9532
J. Phys. Chem. B 2008, 112, 9532–9539
Effect of the Air-Water Interface on the Structure of Lysozyme in the Presence of Guanidinium Chloride Adam W. Perriman,† Mark J. Henderson,‡ Christian R. Evenhuis, Duncan J. McGillivray,§ and John W. White* Research School of Chemistry, The Australian National UniVersity, Canberra, ACT 0200, Australia ReceiVed: January 14, 2008; ReVised Manuscript ReceiVed: April 29, 2008
We report observations of the changes in the surface structure of lysozyme adsorbed at the air-water interface produced by the chemical denaturant guanidinium chloride. A primary result is the durability of the adsorbed surface layer to denaturation, as compared to the molecule in the bulk solution. Data on the surface film were obtained from X-ray and neutron reflectivity measurements and modeled simultaneously. The behavior of lysozyme in G.HCl solutions was determined by small-angle X-ray scattering. For the air-water interface, determination of the adsorbed protein layer dimensions shows that at low to moderate denaturant concentrations (up to 2 mol L-1), there is no significant distortion of the protein’s tertiary structure at the interface, as changes in the orientation of the protein are sufficient to model data. At higher denaturant concentrations, time-dependent multilayer formation occurred, indicating molecular aggregation at the surface. Methodologies to predict the protein orientation at the interface, based on amino acid residues’ surface affinities and charge, were critiqued and validated against our experimental data. 1. Introduction Exposing proteins to increasing concentrations of chemical denaturants is performed routinely to provide information on their stability. However, most research has focused on the effects of chemical denaturants on globular proteins in solution rather than those adsorbed at the air-water interface. The air-water interface presents a highly asymmetric environment that impacts protein stability and may alter their resistance to chemical denaturation. We explored the changes in the structure of lysozyme adsorbed at the air-solution interface when exposed to increasing concentrations of guanidinium chloride (G.HCl) using neutron and X-ray reflectometry. These changes are compared with the protein’s response to denaturant in bulk solution, using small-angle X-ray scattering (SAXS). We also developed a methodology to enable prediction of the orientation of a protein at the air-water interface, which we validate against our experimental data. Many proteins are amphipathic, which gives rise to an enthalpically driven accumulation of molecules at the air-water interface. The orientation and packing density of protein molecules at this interface are dependent on the distribution of the protein’s hydrophilic and hydrophobic regions as well as the positions of any charged side chains.1 Adsorption of a protein to the air-water interface may result in changes to the protein structure, including unfolding, which is dependent on factors such as the strength of stabilizing factors such as disulfide bonds in the tertiary structure. At the extreme, a previous study of myoglobin at the air-water interface showed near-total disruption of the protein’s tertiary structure when adsorbed at the * To whom correspondence should be addressed. E-mail: jww@ rsc.anu.edu.au; tel.: +61 2 6125 3578; fax: +61 2 6125 4903. † Current address: School of Chemistry, University of Bristol, BS8 1TS, U.K. ‡ Current address: Laboratoire de Physique de l’Etat Condense ´ , UMR 6087 CNRS, Boulevard Olivier Messiaen, 72085 Le Mans, France. § Current address: The Department of Chemistry, The University of Auckland, Private Bag 92019, Auckland, New Zealand.
air-water interface, which can be viewed as surface-induced denaturation.2 Myoglobin, however, has no disulfide bonds. Another effect of adding denaturants to protein solutions is that protein aggregation may occur. This is of great interest because (a) protein aggregation causes amyloidal plaques found in disease states such as Creutzfeld-Jacob disease (CJD) and (b) aggregation causes the formation of disordered inclusion bodies during overexpression of proteins by recombinant DNA methods.3–5 On the other hand, chemical denaturants also are used to solubilize inclusion bodies, releasing the desired protein into solution in a denatured state, which may then refold in nondenaturing solution conditions.6,7 Lysozyme was selected for this study because of the robustness of this protein toward chemical denaturation and because its three-dimensional structure is well-known, having been determined by X-ray crystallography in 1965.8 Lysozyme is a hydrolase involved in cleaving the glycosidic linkage of polysaccharides present in bacterial cell walls.9 It is a compact, water-soluble globular protein of two structural domains with 129 amino residues and a molecular weight of 14.3 kg mol-1. The interior of the structure is extremely hydrophobic, containing 40% aromatic side chains.10 Four disulfide bonds allow the lysozyme to maintain a rigid globular structure.11 The degree of structural modification that a lysozyme undergoes upon adsorption to the air-water interface is not clear. Malmsten defines a lysozyme as a hard or particle-like protein where few or no structural alterations occur upon adsorption.12 Lu et al. examined the effect of pH on the equilibrium structure of lysozyme at the air-water interface, assuming that the protein remains in its native statesan assumption justified by their ability to model the protein at the interface using only this shape and changes in orientation.13 Postel et al., however, reported R-helix to β-sheet transitions in the secondary structure of lysozyme upon adsorption at the air-water interface, and they postulated partial protein unfolding.14
10.1021/jp800354r CCC: $40.75 2008 American Chemical Society Published on Web 07/11/2008
