Adsorption of Human Carbonic Anhydrase II Variants to Silica

The surface adsorption behavior of protein variants of the enzyme human carbonic anhydrase II ( ... All protein variants initially adsorbed to the par...
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8470

Langmuir 2000, 16, 8470-8479

Adsorption of Human Carbonic Anhydrase II Variants to Silica Nanoparticles Occur Stepwise: Binding Is Followed by Successive Conformational Changes to a Molten-Globule-like State Martin Karlsson,† Lars-Go¨ran Mårtensson,† Bengt-Harald Jonsson,‡ and Uno Carlsson*,† IFM-Department of Chemistry, Linko¨ ping University, SE-581 83 Linko¨ ping, Sweden, and Department of Biochemistry, Umeå University, SE-901 876 Umeå, Sweden Received February 25, 2000. In Final Form: June 29, 2000 The surface adsorption behavior of protein variants of the enzyme human carbonic anhydrase II (HCA II) to silica nanoparticles has been investigated. Various destabilized mutants were produced by sitedirected mutagenesis of amino acids located in the interior of the protein. The silica particles induced a molten-globule-like state in all of the variants. All protein variants initially adsorbed to the particles, and then underwent conformational rearrangements in a stepwise manner, as indicated by the loss of activity and the subsequent loss of tertiary structure. Activity, CD, and ANS fluorescence measurements showed that a decrease in the global stability of the protein is strongly correlated to increased rates of conformational change following particle adsorption. In contrast to unfolding processes induced by chemical denaturants or heat, in the transition to the molten-globule-like state induced by the silica particles, the active site region unfolds before the majority of the tertiary interactions are broken.

1. Introduction Proteins have a strong tendency to adsorb spontaneously and irreversibly to most solid surfaces.1 In some cases protein adsorption is disadvantageous, e.g., in various fouling processes, whereas in other situations this is a desirable process, e.g., in various biosensor applications. A detailed understanding of protein adsorption mechanisms is a prerequisite for any optimization of conditions in all such cases. It is well-known that proteins frequently undergo conformational changes in solution, and such structural rearrangements are well characterized. Although protein adsorption onto solid surfaces from aqueous solutions also leads to conformational changes, these changes have been much less well characterized in structural detail.1,2 The disruption of the native structure of the protein, leading to entropic gain, is thought to be one of the major driving forces of protein adsorption. Although protein adsorption on solid surfaces has been thoroughly studied, the mechanisms of adsorption are not fully understood. For instance, structural rearrangements may be induced by the physicochemical properties of the solid surface. On the other hand, conformational changes also depend on the intrinsic properties of the protein. Norde3 has demonstrated that various intrinsic characteristics of the protein determine the degree of structural change that occurs upon adsorption and has drawn distinctions between structurally stable, “hard”, and labile, “soft”, proteins with respect to their interactions with hydrophilic surfaces. * Corresponding author. † Linko ¨ ping University. ‡ Umeå University. (1) Norde, W.; Haynes, C. A. In Proteins at Interfaces II, Fundamentals and Applications; Horbett, T. A., Brash, J. L., Eds.; American Chemical Society: San Diego, CA, 1995; pp 26-40. (2) Norde, W. In Biopolymers of Interfaces; Malmsten, M., Ed.; Marcel Dekker: New York, 1998; pp 27-54. (3) Norde, W. Adv. Colloid Interface Sci. 1986, 25, 267-334.

With protein engineering it is possible to alter certain intrinsic properties, such as the global stability of specific proteins. We found previously, for instance, that N-terminally truncated mutants of human carbonic anhydrase II (HCA II) initially undergo greater conformational changes upon surface adsorption than the wild-type enzyme.4 The lower structural stability of the truncated proteins was assumed to be the main cause for the greater conformational change found with these proteins compared to that of the unmutated protein. The local alteration of the protein surface caused by the truncations was, on the other hand, not considered important. In the present study we used site-directed mutagenesis to produce a set of point-mutated HCA II variants differing in stability, to unequivocally determine the cause of the differences in adsorption behavior. These point mutations are located in the interior of the protein. Thus, the substituted amino acid should not directly participate in the adsorption process. Furthermore, in one position (pos. 56) we replaced the wild-type amino acid by three different amino acids in order to discriminate between effects on stability and effects arising from the chemical properties of the inserted amino acid. Previous studies on the effect of destabilizing mutations on adsorption have also been performed on the enzyme T4 lysozyme.5,6 In those studies it was shown that the structural stability is a key factor in determining the extent of conformational change upon adsorption. Further, the initial rate of conformational change was more rapid for the less stable protein variants. HCA II has many advantages as a model for studying the processes involved in protein adsorption to interfaces, since several conformational states it adopts have been characterized during extensive studies of its refolding and (4) Billsten, P.; Freskgård, P.-O.; Carlsson, U.; Jonsson, B.-H.; Elwing, H. FEBS Lett. 1997, 402, 67-72. (5) McGuire, J.; Wahlgren, M.; Arnebrant, T. J. Colloid Interface Sci. 1995, 170, 182-192. (6) Billsten, P.; Wahlgren, M.; Arnebrant, T.; McGuire, J.; Elwing, H. J. Colloid Interface Sci. 1995, 175, 77-82.

