Anomalous Effect of Poly(ethylene)Glycol on Intermolecular Interaction

Institute for Human Science and Biomedical Engineering, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Central 6, Ts...
2 downloads 0 Views 2MB Size
Download PDF
CRYSTAL GROWTH & DESIGN

Anomalous Effect of Poly(ethylene)Glycol on Intermolecular Interaction and Protein Molecule Association

2009 VOL. 9, NO. 5 2517–2524

Kazuo Onuma,*,† Naoki Furubayashi,‡ Fujiko Shibata,‡ Yoshiko Kobayashi,‡ Sachiko Kaito,‡ Yuki Ohnishi,‡ and Koji Inaka‡ Institute for Human Science and Biomedical Engineering, National Institute of AdVanced Industrial Science and Technology, 1-1-1 Higashi, Central 6, Tsukuba, Ibaraki 305-8566, Japan, and Maruwa Foods and Biosciences Inc., 170-1, Tsutsui-cho, Yamatokoriyama, Nara 639-1123, Japan ReceiVed January 8, 2009; ReVised Manuscript ReceiVed February 10, 2009

ABSTRACT: Taka-amylase A (TAA) proteins purified from Aspergillus oryzae were investigated using dynamic and static light scattering for solutions containing poly(ethylene)glycol (PEG) with a molecular weight of 8000 as an association-inducing reagent. The hydrodynamic TAA monomer radius at a zero protein concentration gradually decreased from 3.2 to 2.0 nm with an increase in the PEG concentration from 0 to 12.5% (w/v). The molecular weight of TAA monomers at 0% PEG, 49.0 kDa, decreased to 31.0 kDa at 12.5% PEG. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the monomer-band position decreased with an increase in the PEG concentration. While these findings suggest PEG-induced fragmentation of monomers, circular dichroism measurements showed no difference between 12.5 and 0% PEG solutions in the secondary structure of TAA monomers. Analytical ultracentrifugation with a 12.5% PEG solution gave a TAA molecular weight of 51.0 kDa, almost equal to that for a 0% PEG solution measured using static light scattering. These results suggest that a PEG-induced network locally formed in the solution and affected the dynamics and the scattering intensity of the TAA monomers. The existence of such a solution structure makes it impossible to explain the behavior of monomers on the basis of simple attractive depletion interaction by PEG.

1. Introduction The use of an appropriate association-inducing reagent is the key to successful protein crystallization. In early research efforts, inorganic salts such as ammonium sulfate and sodium chloride were used to crystallize proteins.1–7 These salts screen the surface charge of protein molecules, thereby increasing the relative contribution of the van der Waals attractive force to molecule association. Salt-specific bridging between protein molecules is an important effect in protein crystallization.8,9 Although several proteins have been successfully crystallized using inorganic salts, some proteins, especially high-molecularweight ones, cannot be crystallized using inorganic salts even when the salt concentration is high enough to screen the surface charge. To overcome this, polymers have been considered as crystallizing reagents instead of, or along with, inorganic salts.10,11 Poly(ethylene)glycol (PEG) is the most promising polymer used as a crystallizing reagent.12–18 PEG is a neutral-charged reagent, so it has less effect on the electrostatic repulsion of protein molecules. Local concentration difference of PEG caused osmotic pressure, and it induces an attractive depletion force between protein molecules. Because the strength of force can be controlled by varying its concentration and molecular weight, PEG is now widely used in the research on protein crystallization. We estimate that PEG has been used for more than 70% of the successfully crystallized proteins. We previously investigated the light scattering of takaamylase A (TAA) purified from Aspergillus oryzae using ammomiun sulfate as an association-inducing salt.19 TAA barely crystallizes at 20 °C even though the concentration of ammonium sulfate is high. This is due to the small Hamaker constant of its monomers. To obtain TAA single crystals using * To whom correspondence should be addressed. Tel: +81-29-861-4832. Fax: +81-29-861-6149. E-mail: [email protected]. † National Institute of Advanced Industrial Science and Technology. ‡ Maruwa Foods and Biosciences Inc.