Effect of Air-Water Interface on Lysozyme Structure Lysozyme is also robust against chemical denaturation, for example, resisting changes to its conformation in the presence of urea at concentrations up to 3 mol L-1.15 When the protein does undergo chemical denaturation, the two structural domains of the protein unfold cooperatively rather than independently.16 2. Experimental Procedures 2.1. Materials. Hen egg white lysozyme of purity greater than 95% (Sigma, L6876) and G.HCl (Aldrich, L177253) were used as received. Stock protein solutions were prepared in phosphate buffer (50 mM H2PO4-/HPO42-) at pH 6.5, maintained at 4 °C, and used within 1 day of preparation. Stock G.HCl and stock protein solutions (both twice the final concentration) were combined and mixed thoroughly by swirling for 2 min immediately before being poured into PTFE troughs, giving final protein concentrations of 10 mg mL-1 and five concentrations of G.HCl between 0 and 3.5 mol L-1. The troughs were hermetically sealed and temperature-controlled, maintained under a nitrogen atmosphere at 25 °C. Neutron and X-ray reflectivity measurements were recorded after a minimum equilibration time of 60 min. Solutions containing the highest denaturant concentration also were measured over a longer time to investigate any slow kinetic processes. Solutions used for neutron reflectometry were produced using air contrast matched water (ACMW) that was previously deaerated with high purity nitrogen. ACMW is a heavy/light water mixture that has a coherent neutron scattering length density (nSLD) of zero. ACMW has the same neutron refractive index as air, and so the undecorated air-water interface of this liquid is invisible to neutrons, which emphasizes an adsorbed protein layer. 2.2. Reflectometry Measurements. The neutron reflectivity was measured at the CRISP reflectometer of the ISIS spallation neutron source at the Rutherford Appleton Laboratory, Oxfordshire, U.K.17 Multiple angles of incidence were used to increase the measured Qz range, where the momentum transfer perpendicular to the surface Qz is 4π/λ sin θ, θ is the incident angle of the neutrons on the surface, and λ is the neutron wavelength. The angles used were 0.35, 0.50, 0.80, and 1.50°, giving an effective Qz range of 0.015-0.15 Å-1, limited at high Qz by sample-dependent incoherent background scattering. A solution containing only G.HCl at 3.5 mol L-1 was measured to test the surface activity of the denaturant and allowed the calculation of the subphase X-ray scattering length density (xSLD) of solutions containing denaturant. X-ray measurements were made on an angle dispersive instrument at the rotating anode source of the Research School of Chemistry, Australian National University (ANU).18 Cu KR radiation (λ ) 1.542 Å) was selected using a graphite (002) monochromator. Measurements were made at angles of incidence in the range 0.0° < θ e 3.4° (0 < Qz e 0.48 Å-1). The reflectivity data were modeled using CX-MULF, a program incorporating the optical transfer matrix method of classical optics, and refined using the Marquardt least-squares minimization algorithm. Uncertainties in parameters were derived from the matrix of covariance of the fit.18,19 Model parameters used in this program are τ, the film thickness (Å); Nbx, the X-ray SLD (Å-2); Nbn, the neutron SLD (Å-2); and σ, the Gaussian interfacial roughness (Å) of a series of homogeneous slabs. X-ray and neutron data sets were initially fitted individually, and then the models were corefined. When refined independently, at least a two-layer model was needed to
J. Phys. Chem. B, Vol. 112, No. 31, 2008 9533 adequately describe X-ray data sets, whereas a model that contained one fewer layer was sufficient for neutron data sets due to their more limited Qz resolution. When corefining the neutron and X-ray models, the total thickness of the X-ray layers was constrained to be equal to that of the lower resolution neutron measurements. This approach is discussed in detail by Henderson et al.20 2.3. SAXS Measurements. SAXS measurements were performed on a camera located at the rotating anode source of the Research School of Chemistry, ANU.21 The camera has a pinhole geometry operating at a wavelength of λ ) 1.5482 Å, focused on a 1-D wire detector at a sample-detector distance of 1638 mm and an effective Q range of 0.02-0.2 Å-1. Samples were produced at 10 mg mL-1 in 50 mM phosphate buffer at pH 6.85, with G.HCl at concentrations between 0 and 3.5 mol L-1, and measured at 22 °C in 1.5 mm glass capillaries. SAXS data were fitted assuming no interparticle interactions using an ellipitical form factor,22 using the NIST Igor Pro-based analysis package.23 2.4. Methodology. The average volume occupied by a protein at the air-water interface (Vprot, Å3) provides information on the packing density of the protein in the surface layer. This value can be evaluated directly from neutron reflectometry experiments from control protein solutions with no denaturant present in ACMW, according to eq 1
Vprot )
∑ bprot Nbn
(1)
where ∑bprot is the sum of scattering lengths of the elements that make up the protein (Å), allowing for any isotopic hydrogen exchange with ACMW, and Nbn is the measured neutron SLD of the layer (Å-2). The surface excess (Γ, mg m-2) of a protein at the air-water interface also can be determined directly under these conditions using eq 2
Γ)
1023MwNbnτ NA
∑ bprot
(2)
where Mw is the molecular weight of the protein (kg mol-1), τ is the thickness of the layer (Å), and NA is Avogadro’s number.2 If reflectometry experiments are performed using X-rays only, the surface excess can evaluated using eq 3
Γ)
∑ φprotFprot
(3)
where Fprot is the physical density of the protein (g cm-3). The volume fraction of the protein φprot, is given by eq 4
φprot )
Nbx_sub - Nbx Nbx_sub - Nbx_prot
(4)
where Nbx_sub is the X-ray SLD of the subphase (Å-2), Nbx is the measured X-ray SLD of layer (Å-2), and Nbx_prot is the calculated X-ray SLD of the protein (Å-2). To calculate Nbx_prot, the solvent-excluded volume of a single protein molecule must be known. For the present study, the volume of lysozyme was calculated using the method described by Jacrot.24 For protein solutions containing G.HCl, the surface excess of protein can be calculated from combined neutron and X-ray measurements, without dependence on an estimate of the protein volume. First, it is necessary to calculate the neutron scattering length of the subphase (Σbn_sub, Å) using eq 5
9534 J. Phys. Chem. B, Vol. 112, No. 31, 2008
∑ bn_sub )
Nbn_sub〈Mw 〉 FsubNA
Perriman et al.