10.1021/la0002738 CCC: $19.00 © 2000 American Chemical Society Published on Web 10/03/2000

Surface Adsorption Behavior of HCA II

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2. Materials and Methods

Figure 1. Overall structure of HCA II with mutated positions indicated. The 3D structure is based on the crystal structure determined by Håkansson et al.10

unfolding reactions.7 Thus, these states can be used as reference states in comparisons with conformations induced by adsorption. HCA II is a monomeric enzyme, consisting of 259 amino acid residues,8 with a molecular weight of 29.3 kDa. It is largely composed of β-sheets and is bisected by 10 β-strands that span the entire molecule9,10 (Figure 1). The unfolding of the protein has been demonstrated to be a three-state process that includes formation of a stable equilibrium intermediate of the molten-globule type at moderate concentrations of denaturant.11 In addition, an ensemble of compact conformational states exists after the rupture of the molten-globule state under very strongly denaturing conditions.11,12 In this study we used colloidal silica nanoparticles (9 nm average diameter) to investigate the conformation of the protein at the solid/liquid interface. One advantage of using such ultrafine particles is the very low levels of light scattering these particles induce, which allows spectrophotometric analysis of the structural alteration of the adsorbed proteins.4,13 Thus, CD and fluorescence spectroscopy were used to monitor the adsorption process in order to evaluate the dependence of protein stability on the rate and extent of conformational changes induced by surface adsorption. The results of our studies indicate that the protein molecules first rapidly bind to the particle surface and then undergo conformational changes in a stepwise manner: the active site ruptures before the rest of the tertiary structure does. The kinetics of the conformational changes are affected by the stability of the protein variants. However, the extent of conformational changes is not affected by protein stability. In all cases a moltenglobule-like state is finally attained upon adsorption. (7) Carlsson, U.; Jonsson, B.-H. Curr. Opin. Struct. Biol. 1995, 5, 482-487. (8) Henderson, L. E.; Henriksson, D.; Nyman, P. O. J. Biol. Chem. 1976, 251, 5457-5463. (9) Eriksson, A. E.; Jones, T. A.; Liljas, A. Proteins: Struct., Funct., Genet. 1988, 4, 274-282. (10) Håkansson, K.; Carlsson, M.; Svensson, L. A.; Liljas, A. J. Mol. Biol. 1992, 277, 1192-1204. (11) Mårtensson, L.-G.; Jonsson, B.-H.; Freskgård, P.-O.; Kihlgren, A.; Svensson, M.; Carlsson, U. Biochemistry 1993, 32, 224-231. (12) Svensson, M.; Jonasson, P.; Freskgård, P.-O.; Jonsson, B.-H.; Lindgren, M.; Mårtensson, L.-G.; Gentile, M.; Bore´n, K.; Carlsson, U. Biochemistry 1995, 34, 8606-8620. (13) Kondo, A.; Mihara, J. J. Colloid Interface Sci. 1996, 177, 214221.

2.1. Chemicals. 8-Anilino-1-naphthalenesulfonic acid (ANS) was purchased from Sigma. Guanidine hydrochloride (GuHCl) was obtained from Pierce and was of sequanal grade. The concentration was determined refractometrically.14 The colloidal, negatively charged, silica particles (food-grade quality) were kindly provided by EKA-Nobel, Stenungsund, Sweden. They were used without further modification apart from dilution in buffer. The stock solution contained 5.1 × 1017 particles/mL with an average diameter of 9 nm. All other chemicals were of reagent grade. 2.2. Production and Purification of Mutated Protein. Site-directed mutagenesis, protein production, and purification were performed as described in Freskgård et al.15 Protein concentrations were determined from absorbance at 280 nm using 280nm ) 54 800 M-1 cm-1 for HCA IIpwt16 and for all mutants in which no tryptophan was removed. For the W97C mutant 280nm ) 49 000 M-1 cm-1 was used.17 2.3. Stability Measurements. To determine the stability of the HCA II mutants, the protein (0.85 µM) was incubated overnight in various concentrations of GuHCl containing 0.1 M Tris-H2SO4, pH 7.5. The red-shift of the intrinsic tryptophan fluorescence maximum was used to monitor the unfolding of the protein. Fluorescence spectra were obtained on a Hitachi F-4500 spectrofluorimeter equipped with a thermostated cell. The spectra were recorded in a 1-cm quartz cuvette at 23 °C. The excitation wavelength was 295 nm, and 3 accumulative emission spectra were recorded in the wavelength region 310-450 nm. Fivenanometer slits were used for both excitation and emission. The midpoint concentrations of denaturation were determined according to Garvey and Matthews.18 The fits were done by using a nonlinear least-squares fitting program, Table Curve (Jandel Scientific). 2.4. Chemical Denaturation Kinetics. In experiments where the kinetics of chemical denaturation were monitored, 150 µL of 8 M GuHCl was mixed with 850 µL of 10 µM protein (HCA IIpwt) in 10mM Na-borate buffer, pH 8.5, giving a protein concentration of 8.5 µM in 1.2 M GuHCl, a concentration of denaturant at which the HCA IIpwt is in its intermediate, moltenglobule, state. Immediately after mixing, the samples were either used for CD trace measurements, monitored at 270 nm, or used for CO2 hydration activity measurements. 2.5. Adsorption Procedure. All experiments were performed in 10 mM Na-borate buffer, pH 8.5, at 23 °C. The protein samples were filtered (Millipore; pore size 0.45 µm) prior to use. The protein molecules were mixed with an equal amount of particles to obtain a 1:1 ratio of protein-to-particle (8.5 µM) in the kinetic experiments. When the particle concentration dependence on adsorption was studied, a protein concentration of 0.85 µM was used. Enzyme activity and CD spectrum were measured on the protein prior to mixing with the particles. 2.6. Dynamic Light-Scattering Measurements. Dynamic light-scattering (DLS) measurements were conducted using a Brookhaven BI-90 instrument, equipped with a 2-W argon laser (Lexel Corp.). The measurements were performed at a wavelength of 488 nm. A laser power of 700 mW and a scattering angle of 90° were used. Sample path length was 1 cm. The distilled water, in which the particles were dissolved, was repeatedly filtered (Millipore; pore size 0.45 µm) to remove dust particles. 2.7. Enzyme Activity Measurements. The loss of CO2 hydration activity was measured upon mixing the enzyme with the nanoparticles. Aliquots were withdrawn during the reaction, and the CO2 hydration activity was assayed as described elsewhere.19,20 The protein concentration was 0.25 mg/mL (8.5 µM). (14) Nozaki, Y. Methods Enzymol. 1972, 26, 43-50. (15) Freskgård, P.-O.; Mårtensson, L.-G.; Jonasson, P.; Jonsson, B.H.; Carlsson, U. Biochemistry 1994, 33, 14281-14288. (16) Nyman, P. O.; Lindskog, S. Biochim. Biophys. Acta 1964, 85, 141-151. (17) Mårtensson, L.-G.; Jonasson, P.; Jonsson, B.-H.; Freskgård, P.O.; Svensson, M.; Carlsson U. Biochemistry 1995, 34, 1011-1021. (18) Garvey, E. P.; Matthews, C. R. Biochemistry 1989, 28, 20832093. (19) Rickli, E. E.; Ghazanfar, S. A. S.; Gibbons, B. H.; Edsall, J. T. J. Biol. Chem. 1964, 239, 1065-1078.

