Protein Salting Out Observed at an Air−Water Interface - The Journal

LETTER pubs.acs.org/JPCL

Protein Salting Out Observed at an Air
Water Interface Yohko F. Yano,*,†,§ Tomoya Uruga,‡ Hajime Tanida,‡ Yasuko Terada,‡ and Hironari Yamada† † ‡

Research Organization of Science & Engineering, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu-shi, Shiga 525-8577, Japan Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo-cyo, Sayo-gun, Hyogo 679-5198, Japan

bS Supporting Information ABSTRACT: A protein salting-out process is directly observed at an air
water interface. By using time-resolved X-ray specular reflectivity and off-specular diffuse scattering, we identified several key stages in the adsorption of hen egg white lysozyme in a concentrated NaCl solution, (1) adsorption-induced unfolding, (2) monolayer formation with unfolded proteins, (3) protein refolding, and (4) island formation with the refolded proteins. Stages 3 and 4 are not observed either at the isoelectric point or in the salt-free solution, suggesting that they are induced by screening of the positive charges in the lysozyme by chloride ions. It is considered that the hydrated salt ions act to minimize the water-accessible surface area of the protein, not only enhancing protein dehydration (stages 1 and 2) but also assisting in protein refolding and association (stages 3 and 4). These results provide insight into the early stages of protein crystal nucleation. SECTION: Macromolecules, Soft Matter

P

rotein crystallization is typically realized by the addition of a salt or an organic solvent to a supersaturated protein solution to decrease the protein solubility. “Salting out” is a method for separating proteins, in which the hydrated salt ions reduce the number of water molecules available for interaction with the proteins. Protein solubility is a macroscopic property resulting from various molecular interactions, including protein
protein, protein
ion, ion
water, and water
protein interactions, and depends on temperature, pH, ion species, and concentration. At low salt concentrations, a “salting-in” region exists, where favorable interactions occur between the protein surface charges and the salt ions in solution to increase protein solubility, which is sometimes associated with protein unfolding. At higher salt concentrations, the repulsive electrostatic interactions are screened, resulting in protein stabilization, and protein
protein interactions such as hydrophobic interactions and dispersion forces can drive aggregation and precipitation.1
4 The effect of ion species on the solubility of proteins is classified by the well-known Hofmeister series, defined by the concentration of a particular salt needed to precipitate proteins from whole egg white.5,6 The series are usually given in terms of the ability of the ions to stabilize the structure of proteins. Although Hofmeister ion effects on protein stability are widely reported in protein research, the mechanism is not entirely clear to date. Most previous research on Hofmeister ion effects on protein
protein interactions has been performed in bulk liquids by varying ion species and concentration.4,7
11 The surface tension, that is, the surface free energy per area, can be regarded as the work in bringing a molecule from the interior of a liquid to the surface and is strongly affected by molecular forces and configurations.12 When salt ions dissociate r 2011 American Chemical Society

in water, the magnitude of the surface tension increment indicates the energy required to separate the ions from the water molecules. The molar surface tension increment of the salt has been known to follow the rank order of the Hofmeister series.13
15 Several recent experiments and computer simulations16 looking at interfaces have revealed that the propensity for salt anions to adsorb at an air
water interface follows an inverse Hofmeister series, that is, strongly hydrated anions effective in precipitating proteins do not adsorb at the air
water interface and vice versa.17 These results suggest that studying the surface adsorption of proteins in a salt solution could provide valuable insight into the salting out of proteins generally. Proteins adsorb at air
water interfaces as surfactants. The effect of salt on protein adsorption under equilibrium conditions has been studied by surface pressure measurements, surfacespecific spectroscopies, and neutron reflectometry.18
22 The amount of adsorption and the thickness of the interfacial layer are strongly dependent on the salt concentration, ion species, and pH. It has been clarified that small changes in conditions may drastically affect the intra- and intermolecular arrangements, making prediction of these systems difficult. Investigating the adsorption process would help to reveal the role of salt on protein adsorption. Herein, we examine the protein adsorption process in a concentrated salt solution using time-resolved X-ray reflectometry to investigate the salting-out phenomenon at the air
water interface. Previously, we have studied the adsorption of hen egg Received: January 24, 2011 Accepted: April 4, 2011 Published: April 11, 2011 995

