example, means 2 × 103): (a, top) W16C, (b, bottom) W97C. Protein (8.5 µM) was resuspended in 10 mM phosphate buffer (pH 7.5) was used.
Discussion Differential scanning calorimetry is a useful method for assessing some important aspects of the conformational state of proteins when adsorbed to solid surfaces, which has also been previously reported.19,20 One important result from the present study was that the W16C and W97C mutants attained a molten globulelike state upon adsorption, according to CD (Figures 3 and 4) and fluorescence data (Table 2). Previously, it has also been shown that the truncated variants of HCAIIpwt obtain a molten globule-like state upon adsorption.11 The molten globule state is generally described as a kinetic and equilibrium folding intermediate, which, in moderate concentrations of denaturing agents such as GuHCl or at low pH, is stable. It is further characterized by a fluctuating tertiary structure, with more prominent exposure of hydrophobic amino acids in patches, and a larger volume than the native state, still maintaining the
Particle-Adsorbed Carbonic Anhydrases
Langmuir, Vol. 15, No. 19, 1999 6399
Figure 4. Near-UV circular dichroism of HCAII variants in the absence of particles (solid line) and after adsorption to nanoparticles (broken line): (a, top) W16C, (b, bottom) W97C. Protein (17 µM) was resuspended in 10 mM phosphate buffer (pH 7.5) was used. Table 2. Fluorescence Emission Maxima (nm) for Intrinsic Tryptophan Fluorescence HCAII variant
without nanoparticles
with nanoparticles
1.5 M GlHCl
W16C W97C
335 337
343 343
342 346
secondary structure.25 However, independent of the structural characteristics of the molten globule state, it does not exist without an external perturbation, such as temperature, denaturing agent, or pH, affecting the energy landscape determining the conformational free energy minimum for the protein in question. Hence, it is actually not unlikely that a perturbation induced by a solid surface (in this case the silica nanoparticles) might change the (25) Ptitsyn, O. B. Adv. Protein Chem. 1995, 47, 83. (26) Kraulis, P. J. J. Appl. Chrystallogr. 1991, 24, 1192.
conformation of a protein to a state that resembles some of those that characterize the molten globule state. However, in comparison to a protein in the molten globule state in solution an adsorbed protein is attached by several contact points directly to the surface. We therefore specifically point out that the state(s) of the mutated and truncated variants of HCAII identified from the CD and intrinsic Trp fluorescence measurements must be referred to as molten globule-like. Even more interesting become these results when compared to those obtained from adsorption of HCAIIpwt, which, according to the DCS measurements (see Figure 2), seems to adopt a conformation which differs from both the molten globule-like states described above and the native state in solution. Since the most important difference between HCAIIpwt and the altered variants is their global stability, these results indicate that the global stability of the protein in solution is an important parameter for the nature and degree of conformational changes upon adsorption. This interpretation is further supported by the fact that, for the mutants investigated, distinctly different regions of HCAIIpwt were altered (Figure 1), though reported to have similar stability in solution.6,15,16 If local interactions are of importance, it is likely that these variants would have adopted more different states upon adsorption. However, the significance of specific regions or amino acids on the protein surface for the adsorption process cannot be fully inferred from the present results. Such information would require a systematic mutational study where an amino acid or a region on the protein surface has been given designed properties. Previous CD and fluorescence measurements suggested that the structure of HCAIIpwt was only marginally affected by the adsorption to the nanoparticles.11 The data from that study indicated that adsorption of HCAIIpwt is accompanied by no or very minor conformational changes. However, the present DSC data showed that the stability (Tm) of adsorbed HCAIIpwt is considerably lowered, suggesting that the influence of the surface alters the properties of the protein molecules. In conclusion, these data taken together show that a specific state of HCAIIpwt after adsorption is created, characterized by a roughly maintained tertiary structure, but in which a considerable strain is placed on the protein molecule leading to a destabilization. Acknowledgment. We thank Geng Wang for help with the DSC instrument and Marie Wahlgren for her assistance. We would also like to thank Dick Andersson for production and purification of the W97C mutant. This work was supported by the Swedish Research Council for Engineering Science (TFR) (P.B. and H.E.), Strategic Research on Marine Biofouling (MASTEC) (H.E.) and the Swedish Natural Science Research Council (NFR) (B.H.J. and U.C.) LA980288U