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Table 1. Characteristics of the Protein Variants

protein HCA IIpwt (C206S) M241L S56C W97C S56N S56F

fractional surface accessibilitya

positionb

0.01

β-7

0.96

0 0 0 0 0

β-2 β-4 β-2 β-2

0.75 0.69 0.47 0.35 0.31

CmNI

(M)c

destabilization (kcal/mol)

1.7 2.2 3.8 5.0 5.4

activity CmIU

(M)c

CO2 hydration

esterase

2.24d

100

100

1.95e 2.25e 1.94d 1.95e 2.05e

78 65 51 81 100

86 n.d. n.d. 99 92

a Computed using the program SAVOL in the biopolymer module of SYBYL (Tripos, Inc.). Fractional values are the surface areas of the residues relative to those for the exposed residues in the tripeptide Ala-X-Ala, where X denotes the residue of interest. b β-strand positions according to Mårtensson et al.11 c CmNI and CmIU represent the transition midpoint concentrations (GuHCl) for the transitions from the native to the molten-globule intermediate and from the molten-globule intermediate to the unfolded enzyme, respectively. d Determined by measuring changes in tryptophan absorption at 292 nm as described previously.11 e Determined by measuring changes in the tryptophan fluorescence emission peak, as described in Materials and Methods.

Carbonic anhydrase in vitro also possesses esterase activity that can be monitored spectrophotometrically.21 The esterase activity of HCA II was determined after the gel permeation chromatography analysis (see section 2.10). The assay solution contained 1.2 mM p-nitrophenyl acetate as a substrate dissolved in 0.05 M Tris-H2SO4, pH 8.5 (ionic strength 0.1 M; adjusted with Na2SO4). Product formation was registered by monitoring the absorbance change at 348 nm, using 348nm ) 5540 M-1cm-1. The second-order rate constant, k’, was calculated from the equation v ) k’[E][S].21 2.8. CD Measurements. CD spectra were recorded on a spectrodichrograph (Jobin-Yvon Instruments SA, Longjumeau, France), employing constant N2 flushing. The instrument was calibrated with an aqueous solution of d-10-(+)-camphorsulfonic acid at 290 nm. Each CD spectrum represents the average of three scans obtained by collecting data at 0.5-nm intervals with an integration time of 2 s. The protein spectra were corrected by a spectrum of a reference solution lacking the protein but otherwise identical. The ellipticity is reported as mean residue molar ellipticity ([θ], in deg ‚ cm2 ‚ dmol-1) according to the equation