(5)
where Nbn_sub is the subphase neutron SLD, Fsub is the physical density of the subphase, and 〈Mw〉 is the molecular weight average of all components in the subphase. The average volume occupied by a protein can now be calculated using eq 6
Vprot )
(∑ Zprot - n〈∑ Zsub〉)
r0
Nbx
(6)
where r0 is the classical electron radius (2.8 × Å), ∑Zprot is the number of electrons the protein molecule, 〈∑Zsub〉 is the average of the sum of electrons in the solution, and n is given by eq 7 10-5
n)
∑ Zprot - Nbx ∑ bprot Nbx ∑ bn_sub - r0Nbn〈∑ Zsub 〉 r0Nbn
(7)
Now that the average volume has been evaluated, the surface excess can be calculated using a modification of eq 2
1023Mwτ Γ) NAVprot
(8)
2.5. Prediction of Protein Orientation. Protein orientation at the air-water interface may be predicted by minimizing the molecular-surface interaction energy estimated from models of the surface affinities of various amino acids with water and air, under the assumption of insignificant perturbation of the protein crystal structure (derived from PDB structure 1VDQ8). We use here two different methods of estimating this surface affinity. The hydropathy index (HI) method estimates these affinities by multiplying the HI (taken from Kyte and Doolittle25) for each given amino acid residue by the solvent accessible surface area (SASAres) of that residue in the structure.25 SASAres was calculated using the program PSPC (protein surface properties calculator),26 which employs Connolly’s algorithm.27 A pitfall associated with the calculation is the possibility of compounding properties that are correlated. Specifically, those amino acid residues with large HI values tend to also have large SASA values. This effect was reduced by normalizing SASAres using the SASA of the free amino acid (SASAfa). Thus, the surface affinity of each residue in the HI method (SAHaa) can be defined using eq 9
SAHaa )
SASAres HI SASAfa
(9)
The charged-only (CO) method estimates surface affinities considering only the charged residues and their high affinity for the solvent. To apply the CO method, it was necessary to find accurate pKa values of the amino acid residues in the lysozyme molecule so that the charge carried by each residue at the measurement pH can be calculated. Calculated and experimental pKa values for lysozyme by Yang and Honig were used in this study.28 From these values, the solution affinity was determined simply by the charge on a residue that can be calculated from the Henderson-Hasselbalch equation,29 and the surface affinity of each residue under the CO method (SACaa) can be defined as
SACaa ) (1 + faa)-1
(10)
where faa is the fraction of each residue charged at the measurement pH.