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30, for which the native LSZ molecule (elliptical in shape with approximate dimensions of 30  30  45 Å3) is oriented with its long axis parallel to the air
water interface (side-on). In all conditions, the electron density profiles for the initially adsorbed LSZ are highly distorted as compared to the native configuration of a flatter structure at the interface. According to previous work, in the absence of added salt at pH 7, the electron density profile at z < 20 Å corresponds to a first layer of initially adsorbed LSZ molecules, whereas that at z > 20 Å corresponds to a second layer of LSZ molecules adsorbed beneath the first layer. As shown in Figure 2a, the LSZ molecule initially unfolds and lays flat on the surface to form a monolayer, and subsequently, LSZ molecules gradually adsorb underneath the first layer to form a multilayer, with a lower density molecular configuration. However, the density profile of the first layer does not change during the adsorption process. This time-dependent behavior reproduces previous results in which a potassium-based buffer solution was used instead of sodium.31 For the 2 M NaCl solution, however, the electron density profile for the first layer gradually decreases during the adsorption process (Figure 2d) and approaches that of the native structure at 122 min after injection. In the absence of added salt at pH 3, the amount of adsorption is much less than that at pH 7 as a result of enhanced electrostatic repulsion between the LSZ molecules; the density profile in the first layer increases during adsorption, whereas the second layer formation is suppressed (Figure 2b). This behavior is very similar to that observed for the 0.01 mg/mL LSZ solution at pH 7.23 It is known that the adsorption rate decreases with a decrease in the LSZ bulk concentration, and subsequently, the molar area occupied by one adsorbed LSZ molecule becomes larger to increase the surface coverage.32 On the other hand, at low pH, the LSZ molecule with a high net charge raises the activation energy of adsorption33 and subsequently decreases the adsorption rate. Although the origins of the two cases are different, decreasing either the LSZ bulk concentration or the pH gives the same adsorption behavior. The present result indicates that for the salt-free solution, the positively charged LSZ molecule adsorbs at the air
water interface as a monomolecular structure, and the configuration depends only on the amount at the surface. In contrast, for the 2 M NaCl solution, the density profile of the first layer gradually decreases over time to approach the native profile (Figure 2e), as already shown in the case at pH 7. At pH 11.5, the isoelectric point, the adsorption behavior for the salt-free solution is very similar to that at pH 7, whereas for the 2 M NaCl solution at this pH, the surface excess drastically decreases because formation of the second layer is suppressed (Figure 2f). This result is in good agreement with previous results by neutron diffraction studies under equilibrium conditions,21 in which the surface excess and the layer thickness drastically decrease with increasing salt concentration at the isoelectric point. As we have seen above, LSZ refolding is only observed in concentrated salt solutions at pH 3 and 7, at which pH values, the LSZ carries a net positive charge. The LSZ refolding may be a result of chloride ions shielding the positive charge of the LSZ.34 For the LSZ injected into the salt-free solution at pH 7, the offspecular diffuse scattering intensity follows the capillary wave model accurately. However, for the 2 M NaCl solution at pH 7, the diffuse scattering intensity gradually increases during the adsorption process. The excess scattering intensity after subtraction of the capillary wave contribution28,29,35 is shown in Figure 3. The intensity at qy = 0, Iexcess(0), shows a maxima at 77 min. We

Figure 1. (a) Time dependence of the intrinsic structure factor normal to the surface |Φ(qz)|2 for LSZ injected into a buffer solution in a saltfree solution at pH 7. The continuous lines show best fits to the data using three-box models. (b) The same conditions as those in (a) with 2 M NaCl solution.

white lysozyme (hereafter, LSZ) at an air
water interface in the absence of added salt to investigate the mechanism of adsorption-induced protein unfolding.23 We found that the LSZ molecule initially adsorbed by adopting a flat, unfolded structure as a result of hydrophobic interactions with the gas phase. In contrast, as adsorption continues, the LSZ molecules gradually adapt their configuration to the surroundings. In the present study, real-time structural information is obtained for both the intramolecular conformation and intermolecular association of LSZ at an interface using X-ray off-specular diffuse scattering in combination with X-ray reflectivity.24
26 We demonstrate the power of the present technique in visualizing such surface phenomena. Figure 1a and b shows the intrinsic structure factor |Φ(qz)|2, which is the Fourier transform of the laterally averaged electron density profile in the surface normal direction ÆF(z)æxy
2

1 Z DÆFðzÞæxy iqz z


2 e
jΦðqz Þj ¼
Bulk dz ð1Þ

F Dz Analytically, the structure factor is derived from the X-ray reflectivity data divided by the Fresnel reflectivity and capillary wave contribution.27
29 As shown in Figure 1a and b, the broad peak observed in every profile at around qz = 0.2 Å
1 originates from a LSZ monolayer 20 Å thick, in accordance with previous work for LSZ in the absence of added salt at pH 7.23 The small peak at qz < 0.1 Å
1 indicates a second layer formed beneath the monolayer as adsorption continues. For the LSZ solution with added salt, the intensity at qz > 0.3 Å
1 decreases during adsorption, as shown in Figure 1b, suggesting that the density of the first layer decreases. The |Φ(qz)|2 was fitted using a three-slab model to obtain the electron density profile F(z). The surface excess (adsorbed amount) Γ of LSZ was obtained by integrating the electron density profile (computational details and results are shown in Supporting Information). Figure 2 shows the time dependence of the electron density profile for LSZ injected into a buffer solution at pH 3, 7, or 11.5 (close to the isoelectric point of 11.35) with and without the addition of 2 M NaCl. The gray area in each figure corresponds to the simulated profile taken from ref 996