[θ] ) [θ]obs × mrw/10lc where [θ]obs is the ellipticity (deg), mrw is the mean residue molecular weight (molecular weight 29 300 and 259 amino acid residues), c is the protein concentration (g/mL), and l is the optical path length of the cell (cm). Immediately after the protein-particle mixing, the solution was filtered (Millipore; pore size 0.45 µm) to obtain a clear solution for the CD measurements. Far-UV CD spectra were obtained by scanning the proteins (17 µM) in 10 mM Na-borate buffer, pH 8.5, in a 0.1-mm quartz cell. Near-UV CD spectra were recorded using the same buffer in a 5-mm quartz cell. A protein concentration of 8.5 µM in 10 mM Na-borate buffer, pH 8.5, was used in the kinetic measurements, and time scanning was performed at 270 nm in a 10-mm quartz cell thermostated at 23 °C. 2.9. ANS Binding. A 10-fold molar excess of ANS was used over protein (8.5 µM), and the time course of ANS binding to the protein upon adsorption was registered by fluorescence measurements. ANS was added to the adsorbing protein at various time points during the adsorption process, and fluorescence emission spectra, between 450 and 650 nm, were recorded immediately after the addition of ANS. The change in ANS emission during adsorption was calculated at 470 nm after subtraction of the fluorescence from ANS and protein without particles. Addition of particles to ANS did not affect the fluorescence spectrum of ANS. The samples were excited at 360 nm, and excitation and emission bandwidths were 5 and 10 nm, respectively. The cuvette length was 1 cm, and the spectrofluorimeter was thermostated at 23 °C. (20) Freskgård, P.-O.; Bergenhem, N.; Jonsson, B.-H.; Svensson, M.; Carlsson, U. Science 1992, 258, 466-468. (21) Whitney, P. L.; Fo¨lsch, G.; Nyman, P. O.; Malmstro¨m, B. G. J. Biol. Chem. 1967, 242, 4206-4211.

2.10. Gel Permeation Chromatography. The HCA II variants were, after adsorption to the particles, chromatographed on a gel permeation column (Sephacryl S100 HR, 18 × 350 mm, Amersham Pharmacia Biotech, Sweden). The Bio-Rad BioLogic LP chromatography system (Hercules, CA) was used for the separations. One milliliter of the protein-particle solution (68 µM of each) was applied to the column. The flow rate was 1 mL/min, and fractions containing 1 mL were collected. 2.11. Data Analysis. The kinetics of CD and activity measurements were fitted to the sum of two exponential terms by the use of a nonlinear least-squares program (TableCurve, Jandel Scientific).

3. Results and Discussion 3.1. Particle Properties and Conditions. Colloidal silica nanoparticles were used in this study to provide the solid surface for the adsorption studies. The nanoparticles are negatively charged and have an average diameter of 9 nm, which is approximately twice the diameter of the HCA II protein used in the investigation. In most experiments involving nanoparticles, a 1:1 molar ratio of protein-to-particles was used to ensure that there was sufficient surface area available for all the protein molecules to be adsorbed. In further trials, however, other particleto-protein ratios were used in order to evaluate the possible dependence on particle or protein concentration. Dynamic light-scattering analysis revealed that a fraction of the nanoparticles formed aggregates at pH 7.5 (in 0.1 M Na-phosphate buffer), while no aggregates were detected at pH 8.5 (in 10 mM Na-borate buffer) (data not shown). Therefore, all subsequent protein adsorption measurements were performed at pH 8.5. 3.2. Effect of the Mutations on Assayed Properties of HCA II. A pseudo-wild-type form of HCA II (HCA IIpwt) was used as a template for all mutations. This pseudowild-type of the enzyme is functionally and structurally indistinguishable from the wild-type form,22 and its stability toward denaturation is practically identical.11 The stability of the engineered variants with regard to GuHCl denaturation is shown in Table 1. Stability was monitored by measuring the changes in absorbance at 292 nm or the change in intrinsic Trp fluorescence (redshift) that occurred upon unfolding. Since there are seven Trp residues, which are fairly evenly distributed throughout the 3-D structure of HCA II,9 these spectral changes can be considered to reflect overall conformational changes in the molecule (global transitions). As has been demonstrated for other mutants,11,12,22 all the newly engineered mutants unfolded in two, wellseparated stages, with the occurrence of a stable inter(22) Mårtensson, L.-G.; Jonsson, B.-H.; Andersson, M.; Kihlgren, A.; Bergenhem, N.; Carlsson, U. Biochim. Biophys. Acta 1992, 1118, 179186.

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Figure 2. CD spectra of HCA II variants. Panels: A, far-UV; B, near-UV. HCA IIpwt ()); M241L (- - -); S56C (- - -); W97C (- - - -); S56N (- - - -); S56F (s).

mediate (I) of a molten-globule type. The enzyme activity was lost concomitantly with the first unfolding transition, which is also in agreement with the earlier studies. The experiments showed that the greatest effects were reflected by the first unfolding transition, at which the midpoint concentration of denaturation (CmNI) varied between 0.75 and 0.31 M GuHCl, compared to 0.96 M GuHCl for HCA IIpwt. These values reflect how stable the native state (N) is against a transition to the moltenglobule state. As noted earlier, the second unfolding transition (CmIU), during which the molten-globule state is ruptured, appeared to be less sensitive to the various substitutions. The reason for this probably lies in the fact that in the native state all the replaced side chains are involved in specific tertiary interactions. However, in the moltenglobule state the motions of these side chains are less constrained, leading to side-chain interactions that are weaker and less dependent on the specific side-chain properties. The decreases observed in CmNI are difficult to accurately convert into changes in free energy. Ideally, the stability of the mutants should be compared in the absence of denaturant. This, however, requires rather long extrapolations, since stability data can only be obtained in the relatively narrow transition zone. As a consequence, such extrapolated values have been shown to suffer from quite high levels of uncertainty.23 An alternative strategy is to compare the free energies where they can be most accurately measured.24-26 Previously, we performed free energy calculations for a series of HCA IIpwt mutants. There we chose to estimate the changes in stability at a concentration of denaturant that was in the middle of the overlapping data in the various transition zones.11 Using these data we found that a decrease of CmNI of 0.1 M GuHCl corresponds to a mean destabilization of 0.82 kcal/mol of the native state relative to the molten-globule state. This value gives a stability of 7.9 kcal/mol for HCA IIpwt, which is in fairly good agreement with previously extrapolated values of the native conformation’s stability in water compared to the molten-globule intermediate (7.8, 6.8, and 7.9 kcal/mol are three estimates, derived using different methods of (23) Pace, C. N.; Vanderburg, K. E. Biochemistry 1979, 18, 288-292. (24) Cupo, J. F.; Pace, C. N. Biochemistry 1983, 22, 2654-2658. (25) Matthews, C. R. Methods. Enzymol. 1987, 154, 498-511. (26) Kellis, J. T.; Nyberg, K.; Fersht, A. R. Biochemistry 1989, 28, 4914-4922.

