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High-Resolution 2D 1H-15N NMR Characterization of Persistent Structural Alterations of Proteins Induced by Interactions with Silica Nanoparticles Martin Lundqvist,† Ingmar Sethson,‡ and Bengt-Harald Jonsson*,† Division of Molecular Biotechnology, IFM, Linko¨ ping University, SE-58183 Linko¨ ping, Sweden, and Department of Organic Chemistry, Umeå University, 901 87 Umeå, Sweden Received March 2, 2005. In Final Form: April 22, 2005 The binding of protein to solid surfaces often induces changes in the structure, and to investigate these matters we have selected two different protein-nanoparticle systems. The first system concerns the enzyme human carbonic anhydrase II which binds essentially irreversibly to the nanoparticles, and the second system concerns human carbonic anhydrase I which alternate between the adsorbed and free state upon interaction with nanoparticles. Application of the TROSY pulse sequence has allowed high-resolution NMR analysis for both of the protein-nanoparticle systems. For HCAII it was possible to observe spectra of protein when bound to the nanoparticles. The results indicated that HCAII undergoes large rearrangements, forming an ensemble of molten globule-like structures on the surface. The spectra from the HCAI-nanoparticle system are dominated by HCAI molecules in solution. A comparative analysis of variations in intensity from 97 amide resonances in a 1H-15N TROSY spectrum revealed the effects from interaction with nanoparticle on the protein structure at amino acid resolution.
Introduction Proteins’ interactions with, and adsorption to, solid surfaces is of interest in a large variety of fields such as protein purification, the food industry, and medicine.1 In recent decades research has considerably deepened knowledge about the mechanisms of protein adsorption from a macroscopic perspective. Differences in adsorption patterns of different proteins to the same kind of surfaces under identical conditions have been shown to be due to differences in their chemical properties (surface charges, hydophobicity, etc.) and their conformational stability.2-6 The knowledge obtained has been mostly based on CD,7 fluorescence,8 and IR9 analyses and other methods that give valuable information on secondary structure and gross alterations in tertiary structure, but limited information on conformations at individual amino acid residues. However, to characterize the conformational consequences of proteins’ interactions with a solid phase more comprehensively at high resolution, a method that generates data from many individual atoms throughout the entire protein structure is required. Two-dimensional 1H-15N NMR can give high-resolution data from all coupled H and N atoms in a protein; i.e., the NMR signal of a amide proton is connected with the nitrogen that is directly attached to it by magnetization transfer. Therefore, the signal (resonance) from the amide proton and the nitrogen nuclei are correlated and together produce a 2-dimensional †
Linko¨ping University. Umeå University. * Corresponding author: e-mail,
[email protected].
‡
(1) Nakanishi, K.; Sakiyama, T.; Imamura, K. J. Biosci. Bioeng. 2001, 91, 233. (2) Arai, T.; Norde, W. Colloids Surf. 1990, 51, 1. (3) Bhaduri, A.; Das, K. P. J. Dispersion Sci. Technol. 1999, 20, 1097. (4) Kondo, A.; Urabe, T. J. Colloid Interface Sci. 1995, 174, 191. (5) Norde, W.; Haynes, C. A. Proteins Interfaces II 1995, 602, 26. (6) Karlsson, M.; Mårtensson, L. G.; Jonsson, B. H.; Carlsson, U. Langmuir 2000, 16, 8470. (7) Billsten, P.; Freskgård, P. O.; Carlsson, U.; Jonsson, B. H.; Elwing, H. FEBS Lett. 1997, 402, 67. (8) Czeslik, C.; Jackler, G.; Royer, C. Spectrosc.: Int. J. 2002, 16, 139. (9) Tarasevich, Y. I.; Monakhova, L. I. Colloid J. 2002, 64, 482.