Regardless of the method employed to calculate the properties of the individual amino acids in the three-dimensional structure, these properties must be added to produce a directional model for molecule-surface interactions. The approach used here was to rotate the protein molecule to effectively give the maximum number of hydrophobic residues at the surface, and hydrophilic residues toward the solution, as determined by maximizing eq 11
SAaa,r(zr - zmin) ) max zmax - zmin r)∞ r)1
∑
(11)
where the z-axis is perpendicular to the interface, zr gives the coordinate of each residue, and zmax and zmin, respectively, define the furthest and closest points of the protein to the subphase. The rotations of the whole molecule and maximization of eq 11 were performed using the program Matlab, allowing the molecule to orientate in any direction. To compare the predicted orientations with the reflectometry data, a one-dimensional X-ray SLD profile normal to the surface was calculated for the optimum solutions using amino acid residue positions and volumes. The area per molecule of the lysozyme at the interface was fixed from the corefined neutron and X-ray data models, and the residual volume at the surface was filled with water. The profile was then sliced into individual slabs, and the thickness was chosen to approximately match the X-ray instrumental resolution (∼5 Å) or match the two layer model structure used for modeling experimental data. 3. Results 3.1. Structure Dependence on Denaturant Concentration. Figure 1 shows the RQz4 versus Qz reflectivity profiles for neutron and X-ray experiments at the extremes of denaturant concentration, 0 and 3.5 mol L-1 G.HCl, illustrating the effect of chemical denaturation on the reflectivity from lysozyme. The neutron data recorded on ACMW all reach the sample-dependent background at Qz ≈ 0.15 Å-1. A single layer model for neutron data, and a two layer model for X-ray data, was sufficient to extract the layer thickness and SLD for solutions with [G.HCl] e 2 mol L-1. An extra layer for both neutrons and X-rays was required to describe the reflectivity from protein solutions containing the highest denaturant concentration of 3.5 mol L-1. Table 1 presents a summary of the surface parameters resulting from the corefinement of neutron and X-ray models over the denaturant range studied. Figure 1c displays the real-space X-ray SLD profiles resulting from the models to the reflectivity data in Figure 1a,b. For the control experiment, in the absence of G.HCl, the data are in good agreement with those of Lu et al. with a total layer thickness of the order of 47 Å,1 made up in X-ray SLDs of a denser layer of 19 Å near the surface and a broader less dense second layer of 28 Å. At the other extreme, a G.HCl concentration of 3.5 mol L-1, the denaturant caused an increase in both the X-ray SLD of the subphase and the two upper protein layers. The formation of an extra diffuse 70 Å layer also was observed at this denaturant concentration, signifying protein multilayer formation. Figure 2 shows the dependence of the total surface layer thickness and surface excess on G.HCl concentration, in the range before multilayer formation is detected. The surface excesses were evaluated using both neutron and X-ray data and are therefore independent of assumptions of the structural state of the protein. At the lowest measured denaturant concentration, 0.2 mol L-1, the presence of G.HCl caused a reduction in the protein thickness and surface excess. As the denaturant con-
Effect of Air-Water Interface on Lysozyme Structure
J. Phys. Chem. B, Vol. 112, No. 31, 2008 9535 layer, due to an increase in the air-film roughness. Figure 3b shows the real-space X-ray SLD profiles generated from the modeled data. After 30 min, the surface structure consists of two regions with thickness parameters and surface excesses similar to those for 10 mg mL-1 lysozyme containing 2 mol L-1 G.HCl (Table 1). After 2 h, the formation of a third diffuse layer occurred. This resulted in an increase in the total surface excess of 1 mg m-2 (Table 2). After 18 h, there was no further change in the total surface excess, although there was a reduction in the layer thickness, and an increase in the X-ray SLD, of the layer closest to the subphase. 3.3. Bulk Solution Structures from SAXS. The SAXS measurements on the protein in bulk buffer solution show that the protein exists in solution in an ellipsoidal shape, with observed radii in the absence of denaturant of 10 and 26 Å (see Table 3), similar to that previously observed by other authors in similar conditions.30–33 In the presence of 0.2 mol L-1 G.HCl, this shape is slightly swollen, with the minor axis radius increasing to 13 Å, and the contrast of the protein to the bulk (∆Nbx) decreasing, indicating partial protein unfolding with concomitant increased solvation. By 1 mol L-1, however, the protein already shows signs of aggregation, with the volume of the protein increasing and the contrast (and hence protein density) also increasing, and this trend continued as the denaturant concentrations increased, although the aggregate structures are still well-described using an ellipsoidal model.
Figure 1. Effect of G.HCl on the surface structure of 10 mg mL-1 lysozyme solutions at the air-water interface. (a) RQz4 vs Qz neutron reflectivities and fits (line) in ACMW containing no G.HCl (black squares) and 3.5 mol L-1 G.HCl (gray triangles); (b) RQz4 vs Qz X-ray reflectivities and fits (line) of solutions containing no G.HCl (black squares) and 3.5 mol L-1 (gray triangles) G.HCl; and (c) real-space X-ray SLD profiles of solutions containing no denaturant (solid line) and 3.5 mol L-1 G.HCl (dashed line) resulting from corefinement of the data in panels a and b. All measurements were made 2 h after surface formation.