measurement).11,22 On the basis of the above-mentioned calculations, estimated destabilizations of the native state versus the molten-globule state of the various mutants are also given in Table 1. Clearly it is possible to engineer destabilizations in the range 1.7-5.4 kcal/mol, by the selected point mutations. The noted differences in stability of the variants do not seem to reflect any large disturbance in the protein conformation since the mutations have only minor effects on the catalytic capacity, as measured by either the CO2 hydration or the esterase activities of the enzyme. Thus, although the W97C mutant is least active, it still exhibits considerable specific CO2 hydration activity, equivalent to 51% of that of HCA IIpwt. The almost identical near-UV CD spectra of all but the W97C mutant (Figure 2) also clearly demonstrate that the tertiary structures of the variants are unaffected by the mutations. The CD spectrum of the W97C mutant has a different appearance, owing to the substitution of the Trp residue. It was earlier shown that W97 is the major contributor to the near-UV CD spectrum.15 However, NMR measurements have confirmed that this mutation causes only minor conformational changes in the molecule.27 The far-UV CD spectra of all mutants (Figure 2) are also typical of the native conformation of HCA II.15 3.3. Formation of a Molten-Globule-like State upon Adsorption of Destabilized HCA II Variants. A molten-globule intermediate is generally considered to have a somewhat larger radius than the native conformation of a protein,28 as demonstrated, for instance, by bovine carbonic anhydrase.29 In addition, most of the tertiary interactions are broken, while most of the secondary structure remains intact.11,28 Often, hydrophobic patches are formed in molten-globule states by amino acid residues that are otherwise buried in the native state. For HCA II it has been shown that the molten-globule state is not significantly more hydrated than the native state.30,31 The remarkably complex near-UV CD spectrum of the native conformation (Figure 2B) arises mainly from the seven Trp residues in the protein molecule because these (27) Mårtensson, L.-G.; Jonasson, P.; Freskgård, P.-O.; Svensson M.; Carlsson U.; Jonsson, B.-H. Biochemistry 1994, 34, 1011-1021. (28) Ptitsyn, O. B. Adv. Protein Chem. 1995, 47, 83-229. (29) Uversky, V. N. Biochemistry 1993, 32, 13288-13298. (30) Denisov, V. P.; Jonsson, B.-H.; Halle, B. Nat. Struct. Biol. 1999, 6, 253-260. (31) Jonasson, P.; Kjellsson, A.; Sethson, I.; Jonsson B.-H. FEBS Lett. 1999, 445, 361-365.

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Figure 3. CD spectra of HCA II variants adsorbed to silica particles. The CD spectra were recorded after all mutants had been inactivated by the adsorption. Panels: A, far-UV; B, near-UV. HCA IIpwt ()); M241L (- - -); S56C (- - -); W97C (- - - -); S56N (- - - -); S56F (s). The CD spectrum of HCA IIpwt was recorded after 5-month incubation with the particles, when 5% activity remained.

aromatic residues are locked in an asymmetric environment.15,32 Therefore, the near-UV CD spectrum is a very sensitive “fingerprint” of the native conformation with its tertiary interactions intact.33 In the GuHCl-induced unfolding transition from the native to the molten-globule state, when measured at equilibrium, the spectral bands of the near-UV CD spectrum disappear in conjunction with the loss in enzymatic activity.32 The various destabilized protein mutants completely lost their enzyme activity at different times after mixing with the particles. A CD spectrum of each HCA II variant was recorded on all samples when the activity had disappeared. Each near-UV CD spectra had lost its ellipticity bands known to be characteristic of the native state (Figure 3B). This implies that all aromatic residues had lost their asymmetric environments and, thus, their tertiary interactions at this stage. The corresponding farUV CD spectra of the adsorbed mutants also underwent significant changes (Figure 3A), becoming very similar to those of the similarly adsorbed truncated variants of HCA II, as reported earlier.4 The kinetics for the loss of enzymatic activity and tertiary structure are presented in section 3.5. The recorded far-UV CD spectra indicate that the adsorbed protein contained a substantial amount of secondary structure, as judged by comparisons with reference spectra.34 This conclusion is supported by our investigation of the effect on the far-UV CD spectra of GuHCl-induced transition from the native to the moltenglobule state.32 The transition to the molten-globule conformation is accompanied by a marked increase in negative ellipticity in the wavelength region around 200 nm. The change does not occur because of a change in the secondary structure components. Rather, it is due to contributions from positive ellipticities of aromatic residues in the native state, which are lost in the moltenglobule state. In conclusion, the CD spectra of the mutants clearly show that they all attained a molten-globule-like state following adsorption. The hydrophobic probe ANS is often used to identify the molten-globule conformation,35 and the emission (32) Bore´n, K.; Carlsson, U. Biochim. Biophys. Acta 1999, 1430, 111118. (33) Andersson, D.; Freskgård, P.-O.; Jonsson B.-H.; Carlsson U. Biochemistry 1997, 36, 4623-4630. (34) Greenfield, N.; Fasman, G. D. Biochemistry 1969, 8, 4108-4116.