NMR spectrum that contains a single signal (a peak in the spectrum) for every amide group in the protein. The position of the signal (the shift) and the dispersion of the signals in the spectrum reflect the local environment of each amide. Changes in the protein structure that occur as a result of environmental changes is reflected in the spectrum as either changes in shift, line width, or volume and can be a combination of them. However, the technique is difficult to apply to samples where the protein molecule is attached to a solid phase. Nevertheless, data concerning a peptide adsorbed to charged colloidal substrates in an aqueous environment, obtained using solution 1H NMR, have been published recently.10,11 The major problem in studying proteins bound to particles is that the complexes tumble very slowly in solution, leading to extreme line broadening in the NMR spectrum. Using nanoparticles with a diameter of 6 nm (which have an approximate weight of 135 kDa) can partly solve this problem when used in combination with an NMR pulse sequence (TROSY12) designed for studies of high molecular weight complexes. The line broadening is dependent on a combination of different relaxation mechanisms. In TROSY experiments the relaxation that cause line broadening is efficiently filtered out, and only a narrow component that essentially is independent of molecular size is observed in the NMR spectrum; i.e., the TROSY methods select this narrow component and therefore have high sensitivity when applied on large complexes. This technique has been successfully applied to large proteins and protein complexes such as 7,8-dihydroneopterin aldolase, a homooctameric protein of 110 kDa,13 and malate synthase G, an 82 kDa monomeric protein.14 (10) Burkett, S. L.; Read, M. J. Langmuir 2001, 17, 5059. (11) Read, M. J.; Burkett, S. L. J. Colloid Interface Sci. 2003, 261, 255. (12) Pervushin, K.; Riek, R.; Wider, G.; Wuthrich, K. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 12366. (13) Salzmann, M.; Pervushin, K.; Wider, G.; Senn, H.; Wuthrich, K. J. Am. Chem. Soc. 2000, 122, 7543. (14) Tugarinov, V.; Hwang, P. M.; Kay, L. E. Annu. Rev. Biochem. 2004, 73, 107.
10.1021/la050569j CCC: $30.25 © 2005 American Chemical Society Published on Web 05/24/2005
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In this study we used the human carbonic anhydrase isoenzymes I (HCAI) and II (HCAII), described elsewhere,6,15-23 as model proteins and silica nanoparticles as the solid surface. Notably, the diameters of these two proteins is ∼4.5 nm. Both HCAI and HCAII have been thoroughly investigated by NMR methods, and their backbone atoms have been individually assigned in NMR spectra.24,25 Binding of HCAII to 6 nm silica particles initially leads to complete loss of all NMR signals in a TROSY spectrum. However, measurements after incubation for 4-7 days result in detectable NMR resonances that increase in intensity with incubation time. Additional analysis indicates that the detectable resonances emanate from bound protein in nonnative conformations. HCAI, on the other hand, gives spectra that have nativelike shifts in the presence of particles. A comparison of the stabilities shows that HCAI has approximately 2.5 kcal/mol higher free energy of unfolding than HCAII.17,26 HCAI establishes a dynamic equilibrium between bound and unbound protein when mixed with silica particles.18 At equilibrium the exchange between the two states is slow on the NMR time scale. This allows us to study the effects of the interaction with the solid surface on the protein structure in more detail than would be possible for a process with faster kinetics. That is, the effects on the protein structure of the interaction with the solid surface are prolonged for some time in the unbound protein and can therefore be inferred by monitoring the unbound state with highresolution solution NMR in samples with protein and particles in equilibrium. In studies of other systems elegant H/D exchange experiments have given detailed information, at the level of individual amino acids, about interaction areas between protein and solid surfaces.27,28 The NMR measurements on HCAI show that the interaction with particles results in pronounced differences in the effects on different residues regarding the intensity of their corresponding resonances. The results allow us to characterize effects of the interactions on both deeply buried and surface-exposed amino acid residues in HCAI. However, good conditions for experiments that can give detailed information about which amino acids are involved in the initial steps of the binding to the surface remain to be found. (15) Carlsson, U.; Aasa, R.; Henderson, L. E.; Jonsson, B. H.; Lindskog, S. Eur. J. Biochem. 