centration was increased to 2 mol L-1, no further changes in the surface excess were observed, but at 2 mol L-1, the layer thickness returned to 46 Å with an increase in the solvation of the protein. 3.2. Time Dependence of Interfacial Structure at 25 °C. The X-ray and neutron reflectivity were unchanged over 5 h for protein solutions containing G.HCl concentrations up to 2 mol L-1. However, at the highest denaturant concentration (3.5 mol L-1 G.HCl), time-dependent changes in the reflectivity were observed. Hence, this interface was monitored over a longer time period, using X-rays only. Figure 3a displays reflectivity obtained from solutions containing 10 mg mL-1 lysozyme and 3.5 mol L-1 G.HCl as a function of time, with modeled parameters in Table 2. Over 18 h, there was a decrease in the reflectivity from the protein
4. Discussion 4.1. Denaturant-Induced Orientation Changes in Lysozyme. The control experiments performed on solutions containing 1 mg mL-1 lysozyme yielded total layer thicknesses of 47(1.5) Å (Table 1), with a calculated average volume of a single lysozyme molecule and associated water molecules of 45(0.3) × 103 Å3. The values for the layer thickness and total volume agree well with the crystal structure-derived dimensions of 45 Å × 30 Å × 30 Å (40.5 × 103 Å3).34 The similarity between the dimensions suggests that lysozymes may form a single densely packed layer of protein molecules with near-native tertiary structures when adsorbed at the air-water interface. This is consistent with scanning tunneling microscopy studies that showed that lysozyme forms highly ordered arrays when adsorbed to graphite, with dimensions close to that of the native structure.35 There is a reduction in the surface layer thickness of lysozyme in the presence of 0.2 mol L-1 G.HCl (see Figure 2), while in the bulk, the SAXS data suggest that the protein is actually expanding as it begins to unfold. As the layer thickness decreases, there is also a moderate reduction in the surface excess from 2.4 to 1.8 mg m-2. This suggests that the surface change is the result of a tilt of the molecule at the interface and displacement of protein from the surface, rather than the swelling indicative of denaturation seen in the bulk. Lu et al. reported similar changes in the orientation of lysozyme at the air-water interface as a function of pH, ionic strength, and concentration.13 As the denaturant concentration increases, there are no further changes in the surface excess below 2 mol L-1, and only slight changes in the layer thickness, easily accounted for by slight changes in the protein orientation. At 2 mol L-1, the surface layer begins to expand at the surface. This may indicate the beginnings of surface protein unfolding, with an increase in the solvation of the protein, although the surface excess does not change, and hence, protein aggregation does not appear to have begun. As is also seen in the bulk, the initial stages of protein denaturation involve a swelling of the protein molecule. Over
9536 J. Phys. Chem. B, Vol. 112, No. 31, 2008
Perriman et al.
TABLE 1: Fitting Parameters from Modeling of Data Obtained from 10 mg mL-1 Solutions of Lysozyme as a Function of [G.HCl], All Measured at 2 h after Formation of the Surfacea [G.HCl] (mol L-1) 0.0 0.2 1.0 2.0 3.5
layer
τ (Å)
1 2 1 2 1 2 1 2 1 2 3
18.8 (0.6) 28.3 (0.9) 19.0 (0.6) 21.2 (0.8) 19.1 (0.6) 16.8 (0.6) 20.5 (0.5) 25.8 (0.8) 18.9 (0.9) 28.5 (1) 71 (3)
σair-film (Å)
Nbx (10-6 Å-2)
2.6 (0.4)
11.46 (0.06) 10.39 (0.05) 11.63 (0.07) 10.15 (0.07) 11.43 (0.06) 10.28 (0.07) 11.58 (0.06) 10.08 (0.05) 11.80 (0.09) 10.47 (0.06) 10.10 (0.05)
3.4 (0.5) 3.3 (0.5) 3.8 (0.5) 4.2 (0.5)
Nbn (10-6 Å-2)
Γa (mg m-2)
0.83 (0.01)
2.4 (0.1)
0.75 (0.01)
1.8 (0.1)
0.79 (0.01)
1.6 (0.1)
0.80 (0.01)
1.7 (0.1)
0.94 (0.01)
1.7 (0.1)
0.57 (0.01)
0.5 (0.1)
Γb (mg m-2) 1.7 (0.1) 1.2 (0.1) 1.8 (0.1) 0.7 (0.1) 1.6 (0.1) 0.6 (0.1) 1.9 (0.1) 0.4 (0.1) 1.8 (0.1) 0.8 (0.1) 0.8 (0.1)
a
Data from X-ray and neutron reflectometry were corefined, with layers 1 and 2 combined in the model for the lower resolution neutron data. The surface excess (Γ) was calculated using two methodssfrom neutron and X-ray data combined (Γa) and from X-ray data only (Γb); see section 2.4. for details.
Figure 2. Variation of total lysozyme layer thickness and surface excess for solutions with concentrations of G.HCl e 2 mol L-1.
the same denaturant range, however, there is significant denaturation and aggregation observed in the bulk using SAXS (see Table 3). The implication of these results is that in the asymmetric environment of the solution surface, the effects of G.HCl in denaturing the protein are reduced. As the adsorption of the protein to the surface is slower than the denaturation kinetics,36–38 this reduction in G.HCl effect probably involves reversing the effects of the denaturation by energetically favoring the adsorption of the protein in its native form. This may be through the mechanism of preferentially adsorbing nondenatured protein that exists in solution, similar to the preferential adsorption of monomer protein species observed for β-lactoglobulin,39 or the renaturation of partially denatured protein. The preference for the native form of the protein at the air-water interface may arise through the directing influence of surface-protein interactions, or through protein-protein interactions (which are enhanced through the high local concentration of protein at the interface), or indeed a combination of these two effects. 4.2. Surface Aggregation of Lysozyme. At 3.5 mol L-1 G.HCl, there was a significant increase in the total thickness and surface excess (Table 1) resulting from the formation of a third layer extending into the subphase, signaling the onset of surface aggregation. In the SAXS measurements performed, however, evidence of protein aggregation was seen at G.HCl concentrations as low as 1 mol L-1. Furthermore, over 18 h, the reflectivity from the surface of the 3.5 mol L-1 solution changedsthere was an observed increase in the air-film roughness and in the third protein layer a reduction in the thickness and increase in the X-ray SLD.