spectra of this fluorophore, when added to the destabilized adsorbed protein variants, were typically blue-shifted to wavelengths (around 470 nm) that corresponded to that obtained when ANS was added to the GuHCl-induced molten globule of HCA II (Figure 6 below). This observation further supports the conclusion that all of these variants form a molten-globule-like state after adsorption to the silica particles. There are no indications that any of the adsorbed protein variants have an extended conformation. We have previously shown that a GuHCl-induced molten-globule state of HCA II has a compact hydrophobic interior and most of the dominating β-sheet (β-strands 3-7), located in this core, appear to be intact, whereas the outer β-strands are probably unfolded and the active site region is disrupted.11,12 The results presented here indicate that the adsorbed mutants have properties that are very similar to the GuHCl-induced molten-globule state of HCA II. 3.4. An Initially Nativelike State of Adsorbed HCA II. In contrast to the destabilized mutants, HCA IIpwt was very resistant toward inactivation via adsorption to particles. It retained, for instance, 60% of its enzymatic activity after 48-h incubation with the particles, and it still had 5% residual activity after 5-month incubation. At this late stage no CD bands in the near-UV wavelength region were detected, indicating that the tertiary structure had been ruptured in most of the protein molecules. Earlier, we reported that HCA IIpwt retained most of its native near-UV CD spectrum after 24-h incubation, and the effects on the ANS spectrum upon adsorption of HCA IIpwt were small.4 Our recent DSC (differential scanning calorimetry) measurements indicate that when in this adsorbed state, the protein has a structure that unfolds in a cooperative manner, but with a lower thermal stability than in the absence of particles.36 Thus, it seems as though HCA IIpwt adopts a specific conformation after adsorption with a relatively unaltered tertiary structure. This is then subjected to strain by interactions with the particles, leading to destabilization. To further characterize this state, we performed gel permeation chromatography experiments on the particle(35) Ptitsyn, O. B.; Pain, R. H.; Semisotnov, G. V.; Zerovnik, E.; Razgulyaev, O. I. FEBS Lett. 1990, 262, 20-24. (36) Billsten, P.; Carlsson, U.; Jonsson, B.-H.; Olofsson, G.; Ho¨o¨k, F.; Elwing, H. Langmuir 1999, 15, 6395-6399.

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Figure 4. Gel permeation elution profiles of particle-adsorbed HCA II variants. Panels: A. HCA IIpwt applied to the gel immediately after mixing with nanoparticles; B. HCA IIpwt after 12-h incubation; C. M241L applied to the gel immediately after mixing with nanoparticles; D. M241L and nanoparticles applied to the gel after complete inactivation; E. S56N applied to the gel immediately after mixing with nanoparticles; F. S56N and nanoparticles applied to the gel after complete inactivation; and G. free HCA IIpwt without particles as a reference. The UV absorbance values of the proteins are normalized for comparative purposes. The specific esterase activities (O) of adsorbed protein are shown in panels A-C and E.

adsorbed HCA IIpwt (Figure 4A,B). If the chromatography was performed immediately after mixing HCA IIpwt and the nanoparticles, a fairly broad elution profile was obtained, with a peak that coeluted with the particles (in the void volume, ∼26 mL), and a shoulder between the void volume and the elution volume of the free enzyme

(around 35 mL) (Figure 4A). The peak coeluting with the particles in the chromatogram represents adsorbed protein, since the UV absorbance (A280nm) due to the particles present has been subtracted. The broad tailing peak, however, suggests that a substantial fraction of protein molecules continuously adsorb to, and desorb from, the