1975, 14, 25. (16) Kannan, K. K.; Notstrand, B.; Fridborg, K.; Lo¨vgren, S.; Ohlsson, A.; Petef, M. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 51. (17) Kjellsson, A.; Sethson, I.; Jonsson, B. H. Biochemistry 2003, 42, 363. (18) Lundqvist, M.; Sethson, I.; Jonsson, B. H. Langmuir 2004, 20, 10639. (19) Carlsson, U.; Jonsson, B. H. In The Carbonic Anhydrases; Chegwidden, W. R., Carter, N. D., Edwards, Y. H., Eds.; New Horizons: 2000; p 241. (20) Freskgård, P. O.; Mårtensson, L. G.; Jonasson, P.; Jonsson, B. H.; Carlsson, U. Biochemistry 1994, 33, 14281. (21) Håkansson, K.; Carlsson, M.; Svensson, L. A.; Liljas, A. J. Mol. Biol. 1992, 227, 1192. (22) Hammarstro¨m, P.; Persson, M.; Freskgård, P. O.; Mårtensson, L. G.; Andersson, D.; Jonsson, B. H.; Carlsson, U. J. Biol. Chem. 1999, 274, 32897. (23) Eriksson, A. E.; Jones, T. A.; Liljas, A. Proteins 1988, 4, 274. (24) Venters, R. A.; Farmer, B. T.; Fierke, C. A.; Spicer, L. D. J. Mol. Biol. 1996, 264, 1101. (25) Sethson, I.; Edlund, U.; Holak, T. A.; Ross, A.; Jonsson, B. H. J. Biomol. NMR 1996, 8, 417. (26) Freskgård, P. O.; Carlsson, U.; Mårtensson, L. G.; Jonsson, B. H. FEBS Lett. 1991, 289, 117. (27) Aizawa, T.; Koganesawa, N.; Kamakura, A.; Masaki, K.; Matsuura, A.; Nagadome, H.; Terada, Y.; Kawano, K.; Nitta, K. FEBS Lett. 1998, 422, 175. (28) Engel, M. F. M.; Visser, A.; van Mierlo, C. P. M. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 11316.
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Materials and Methods Silica Particles. The colloidal, negatively charged silica particles used in this study were kindly provided by EKAChemicals, Stenungsund, Sweden. The stock solutions contained 5.09 × 1017 particles/mL with an average diameter of 9 nm and 7.30 × 1017 particles/mL with an average diameter of 6 nm. Before use, they were extensively dialyzed against sample buffer (20 mM Tris, 20-40 mM NaCl, pH 8.4). Both types of particle, which have the same surface charge density according to their manufacturer (personal communication), are stable in solutions at pH greater than 8 and in moderate salt concentrations.29 NMR Experiments. Uniformly 15N-labeled HCAI and HCAII were produced and purified as described by Lundqvist et al.18 The NMR samples consisted of 0.3 mM protein in 20 mM Tris buffer with 40 mM NaCl at pH 8.4 and a 90:10 H2O:D2O ratio. The temperature was held at 30 °C. The protein:particle molar ratio of HCAI samples was 1:1.1 for the 6 nm particles and 1:1.2, 1:0.6, and 1:0.3 for the 9 nm particles. For HCAII the protein: particle ratio was always 1:0.5, with 6 nm particles. The 2D-NMR experiments were performed using a Bruker DRX 600 spectrometer equipped with a triple-resonance (1H/ 13C/15N) probe with XYZ-gradient capabilities using a sensitivityenhanced water flip back TROSY pulse sequence.30 The data collection time was ≈12 h for each spectrum. The 2D data were processed using the program nmrPipe,31 and the spectra were analyzed using the program FELIX.32