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Figure 2. Time dependence of electron density profiles for LSZ injected into a salt-free (a
c) or 2 M NaCl solution (d
f) at (a,d) pH 7, (b,e) pH 3, and (c,f) pH 11.5. The gray area in each figure represents the simulated profile of native LSZ with a side-on orientation taken from ref 30.

Figure 3. Time dependence of the excess scattering obtained from offspecular diffuse scattering for LSZ injected into a buffer solution containing 2 M NaCl at pH 7 [black, 2 min; light green, 24 min; green, 32 min; yellow, 56 min; orange, 77 min; orange (filled circles), 98 min; red, 121 min; red (filled circles), 141 min]. The continuous lines show a best fit to the data using eq 2.

Figure 4. Time dependences of the (a) surface excess Γ (circles) and the (b) Iexcess(0) (crosses) and correlation length ξ (diamonds) for LSZ injected into a 2 M NaCl solution. (c) Schematic drawings of the adsorption process. The different stages (roman numbers) in (a) correspond to those in (c). Chime cartoon representation46 of the X-ray structure of hen egg white lysozyme (Protein Data Bank code 1VDQ) and schematic model showing the hydrophobic regions (green) and positively (blue) and negatively (red) charged groups at pH 7.

analyze the excess scattering by modeling the surface layer inhomogeneity using the following expression25,35   IðqÞ Iexcess ð0Þ
σ22 q2z  e ð2Þ I0 inhmg ð1 þ ξ2 q2y Þ3=2

summarized in Figure 4a and b. The surface excess Γ does not exhibit a monotonous increase, indicating that there are several different stages in the adsorption process. On the other hand, Iexcess(0) and the correlation length ξ show maximum values at the surface excess minima, indicating that the adsorbed LSZ surface layer is initially laterally homogeneous but gradually becomes inhomogeneous. These results can be interpreted as follows: an increase in the positive charge of the surface layer induces migration of the chloride ions to screen the electrostatic repulsion, and hence, the uniform surface layer deforms into islands as a result of the increasing lateral LSZ
LSZ interactions. The maximum value of the correlation length is 80 nm, which

The fitting parameters are Iexcess(0), the correlation length ξ, and the root-mean-square roughness of the layer/gas interface σ2 of an inhomogeneous layer formed on a liquid surface. Because the profile width is related to ξ while the asymmetry of the profile is related to σ2, these parameters can be obtained almost independently of each other. The continuous curves in Figure 3 show the fits to the data. The time dependence of the surface excess Γ, Iexcess(0), and the correlation length ξ for the 2 M NaCl solution at pH 7 are 997

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The Journal of Physical Chemistry Letters corresponds to the average size of the LSZ islands.35,36 On the basis of the electron density profile for the 2 M NaCl solution at pH 7 (Figure 2d), the LSZ molecules in the inhomogeneous layer adopt a folded, compact shape at the point when the surface excess is a minimum. This globular structure is also supported by the N
H residal amide II peak, which is not observed for a saltfree solution,37 being present in the PM-IRRAS spectra (Figure S2, Supporting Information). Addition of salt to the solution is expected to drive the conformational equilibrium toward compact folded states, as indicated by MD simulation.34 After 77 min, the correlation length decreases, in contrast to the surface excess, indicating that the LSZ molecules associate to form the smaller islands. Figure 4c shows a schematic of the adsorption process consisting of four stages, (1) adsorption-induced unfolding mainly caused by hydrophobic interactions with the gas phase, (2) formation of a homogeneous monolayer stabilized by electrostatic repulsion between the positively charged LSZ molecules, (3) protein refolding induced by chloride ion screening, which minimizes the solvent-accessible surface area of LSZ, and (4) island formation induced by hydrophobic attraction and dispersion forces38
40 between the LSZ molecules. The salt ions enhance dehydration of the protein in stage 1, whereas they assist in protein refolding and association in stages 3 and 4. Ultimately, these phenomena can be interpreted as the salt ions acting to minimize the water-accessible surface area of the protein.41 At the isoelectric point, protein adsorption is suppressed upon addition of the salt,21 as shown in Figure 2f. While the time dependence of the surface excess for the 2 M NaCl solution at pH 11.5 resembles that in the salt-free solution at pH 3 (Figure S1, Supporting Information), the electron density profiles are quite different; the bottom part of the protein layer extends into the water phase for the 2 M NaCl solution at pH 11.5, whereas this extrusion is not as significant in the salt-free solution at pH 3. The salt-in effect, which usually occurs at low salt concentrations or near the isoelectric point,42 may enhance LSZ denaturation and suppress the surface adsorption. In conclusion, we have demonstrated that protein salting out can also be observed at an air
water interface. The present LSZ concentration in the bulk solution is only 2
3% of that used in crystallization, and subsequently, the positive charge on the LSZ in bulk solution is insufficient for chloride screening to be a factor. The adsorption behavior for the positively charged LSZ in the concentrated salt solution was found to follow distinct stages. In stage 1, the initially adsorbed LSZ molecules unfold to become flatter than those in the salt-free solution because the salt enhances the LSZ dehydration. As adsorption continues and the electrostatic repulsion between the LSZ molecules becomes greater, screening by the chloride ions occurs (stage 2). In protein crystallization, in which the crystal nucleation rate increases with protein concentration,43,44 the phenomena of salt-induced protein dehydration, conformational stabilization, and association would occur simultaneously. Thus, by investigating the air
water interface, we can help clarify the contribution of added salt to protein crystal nucleation.