Figure 3. (a) RQz4 vs Qz X-ray reflectivity profiles and (b) resulting real-space X-ray SLD profiles obtained from 10 mg mL-1 lysozyme solutions containing 3.5 mol L-1 G.HCl with time.
This may be caused by slow aggregation processes leading to the formation of large aggregated structures at the interface. With reflectometry alone, however, it is not possible to characterize these large heterogeneous structures at the air-water interface. G.HCl has indeed been shown to induce amyloid fibril formation from lysozyme in bulk at 50 °C, where the maximum growth rates and minimum lag times (>1 h) occurred at 3 mol L-1 G.HCl (near the minimum of the osmotic second virial coefficient (OSVC) curve).40 At this denaturant concentration, the lysozyme molecule in bulk solution is partly unfolded, and previously buried hydrophobic groups are exposed.41 Exposure of these groups increases both the affinity of the protein for the air-waterinterfaceaswellaspromotehydrophobicprotein-protein interactions.
Effect of Air-Water Interface on Lysozyme Structure
J. Phys. Chem. B, Vol. 112, No. 31, 2008 9537
TABLE 2: Fitting Parameters from Modeling of Data Obtained from 10 mg mL-1 Lysozyme Solutions Containing 3.5 mol L-1 G.HCl, as a Function of Timea time (h) 0.5 2 18 a
layer
τ (Å)
1 2 1 2 3 1 2 3
20.0 (0.5) 22.3 (0.9) 18.9 (0.9) 28.5 (1) 71 (3) 20.7 (0.7) 21.8 (1.3) 34 (2)
σair-film (Å)
Nbx (10-6 Å-2)
Γ (mg m-2)
3.5 (0.5)
11.66 (0.03) 10.32 (0.03) 11.80 (0.09) 10.47 (0.06) 10.10 (0.05) 11.76 (0.06) 10.61 (0.04) 10.19 (0.04)
1.8 (0.1) 0.5 (0.1) 1.8 (0.1) 0.8 (0.1) 0.8 (0.1) 2.0 (0.1) 0.8 (0.1) 0.6 (0.1)
4.2 (0.5) 4.7 (0.6)
Data were evaluated using X-ray reflectivity only.
TABLE 3: Fitting Parameters from Modeling of SAXS Data Obtained from 10 mg mL-1 Solutions of Lysozyme as a Function of [G.HCl]a [G.HCl] (mol L-1)
ellipsoid minor axis (Å)
ellipsoid major axis (Å)
vol (nm-3)
∆Nbx (10-6 Å-2)
0 0.2 1 2 3.5
10 12.8 15.6 27.3 35.0
25.7 24.2 30.6 26.4 27.7
10 16 31 82 142
1.40 1.22 1.52 1.51 1.78
a
∆Nbx is the contrast difference between the ellipsoid and the surrounding solution.
However, the ability of a protein to form a stable partially unfolded intermediate may in fact determine the likelihood of large-scale aggregation. The formation of inclusion bodies during the refolding of proteins at intermediate denaturant concentrations is common and thought to involve such intermediates.6 The existence of an intermediate in the chemical denaturation pathway of lysozyme has been reported, where the structure of the intermediate was described as having R-domains with a complex native-like secondary structure but distorted tertiary structure.42 Our data suggest that the air-water interface not only works to stabilize the native protein structure but also protects such an intermediate, hindering protein aggregation from occurring at the surface. It is anticipated that aggregation will occur through interactions of exposed hydrophobic regions within proteinssthese regions tend to orient at an air-water interface away from the bulk solution. The aggregation can be seen to be a much slower process (ca. hours) than either protein denaturation or adsorption to the interface. Consequently, the denatured protein intermediate is capable of adsorption at the interface before aggregation occurs and thus is kinetically stabilized. Thus, the air-water interface may provide an opportunity to study such unstable intermediate structures. 4.3. Modeling the Orientation of Lysozyme. To complement the reflectometry study on the orientation of lysozyme at the air-water interface, a protocol was developed to predict the optimum protein orientation (assuming that the protein is unperturbed from its crystal structure) based on the surface affinity of each amino acid residue in the structure.43 Two methods (explained previously), namely, the HI and CO methods, were used. For solutions containing no G.HCl, lysozyme at the air-water interface had a total layer thickness of 47 Å, which includes a dense upper layer 19 Å thick, with a more diffuse layer of 28 Å below (Table 2). The CO method predicted a near-upright protein orientation at the air-water interface, with a z-dimension
Figure 4. Predicted real-space X-ray SLD profile for CO method derived optimum protein orientation, as compared to that derived from modeling experimental data of the protein in the absence of denaturant (black solid line). Predicted profiles were produced at 5 Å resolution (dotted line), experimental resolution (dashed line), and for 3 Å surface penetration (gray solid line). Inset: charged and neutral regions of the surface of lysozyme generated using a 1.4 Å probe radius. Atomic coordinates for the lysozyme structure 1VDQ were sourced from the Protein Data Bank, and the molecular model was generated using visual molecular dynamics (VMD).48,49