Results Spectra from HCAII Bound to Nanoparticles. 2DNMR experiments were conducted with a 1:0.5 ratio of HCAII to 6 nm silica particles. NMR measurements conducted immediately after the protein was mixed with particles showed a complete loss of signals. Measurements after longer incubation times (days) resulted in spectra with ∼20 resonances that grew in intensity with longer incubation times (up to 6-7 days) and then gradually decreased (see Figure 1). To test for the possibility that the change in signal upon prolonged incubation time was due to proteolysis, the protein was desorbed by boiling with a SDS-PAGE loading buffer. Subsequent analyses by SDS-PAGE show only one band corresponding to intact protein. The intensity of the band was virtually identical to the band from an untreated protein sample, showing that no proteolysis occurs within the experimental time. The narrow shift range of those resonances indicates that the bound protein had lost a defined tertiary structure. The results indicate that conformational rearrangements of the bound protein occur over a long time, and equilibrium is not reached within a week under these conditions. Spectra from HCAI in the Presence of Nanoparticles. For HCAI, 2D-NMR experiments were conducted on samples with 1:1.2, 1:0.6, and 1:0.3 ratios of protein to 9 nm silica particles and a 1:1.1 ratio of protein to 6 nm particles. Comparisons of the different spectra in Figure 2 clearly show that the protein has a nativelike conformation and that the total signal from the protein in the spectra declines significantly immediately after addition of silica particles to the sample (a 75-95% reduction, depending on the particles used and the ratio). The amount of signal lost correlates to the concentration of the particles; 1:1.2, 1:0.6, and 1:0.3 protein to 9 nm particle ratios gave 95, 90, and 80% reductions in signal strength, respectively. For the samples with 1:0.3 protein to 9 nm silica particle and 1:1.1 protein to 6 nm silica particle ratios, it was (29) www.ekachemicals.se. (30) Pervushin, K. V.; Wider, G.; Wuthrich, K. J. Biomol. NMR 1998, 12, 345. (31) Delaglio, F.; Grzesiek, S.; Vuister, G. W.; Zhu, G.; Pfeifer, J.; Bax, A. J. Biomol. NMR 1995, 6, 277. (32) www.accelrys.com.
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Figure 1. NMR spectra (acquired with a TROSY pulse sequence) for a 1:0.5 ratio of HCAII and 6 nm silica particles. Spectrum a, HCAII without nanoparticles, shows that the protein in its native form gives a well-dispersed NMR spectrum. Spectrum b, ∼4 days after the addition of particles, shows a few low-intensity nonnative peaks. In spectrum c, ∼5 days after the addition of particles, a few more nonnative peaks become visible, and the intensities of the peaks have increased. Spectrum d, ∼19 days after the addition of particles, shows a decrease in the number and intensity of nonnative. The contour level is set to the same value in the three spectra b-d.
possible to use the resonance assignments by Sethson et al.25 to monitor 97 well-resolved backbone NH resonances and six indole protons from the tryptophans in HCAI. Individual Amino Acids’ NH Backbone Resonances of HCAI Differ in the Intensity Lost in the Initial Phase. The data collection for the first spectrum was initiated 30 min after mixing. Comparison of the changes in signal volume for resonances at various native positions reveals that the reduction in the initial phase of incubation with silica particles differs among the various backbone NH resonances; i.e., interactions with the nanoparticles have different effects at different positions in the protein. The observed difference in the volumes is not an artifact due to data processing because residues that retain both large and small proportions of the initial signal are scattered throughout the spectrum (data not shown). Changes in Spectrum Signal over Time. After the first large reduction of signal volume in the spectrum the native resonances slowly decrease toward noise levels, while other resonances, corresponding to a molten globulelike structure of the protein, rise from noise levels with time. The rate of increase of resonances that indicate a molten globule state correlates well with the rate of loss of tertiary structure and maintained secondary structure measured by near- and far-UV CD.18 The peak indicating a molten globule state that is easiest to observe is the common peak for the indole protons of the six tryptophan side chains. This peak appears almost immediately for samples with a high particle concentration and after 2-3 days with a low particle concentration (1:0.3); i.e., the rate of its appearance depends on the particle concentration.