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particles during the chromatographic process. Interestingly, the adsorbed protein exhibits almost full enzymatic activity (∼90%). This means that adsorption per se does not lead to inactivation of the enzyme. The specific activity of the enzyme at the point corresponding to the elution volume of the free enzyme is 100%, indicating that a minor fraction of HCA IIpwt has not bound to the particles. After 12-h incubation with the particles, the elution profile becomes somewhat narrower (Figure 4B), showing that a small fraction of the adsorbed protein molecules are in exchange with the solution at this stage. Thus, HCA IIpwt becomes more firmly associated to the particle surface after prolonged adsorption. The specific activity of the adsorbed enzyme decreases to about 70-80% that of the native value in the process, similar to the loss of enzymatic activity observed in the kinetic studies of the adsorption process after the same period of incubation (Figure 6A). This shows that a gradual inactivation of the enzyme occurs after the initial binding to the surface of the particles. In addition, the adsorbed, destabilized mutants were analyzed by gel permeation chromatography (selected chromatograms in Figure 4C-F). Samples were chromatographed both immediately after mixing with nanoparticles and after incubating the proteins with particles for a sufficient time to ensure complete inactivation. For all the variants, no significant shoulder in the chromatograms (Figure 4C-F) was detected at the point where the free HCA IIpwt eluted, indicating that no free enzyme was present in solution. Instead, the adsorbed, destabilized protein variants coeluted with the nanoparticles even when the separation was performed immediately after mixing protein and particles, showing that there is a rapid binding process. The specific enzyme activities of these bound variants were lower than that of HCA IIpwt, and the decrease in enzyme activity correlates well with the degree of destabilization of the protein variants. The level of inactivation corresponds reasonably well with the inactivation noted in the kinetic experiments, recorded at the time corresponding to the total time for chromatography and activity measurements (∼30 min.). These studies support the notion that the protein is first adsorbed to the surface of the particles and then undergoes conformational changes leading to an inactive enzyme. When the chromatographic separations were performed, after the enzyme activity had disappeared, the profiles of the elution peak were narrower (Figure 4D,F). This indicates that at this point the proteins were more tightly bound to the particles and that once the conformation of the protein had changed as far as to the molten-globulelike state, the inactivation was irreversible. 3.5. Kinetic Studies of the Adsorption Process. To investigate whether the kinetics of structural rearrangements of the protein during adsorption are affected by the particle concentration, the time course of inactivation was followed for various particle-to-protein concentration ratios in the range 1:1-5:1 (Figure 5). The kinetics of inactivation were found to be almost unaffected by the particle concentration, demonstrating that inactivation of the enzyme via particle adsorption is a first-order process. This suggests that the protein molecules bind rapidly to the particles and then undergo conformational changes leading to disruption of the active site. The large total surface area of the particles (700 cm2/mL in the 1:1 ratio) will, of course, facilitate productive collisions, leading to rapid binding between the protein molecules and the nanoparticles. This mechanism is also supported by the gel permeation experiments, showing that almost fully

Karlsson et al.

Figure 5. Comparison of kinetic traces of loss of tertiary structure after mixing particles and protein (S56N) in the following proportions: 1:1 (-O-), 2:1 (-)-), and 5:1 (-2-).

active HCA IIpwt was bound to the particles directly after mixing. The same experiment on the destabilized variants indicates that this mechanism is independent of the stability of the protein. Thus, adsorption precedes inactivation of the enzyme. The kinetics of different structural rearrangements that follow the adsorption process, after mixing a stoichiometric amount of protein and particles, was monitored by measuring the time dependence of various structural and functional parameters. The time courses of the change in enzymatic activity, near-UV CD ellipticity, and ANS fluorescence intensity of the studied variants are shown in Figure 6, in which the proteins are ordered according to their stability. The calculated rate constants are given in Table 2. The disappearance of the near-UV CD spectrum is monitored at the main ellipticity band at 270 nm, but, as shown for one variant (M241L), the whole near-UV CD spectrum changes uniformly with time (Figure 7). For all variants it should be noted that inactivation precedes the CD change, indicating that the active site is ruptured before the rest of the tertiary structure. This is contrary to the kinetics of chemical denaturation, where the tertiary interactions at the tryptophans are broken prior to loss of activity (Figure 8). The chemical denaturation kinetics are in agreement with the reversed behavior suggested by the results of refolding studies, where activity was regained before the completion of refolding.37 These observations imply that the conformational changes induced by surface adsorption are probably mediated by mechanisms different from those that cause either chemical or thermal denaturation. Thus, the particles appear to be able to induce local conformational changes, which suggests that the particles might bind in the vicinity of the active site. Interestingly, we have showed earlier that the N-terminal minor domain, which forms a “lid” over the active site, is less stable than the rest of the molecule.38,39 The time courses of the increase in fluorescence intensity at 470 nm, when ANS was added at various stages of the adsorption process, were analyzed for all of the adsorbed protein variants (Figure 6). This emission increase reflects formation of the molten-globule state, and the ANS fluorescence time courses coincide, within the limits of experimental error, with those of the near-UV CD change for the destabilized mutants. Thus, the molten-globule state appears to be formed concomitantly with the disruption of the tertiary structure. (37) Andersson, D. Doctoral Thesis, University of Linko¨ping, Sweden, 1999; ISBN 91-7219-643-2, ISSN 0345-7524. (38) Aronsson, G.; Mårtensson, L.-G.; Carlsson U.; Jonsson, B.-H. Biochemistry 1995, 34, 2153-2162. (39) Jonasson, P.; Aronsson G.; Carlsson U.; Jonsson B.-H Biochemistry 1997, 36, 5142-5148.

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Figure 6. Time courses for the change of different conformation-sensitive parameters during particle adsorption. Activity (O), CD ellipticity at 270 nm, registered both continuously (-) and at set time points (-•-), and ANS fluorescence at 470 nm (4). The change in CD ellipticity was measured continuously at 270 nm for 2 h. For longer periods CD spectra were recorded at various times during the adsorption process, and the ellipticities at 270 nm were then taken from these spectra. ANS was added to the adsorbing protein at various times, and fluorescence spectra were recorded immediately after these additions. The continuous curves were calculated from the rate constants given in Table 2. Panels: A. HCA IIpwt; B. M241L; C. S56C; D. W97C; E. S56N; F. S56F.