of 44 Å and the C-terminus pointing toward the subphase. This orientation was refined by rotation through a small angle to match the experimentally observed protein layer thickness (47 Å). Figure 4 displays real-space X-ray SLD distributions calculated from the refined protein orientation at (1) a resolution of 5 Å and (2) two slabs, where only two layers of 20 and 30 Å thickness were used, as in the model used to fit the X-ray data. Also shown is the experimentally derived X-ray SLD profile. Note that, for clarity, the effect of thermal smearing of the surface was omitted from the theoretical X-ray SLD profiles. At 5 Å resolution, a peak can be seen in the profile at 24 Å, representing the approximate position of the internal interface of the molecule, an interface formed predominantly by three R-helices in the upper region of the lower domain. When divided into the two layers used to model the experimental data, the simulated profile agrees with the experiment, in showing a dense upper layer closest to the interface on top of a diffuse layer toward the bulk solution. The difference between simulated and experimental X-ray SLD is, however, significant, particularly in the layer closest to the surface. This anomaly may be reconciled by considering that the lysozyme extends above the air-water interface, as reported recently.1,44 In the proposed orientation, a movement of 3 Å would only expose proline 70 and glycine 71 to air. The modified X-ray SLD profile allowing for 3 Å exposure (two slabs exposed) also is shown in Figure 4. From the good agreement between the calculated and the experimental profiles, particularly in the layer SLDs, we can conclude that the CO method defines the orientation and surface penetration of the protein in the absence of denaturant precisely. On the other hand, there was disagreement between the predictions of the CO and HI methods. When the HI method was applied, a side-on rather than upright orientation of the protein was predicted (see Figure 5), with the C-terminus of the protein closest to the air-water interface, not the subphase. The z-dimension of a side-on orientation perpendicular to the surface (36 Å) is most similar to that observed experimentally in the presence of G.HCl. Thus, the calculated X-ray SLD profile from the HI method was compared to the measured profile from in the presence of 1 mol L-1 G.HCl (Figure 5). Although the
9538 J. Phys. Chem. B, Vol. 112, No. 31, 2008
Figure 5. Predicted real-space X-ray SLD profile for HI method derived optimum protein orientation, as compared to that derived from modeling experimental data of the protein in 1 mol L-1 G.HCl (black solid line). Profiles were produced at 5 Å resolution (dotted line) and at experimental resolution (dashed line). Inset: hydrophilic and hydrophobic regions of the surface of lysozyme generated using a 1.4 Å probe radius. Atomic coordinates for the lysozyme structure 1VDQ were sourced from the Protein Data Bank, and the molecular model was generated using VMD.48,49
Perriman et al. of the protein molecules. Specifically, the CO method accurately predicts the upright protein orientation, while the method using only amino acid hydropathy fails. Indeed, Lu et al. proposed a similar orientation considering only the position of the charged residues at the C-terminus and the observed length of the z-dimension in reflectometry experiments.1 One might consider that as the concentration of salt G.HCl increased, charge screening would reduce the influence of electrostatic forces resulting from charged groups, and the CO method would fail. As the denaturant concentration was increased to 1.0 mol L-1, the protein did change to a side-on or tilted packing arrangement with a reduction in the surface excess. However, predictions based on hydropathy alone are still inaccurate. Thus, it is necessary to consider other nonelectrostatic forces that influence the orientation and packing of the surface layer, particularly protein-protein interactions. The measurement of a protein’s OSVC was used to assess the magnitude of protein-protein interactions over a wide range of solution conditions.45,46 Recent studies showed that the value of the OSVC for lysozyme is positive (repulsive interactions) over a wide range of G.HCl concentrations (0-7 mol L-1).47 Although the values of the OSVC were positive, the magnitude decreased rapidly over low to moderate concentrations (0-3 mol L-1), showing that the repulsion between the protein molecules decreases in the range considered in these experiments. Using a multimolecular approach, where protein-protein interactions were considered, a study of lysozyme at the solid-liquid interface suggested that a side-on orientation was favorable.35 Hence, it seems likely that protein-protein interactions become significant in the presence of G.HCl and that the simple methods used previously that consider only a single molecule need to be expanded to take these into account. 5. Conclusion
Figure 6. Calculated and experimental real-space X-ray SLD distributions for inverted CO-derived orientation, as compared to that derived from modeling experimental data of the protein in 1 mol L-1 G.HCl (black solid line). Profiles were produced at 5 Å resolution (dotted line) and at experimental resolution (dashed line). Inset: charged and neutral regions of the surface of lysozyme generated using a 1.4 Å probe radius. Atomic coordinates for the lysozyme structure 1VDQ were sourced from the Protein Data Bank, and the molecular model was generated using VMD.48,49