LETTER

Three-times crystallized and lyophilized hen egg white lysozyme was purchased from Sigma (Prod. No. L6876) and used as supplied. Protein solutions were made using a phosphate buffer solution (0.02 M NaH2PO4/Na2HPO4) of pH 7 (ionic strength: 0.02 M) using UHQ-grade water. Protein solutions were made to concentrations of 43 mg/mL, from which 1 cm3 portions were added to a 42 cm3 buffer solution of pH 3, 7, or 11.5 with 2 M NaCl in a Langmuir trough45 to give final concentrations of 1 mg/mL. The X-ray reflectivity measurements were performed using the recently developed liquid interface reflectometer at the undulator beamline BL37XU of SPring-8.23,24 The measurements were made at angles of incidence R in the range of 0.005
2.0 for an acquisition time of 150 s at an X-ray energy of 15 keV, which corresponds to qz (=4π sin R/λ) of 0.001
0.5 Å
1. In the experiments, the first measurement was started 4 s after injection of a LSZ solution using a liquid dispenser into a phosphate buffer solution controlled at 25 C.23 The position of the X-ray on the sample was changed horizontally at intervals of 1 s to avoid radiation damage. The surface pressure was monitored throughout using a Wilhelmy plate made of glass mounted on the Langmuir trough. In this system, we exploit the advantages of a two-dimensional pixel array detector to analyze both the X-ray specular reflection and the off-specular diffuse scattering intensities.24,25

’ ASSOCIATED CONTENT

bS

Supporting Information. (1) Surface excess. (2) Experimental details and the results of PM-IRRS measurements. This material is available free of charge via the Internet at http:// pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Phone þ81-6-6721-2332, ext. 4088. Fax þ81-6-6727-4301. Present Addresses

§ Department of Physics, Kinki University, 3-4-1 Kowakae, Higashiosaka City, Osaka 577
8502 Japan.

’ ACKNOWLEDGMENT The synchrotron radiation experiments were performed at the BL37XU beamline at the SPring-8 facility with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposals 2009A1686 and 2009B1308). Y.Y. acknowledges financial support from a Shiseido Female Researcher Science Grant and from a Grant-in-Aid for Scientific Research (No. 22018028) in Priority Area “Molecular Science for Supra Functional Systems” from the Ministry of Education, Culture, Sports, Science and Technology of Japan. ’ REFERENCES (1) Kuehner, D. E.; Engmann, J.; Fergg, F.; Wernick, M.; Blanch, H. W.; Prausnitz, J. M. Lysozyme Net Charge and Ion Binding in Concentrated Aqueous Electrolyte Solutions. J. Phys. Chem. B 1999, 103, 1368–1374. (2) Dumetz, A. C.; Snellinger-O’Brien, A. M.; Kaler, E. W.; Lenhoff, A. M. Patterns of Protein
Protein Interactions in Salt Solutions and Implications for Protein Crystallization. Protein Sci. 2007, 16, 1867–1877.

’ EXPERIMENTAL METHODS The globular protein, lysozyme (hereafter, LSZ), is elliptical in shape with approximate dimensions 30  30  45 Å3. It is regarded as a rigid molecule due to the presence of four disulfide bridges. The isoelectric point is 11.35. 998

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