the protein sample, virtually all of the protein molecules will be attached to the surface. Thus, HCAII was considered a good candidate for high-resolution solution NMR studies of proteins firmly bound to nanoparticles. In a control experiment, HCAII was incubated with nanoparticles for 5 days, after which the particles were removed from the solution by centrifugation. Absorption and NMR measurements on the remaining solution showed no detectable protein, indicating that the concentration of free protein is lower than 0.5 µM. Using this value for calculation of the equilibrium constant shows that the affinity is higher than Ka ) 4 nM, which confirms earlier observations which indicated that HCAII binds strongly to the nanoparticles.6,7 Therefore, it is highly probable that the spectra shown in Figure 1 originate from HCAII bound to the nanoparticles. However, the intensity of the observed spectrum varies with incubation time, and the intensity of the spectrum with the highest intensity is much lower than might be expected from comparison with a spectrum of HCAII in solution. The weakness of the observed intensity may be due to several factors, the most obvious being limitations of the efficiency of the TROSY technique. In theory, when using the TROSY pulse sequence,12 it should be possible to observe a protein molecule that is adsorbed to a silica nanoparticle. However, the field strength (14.1 T) of the 600 MHz instrument used in this study is far from the optimal field strength of 25.8 T that would give a maximum TROSY effect,33 leading to a reduced intensity in our spectra. Another important factor relates to the observation by Karlsson et al.6 and Billsten et al.,7 that when HCAII binds to silica particles it undergoes structural transitions toward a molten globular-like conformation. Interestingly, Zhang et al.34 and Schulman et al.35 have shown that a molten
Discussion HCAII with Particles. Karlsson et al.6 and Billsten et al.7 have shown that HCAII binds strongly to silica surfaces; i.e., when sufficient silica surface is present in
(33) Pervushin, K. Q. Rev. Biophys. 2000, 33, 161. (34) Zhang, O. W.; Forman-Kay, J. D. Biochemistry 1997, 36, 3959. (35) Schulman, B. A.; Kim, P. S.; Dobson, C. M.; Redfield, C. Nat. Struct. Biol. 1997, 4, 630.
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Figure 2. Examples of partial 2D-NMR spectra (acquired with a TROSY pulse sequence) of HCAI. Panels a-e intend to show the effects on the intensities of the individual resonances under the different experimental condition: (a) a 1:1.1 ratio of 6 nm silica particles, (b) a 1:1.2 ratio of 9 nm silica particles, (c) a 1:0.6 ratio of 9 nm silica particles, (d) a 1:0.3 ratio of 9 nm silica particles, and (e) without particles. The contour level is set to the same value in spectra a-e. Panel f shows the full spectrum for HCAI without particles. The data collection started 30 min after mixing particle and protein for all samples.
globule in solution can be described as an ensemble of conformations. NMR is a relatively insensitive method and needs a relatively high concentration of a molecule with one conformation in order to generate a spectrum of it. Hence, the spectra of molten globule-like proteins can differ greatly in intensity, depending on the relative concentration of each conformation and the dynamic characteristics of interconversion between them. Complex chemical exchange in the intermediate exchange region may give rise to line broadening of resonances beyond detection.36,37 The NMR measurements on samples with HCAII bound to nanoparticles were conducted over a period of 20 days, and the results should be evaluated with the above(36) Sudmeier, J. L.; Evelhoch, J. L.; Jonsson, N. B.-H. J. Magn. Reson. 1980, 40, 377. (37) Harris, R. K. Nuclear Magnetic Resonance Spectroscopy, 2nd ed.; John Wiley & Sons: New York, 1986.