z-dimension of the HI predicted orientation agrees well with the measured thickness at this denaturant concentration, the theoretical X-ray SLD profiles in Figure 5 do not agree. Instead, in the two layer model, the X-ray SLD changes from low to high with depth. On the other hand, if the protein is inverted from the HI predicted orientation such that the C-terminus is again facing the subphase (see Figure 6), then the predicted SLD profile matches the experimental model well. The peak in the calculated SLD marking the protein internal interface can be seen at ∼15 Å, followed by a steady decline to 36 Å. When the HI model is divided into two layers to match the modeling of the experimental data, the layer X-ray SLDs are seen to be in accordance with the experiment, without the need to consider exposure of the protein. We suggest that for solutions containing no denaturant, the position of the charged residues, particularly the cluster of charged residues near the C-terminus, dictates the orientation
The air-water interface provides a unique environment to study both orientation of protein molecules as well as protein-protein interactions that lead to aggregation. Reflectometry using X-rays and neutrons showed that the orientation of lysozyme at the air-water interface is dependent on the chemical denaturant, guanidinium hydrochloride, but that the tertiary structure of the protein is not perturbed until concentrations higher than necessary in the bulk. At the highest concentration of denaturant studied, surface aggregation was observed, at a higher onset point than in the bulk. The air-water interface appears to hinder the nucleation for the growth of large structures, through effects on stabilizing the structure of partially unfolded protein molecules as well as limiting their conformational freedom. This implies that the air-water interface can be used as a sensitive probe to study the onset of aggregation and in particular intermediates that are unstable in the bulk. Finally, some straightforward models to predict the orientation of a protein at an interface were developed and tested by comparison with experiment. In the present study, an approach considering only the surface affinity of charges on a single molecule proved adequate in the absence of denaturant but failed when G.HCl was added to the solution. Estimates based on surface affinities calculated using amino acid hydropathy were not found to be reliable in either the presence or the absence of salt. We believe the failure of both methods in the presence of G.HCl relates partially to the screening of previously dominant electrostatic interactions, as well as to the increasing importance of protein-protein interactions. An approach that also takes protein-proteininteractionsintoaccountneedstobedevelopedssuch
Effect of Air-Water Interface on Lysozyme Structure an approach will lead to further valuable insight into the effects of surfaces on protein stability and structure. Acknowledgment. We thank Dr. Stephen Holt at the Rutherford Appleton Laboratory (RAL), U.K., for experimental assistance and the Australian Institute of Nuclear Science and Engineering (AINSE) for funding for A.W.P. through an AINSE Post-Graduate Research Award. Travel to the RAL was funded through the Access to Major Facilities Program administered by the Australian Nuclear Science and Technology Organization. References and Notes (1) Lu, J. R.; Su, T. J.; Thomas, R. K; Penfold, J.; Webster, J. J. Chem. Soc., Faraday Trans. 1998, 94, 3279–3287. (2) Holt, S. A.; McGillivray, D. J.; Poon, S.; White, J. W. J. Phys. Chem. B 2000, 104, 7431–7438. (3) Sipe, J. D.; Cohen, A. S. J. Struct. Biol. 2000, 130, 88–98. (4) Marston, F. A. O. J. Biochem. 1986, 240, 1–12. (5) Schein, C. H. Biotechnology 1989, 7, 1141–1147. (6) Fink, A. L. Fold. Des. 1998, 3, 9–23. (7) Tsumoto, K.; Ejima, D.; Kumagai, I.; Arakawa, T. Protein Expression Purif. 2003, 28, 1–8. (8) Blake, C. C. F.; Koenig, D. F.; Mair, G. A.; North, A. C. T.; Phillips, D. C.; Sarker, D. K. Nature (London, U.K.) 1965, 206, 757–761. (9) Stryer, L. Biochemistry; W. H. Freeman and Company: New York, 1995. (10) Smith, L. J.; Sutcliffe, M. J.; Redfield, C.; Dobson, C. M. J. Mol. Biol. 1992, 229, 930–944. (11) Walsh, M. A.; Schneider, T. R.; Sieker, L. C.; Duauter, Z.; Lamzin, V. S.; Wilson, K. S Acta Crystallogr., Sect. D: Biol. Crystallogr. 1998, 54, 522–546. (12) Malmsten, M. J. Colloid Interface Sci. 1998, 207, 186–199. (13) Lu, J. R.; Su, T. J.; Howlin, B. J. J. Phys. Chem. B 1999, 103, 5903–5909. (14) Postel, C.; Abillon, O.; Desbat, B. J. Colloid Interface Sci. 2003, 266, 74–81. (15) Ibarramolero, B.; Sanchezruiz, J. M. Biochemistry 1997, 36, 9616– 9624. (16) Griko, Y. V.; Freire, E.; Privalov, P. L.; Van Dael, H.; Privalov, G. J. Mol. Biol. 1995, 252, 447–459. (17) http://www.isis.rl.ac.uk. (18) Brown, A. S.; Holt, S. A.; Saville, P. M.; White, J. W. Aust. J. Phys. 1997, 50, 391–405. (19) Penfold, J. Data interpretation in specular neutron reflection. In Neutron, X-ray and Light Scattering; Lindner, P., Zemb, T., Eds.; Elsevier: Amsterdam, 1991; pp 223-236. (20) Henderson, M. J.; Perriman, A. W.; Robson-Marsden, H.; White, J. W. J. Phys. Chem. B 2005, 109, 20878–20886.
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