There is a striking correlation between the rate of conformational unfolding induced by particle adsorption and the global stability of the protein structure, as can be seen in Figure 9 and Table 2. Apparently, the rate of formation of an inactive molten-globule conformation is

generally accelerated if the global stability of the protein is lowered. The only exception to this rule is provided by the M241L mutant, which is more rapidly unfolded than the somewhat less stable S56C mutant. If the particles are able to induce local conformational changes, the

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Table 2. Kinetic Data Associated with the Loss of Enzyme Activity and Tertiary Structure, As Measured by Near-UV CD CD270nm protein HCA IIpwt M241L S56C W97C S56N S56F a

k1

a

(min-1)

A1

0.10 0.38 0.24 0.38 0.93 3.9

0.02 0.20 0.1 0.57 0.72 0.92

a

activity

k2

a

(min-1)

0.008 0.02 0.02 0.04 0.12 0.06

a

A2

0.1 0.54 0.30 0.43 0.29 0.09

k1

a

(min-1)

A1

k2a (min-1)

A2a

1.46 2.4 2.3 3.1 3.1 5.9

0.18 0.37 0.19 0.71 0.81 0.90

0.05 0.03 0.10 0.10 0.20 0.46

0.2 0.38 0.29 0.29 0.20 0.10

a

The rate constants and amplitudes were calculated using a nonlinear fit program (see Materials and Methods).

Figure 7. Near-UV CD spectra of the particle-adsorbed M241L variant. CD spectra were recorded at various stages of the adsorption process. Adsorption times: 0 h, native ()); 0.5 h () ) )); 1 h (- - -); 2 h (- - - -); 3 h (s); 4h (- - -); 1 week, inactive (- - - -).

Figure 9. Kinetic comparisons of the loss of tertiary structure of the destabilized mutants following adsorption, measured as changes in CD ellipticity at 270 nm. The stability of each mutant, in kcal/mol, is given in parentheses for each mutant in the figure.

The kinetic data showed no significant concentration dependence and were best fitted to a first-order reaction (two phases), supporting the conclusion that the structural rearrangements of the protein molecules occur after adsorption to the particles. This interpretation of the kinetic data is further supported by the chromatographic results for HCA IIpwt discussed above, which showed that protein adsorption preceded inactivation of the enzyme. Conclusions

Figure 8. Chemical denaturation. Time courses for the change in different conformation-sensitive parameters during chemical denaturation. Activity (O) and CD ellipticity at 270 nm registered continuously (-).

deviant behavior of the M241L variant might be due to the position of the mutation since the M241L mutation is in a loop region, unlike those of the other variants (Figure 1, Table 1). Furthermore, it is located in the most positively charged region of the protein. Since the nanoparticles are negatively charged, there is reason to believe that the initial adsorption is electrostatic in nature. Thus, the protein might adsorb in a specific orientation, in the vicinity of the M241L mutation. It is also noteworthy that the inactivation and conformational rearrangements of HCA IIpwt are extremely slow. By extrapolation of this trend, it appears that it would be possible to engineer a variant of the enzyme having higher global stability than the wild-type, which would not be affected, or would be only marginally affected, by surface adsorption.

From this study, in which varying global stabilities have been engineered into a protein, it is evident that the rate of conformational rearrangements induced by adsorption is strongly correlated to the global stability of the protein molecule. Thus, the less stable the structure is, the more firmly the protein is associated with the surface and the more rapidly the structure is unfolded. The following model of the adsorption process is supported by our data (Figure 10): initially there is a rapid protein-particle complex formation, followed by irreversible conformational changes, ultimately leading to a molten-globule-like state. The stability of the protein mainly affects the second step in this scheme. Thus, a less stable protein variant is unable to resist the strain that is inflicted upon it when interacting with the particle surface. This conclusion is further strengthened by the fact that various destabilizing substitutions in a specific buried position (pos. 56) follow this trend, ruling out effects from different kinds of structural changes and local chemical properties that could occur when amino acids are mutated in more exposed locations of the molecule. The degree to which the adsorbed protein unfolds is not dependent on its stability, and the protein does not unfold

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Figure 10. Drawing for the course of adsorption and subsequent conformational changes of the protein.

beyond the molten-globule-like state. The reason the different destabilized variants unfold to the same extent is most likely that the stability of the molten-globule conformation is almost unaffected by the mutations, which only reduce the stability of the native state. Interestingly, the particles induce stepwise conformational changes upon adsorption that occur in reverse order, as compared with the stages involved in chemical denaturation of the protein, as demonstrated by the initial rearrangements of the active site followed by breakage of the tertiary interactions. This is an interesting fact one has to take into consideration when comparing global

stability with stability at surfaces, since it clearly shows that denaturation as a consequence of adsorption differs from chemical denaturation. Most probably this is because the protein interacts in a specific orientation with surfaces, while in chemical denaturation the protein is immersed in the denaturing medium. Acknowledgment. This work was financially supported by the Swedish Research Council for Engineering Sciences. LA0002738