mentioned considerations. No spectrum was observed during the first 3 days of incubation. On day four a few resonances appeared, and in the next 3 days the resonances increased in number and grew in intensity. However, upon further incubation the intensity of the spectra slowly decreased. During the first days after mixing the protein apparently adopted too many conformations on the nanoparticles to make it observable with NMR. Later (days 4-7), it seemed that the equilibrium shifted toward fewer conformations, or the flexibility in parts of the structure allowed fast chemical exchange, leading to observable NMR resonances because of line narrowing. The observed chemical shift and the dispersion range clearly indicate that the protein lost its well-defined tertiary structure, which is compatible with a molten globular-like conformation, as inferred from earlier measurements by CD. Upon longer incubations it seems that the protein structure rearranges to form an ensemble of
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other conformations that cannot be observed; i.e., the protein has rearranged into conformations that for most resonances have chemical exchange in the intermediate exchange region (broaden beyond detection). HCAI with Particles. HCAI is a more stable protein (by ∼2.5 kcal/mol) than HCAII17,26 and, in contrast to HCAII, gives a nativelike NMR spectrum (Figure 2) in solutions together with particles. The signal intensity, however, becomes considerably reduced immediately after the particles have been added to the protein sample, and the decrease in intensity correlates with the concentration of particles. Recently, it was established that HCAI bound to the silica particles is in equilibrium with free protein molecules in solution for at least 7 days after mixing with particles and that the interaction with the particles induces alterations in the protein’s native structure.18 However, the structural alterations of HCAI occur much more slowly than the structural alteration of HCAII upon treatment with particles. The extent of structural alteration of HCAI has been shown to depend on the curvature of the particle, and the rate of change depends on the concentration of the particles. It has also been shown that both HCAI’s residence time on the particles and the structural alterations of the protein slightly increase as incubation time with the particles increases.18 Since HCAII, which binds tightly to the surface, generates an extraordinarily low-intensity spectrum due to the tight binding of the molecules, we decided to investigate protein-particle interactions using HCAI, which establishes an equilibrium between bound and free states. In this system the NMR spectrum will be completely dominated by those HCAI molecules that are free in solution. Hence, the spectrum reflects the status of the HCAI molecules that are not interacting with the surface at the moment of measurement. This conclusion is also supported by the fact that the total signal intensity in the observed spectra slowly decreases with incubation time (several days), correlating well with the slow shift, previously observed, in the equilibrium with time from free toward bound protein.18 Notably, these NMR spectra are similar to spectra of native HCAI. However, they differ in intensity at several resonances, and clearly, these spectral differences must be coupled to the conformational rearrangements that have occurred in the bound state. Therefore, it may be possible to deduce information about the bound state from the changes in the free HCAI molecule. For instance, the observation that only 5-30% of the intensity remains in the protein/particle sample compared with native HCAI in solution can be rationalized as follows, for the case with 30% observable signal: ∼25% of the native signal is lost because NMR sensitivity is lower in the presence of particles, as previously shown.18 Therefore, the observed level of ∼30% remaining signal indicates that (30/75) × 100 (%), i.e., ∼40%, of the HCAI molecules are free in solution in the equilibrium between bound and free states, 6 h after mixing. Approximately 40% unbound HCAI is close to the proportion observed for this equilibrium in gel permeation experiments and analytical ultracentrifugation experiments,18 considering that the concentrations of HCAI and particles are higher in the NMR experiments; i.e., the equilibrium is pushed a little toward the bound state in the NMR samples. Those residues that show less than 30% intensity has been affected by the interaction with the particles in a residue-specific manner, and the effect on the conformations at these residues is maintained sufficiently long to be observed in the free HCAI molecule.
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Figure 3. 3-D structure of HCAI, highlighting individual backbone nitrogen atoms of the 25 residues with the strongest remaining signals (blue spheres) and the 25 residues with the weakest remaining signals (red spheres) after interaction with silica particles. The left panel is for a 1:1.1 protein to 6 nm particle sample, and the right panel for a 1:0.3 protein to 9 nm particle sample. The figure was prepared using the program MolMol.38
The lower amount of signal obtained with the 1:0.6 and 1:1.2 protein to 9 nm silica particle ratios is probably due to the shift of the equilibrium toward the bound state because of the higher particle concentration. Perturbations at the Backbone Amide Groups of Individual Amino Acid Residues. The samples with 1:0.3 and 1:1 ratios of protein to 9 and 6 nm silica particles gave very similar NMR spectra (no differences in shifts or line width) immediately after mixing. It was possible to monitor 97 backbone NHs in both of these experiments. The individual signal volume was calculated for all these positions, and it was found that the signals from some residues remain at an intensity that would be expected from the initial concentration of free protein, while the intensity of other signals decreases to much lower levels. To analyze these results in relation to a 3-dimensional structure, the 25 residues that were most affected (i.e., lost most volume) and the 25 residues that were least affected (lost least volume) upon mixing with particles were displayed in a 3D structural representation of the protein (Figure 3). The patterns revealed in the two 3-D representations (Figure 3) are very similar in appearance, implying that the structural alterations induced by the two types of nanoparticle preparation are comparable. Interestingly, the residues in the central β-strands 5-8 (from left to right in Figure 3) appear to be most affected by the interaction with the particles, while residues on the surface of the protein seem to be relatively unaffected. Clearly, the interactions between a HCAI molecule and a silica particle induce conformational changes throughout the HCAI structure. One would expect the residues at the surface of the protein, which must interact with the nanoparticle, to change their conformation upon interaction. However, the NMR results indicate that the effects on surface residues are minor or nonexistent. Thus, the N-terminal, the loops, and the peripheral secondary structures seem to adopt a native conformation rapidly upon dissociation from the nanoparticles. The results also indicate that the central β-strands are strongly affected by the interaction and that the nonnative conformations at these residues have lifetimes that are long enough for the conformation to be maintained in the free protein; i.e., conformational rearrangements among the residues in the central β-strands have quite high-energy barriers in the free protein. This is in good agreement with previously acquired H/D exchange data for HCAI,17,39 (38) Koradi, R.; Billeter, M.; Wuthrich, K. J. Mol. Graphics 1996, 14, 51.
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differences, which indicate that the gradual change in the protein structure starts immediately after mixing. This conclusion is corroborated by near- and far-UV CD results from near identical conditions, showing a gradual change toward the molten globular state within a similar time span.18 The interaction appears to gradually affect the equilibrium between free and bound protein, and the free protein accumulates more extensive conformational changes. The observation of a common peak for all of the tryptophan indole protons indicates that the protein ensemble slowly progresses toward a molten globularlike structure. Conclusions
Figure 4. Spectra (acquired with a TROSY pulse sequence) for HCAI with a 1:1 ratio of 9 nm silica particles after 4 h (upper panel) and 52 h (lower panel) incubation. The arrow indicates the peak for the indole protons.
which showed that the loops and N-terminal are quite flexible under native conditions, while the central β-sheet is rather rigid. Hence, the results in this study show that the nonnative conformation of residues in the central β-sheet does not readily rearrange to the native structure when an HCAI molecule dissociates from a particle, and earlier H/D exchange studies on native protein also show that rearrangements in the central β-sheet have highenergy barriers.17,39 Effects of Prolonged Incubations. The NMR studies of protein-particle samples gathered after prolonged incubation show that the spectra change gradually, as illustrated by comparing the spectrum gathered immediately after mixing with the spectrum gathered after 52 h (Figure 4). A comparison of the spectrum of the native protein with the first spectrum after mixing shows clear (39) Kellsson Lind, A. Doctoral thesis, Umeå University (Umeå), 2002.
The data obtained by studying two closely related proteins (the carbonic anhydrase isozymes HCAI and HCAII, which differ in stability and can be characterized as “hard” and “soft”, respectively) clearly show that the interactions with the nanoparticles have very different impacts on the structure of the two different proteins. The “soft” HCAII binds strongly and rapidly loses its native structure, forming an ensemble of nonnative conformations. In longer incubations (days) the conformational rearrangements continue, leading to the formation of an ensemble of molten globule-like bound protein states. The study of HCAII shows that by use of the pulse sequence TROSY it is possible to obtain high-resolution NMR spectra of a protein bound to nanoparticles in the 140 kDa range. For the “hard” protein HCAI, which binds weakly to the nanoparticles, it is possible to glean information about structural perturbations in the bound state by analyzing high-resolution solution NMR spectra that emanate from free protein molecules in the sample. Despite weak binding to the nanoparticles, it is apparent that the binding perturbs even the central part of the protein. Prolonged incubation leads to a gradual perturbation of the structure toward nonnative tertiary conformations. Acknowledgment. This work was supported by a grant from the Swedish National Science Research Council to B.-H. Jonsson (K5104-5999). Financial support from The Sven and Lilly Lawski Foundation for Scientific Studies to M. Lundqvist is gratefully acknowledged. We thank Ms. Katarina Wallgren for excellent technical assistance. LA050569J