ammonium sulfate, we need to reduce the solution temperature to around 4 °C to increase supersaturation and solution viscosity. Increased viscosity reduces the diffusion coefficient of molecules. Under these conditions, it takes a few weeks to obtain crystals. When we used PEG instead of ammonium sulfate, the crystallization behavior changed drastically. Crystals were obtained at 20 °C, and the growth period was reduced to a few days. However, most of the grown crystals were polycrystals. On the other hand, single crystals are frequently grown under microgravity conditions.20 The suppression of the temperature and/or protein concentration fluctuations of the solutions under microgravity conditions would improve the quality of the grown crystals. However, other reasons are possible because PEGcontaining solutions have very high viscosity even under groundbased conditions, which contributes to reducing these fluctuations. Here, we report our investigation of the dynamic and static light scattering (DLS/SLS) of PEG-containing TAA solutions. Our aim was to clarify the behavior of TAA monomers with changes in the PEG concentration and to correlate findings to the quality of grown crystals. We found that the association model with simple attractive depletion interaction does not explain the effect of PEG on TAA crystallization.

2. Experimental Section 2.1. Sample Preparation and Light Scattering Measurement. TAA was purified in the same manner as in the previous study.19 Proteins gathered from “TAA-1” were used for all measurements. Purified TAA solution was dialyzed to 50 mM of sodium acetate (NaAc)-acetic acid buffer at 20 °C with a pH of 6.0. Two millimolar of CaCl2 was added to this buffer solution. The crystallizing solution was prepared by dissolving PEG (Fluka Inc.; molecular weight of 8000) into the buffer solution. The protein and the crystallizing solutions were passed through a 0.22-µm-pore filter immediately before measurements. These two solutions were mixed at a 1:1 volume ratio and used without filtering for light scattering measurements. The PEG concentration in the final solution was varied from 0 to 20.0% (w/v). The measurement

10.1021/cg900019e CCC: $40.75  2009 American Chemical Society Published on Web 03/16/2009

2518

Crystal Growth & Design, Vol. 9, No. 5, 2009

temperature, the solution cell, and light scattering instruments were the same as those used in the previous study.19 The protocols for the DLS and SLS measurement and analysis are described elsewhere.21,22 In the DLS measurement, the dependence of the diffusion coefficient on the TAA concentration was measured for various concentrations of PEG. The TAA concentration was changed from 0.75 to 40 mg/mL. In the SLS measurement, the molecular weight of the TAA monomers for a 12.5% PEG solution was measured, and time-resolved SLS (TR-SLS) measurements for TAA association were done for a 20.0% PEG solution at a TAA concentration of 25 mg/mL. 2.2. Viscosity Measurement. The solvent viscosities, which are needed to calculate the hydrodynamic radius using the diffusion coefficient data from the DLS measurement, were measured using a rolling-ball viscometer (Anton Paar; AMVn Automated microviscometer). The time for ball-rolling in the capillary glass tube into which the PEG-containing solvents were filled was measured for several tube inclination angles and the time to zero degrees was extrapolated. This was done because solutions with a high PEG concentration can be a non-Newtonian fluids, meaning that measurement at a fixed angle would result in serious errors. 2.3. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis. SDS-PAGE analysis for several kinds of PEG-containing TAA solutions was done under both reduced and nonreduced conditions. A 0.5% (w/v) amount of β-mercaptoethanol was dissolved in the solutions used for the reduced condition. The PEG concentration was varied from 0 to 12.5% for both conditions. TAA solutions collected from several fractions in the TAA-1 were measured for comparison. In addition to using TAA solutions, we used solutions that contained 0.5 and 1.0 M of ammonium sulfate as a crystallizing salt for comparison. 2.4. Circular Dichroism (CD) Measurement. The far-ultraviolet (UV) CD spectra were measured for the TAA solutions with 0 and 12.5% PEG at 20 °C using a spectropolarimeter (Jasco J-820). The protein concentration was 1 mg/mL for both solutions, and that of the buffer was reduced to 1/10 (5 mM) to obtain a spectrum with a high S/N ratio. The solution was placed in a quartz glass cell with a 1-mm light path, which was maintained at 20 °C with an external water bath. 2.5. Analytical Ultracentrifugation. The analytical ultracentrifugation was applied to the 12.5% PEG solution to measure the molecular weight of the TAA monomers for comparison with that for the SLS data. An ultracentrifuge (Beckman Coulter XL-A) was used at 12 000 or 20 000 rpm. Two rotation rates were used to clarify the effect of intermolecular interaction between TAA monomers. Sedimentationequilibrium analysis was applied to the concentration distribution data. This distribution was measured using a spectrophotometer (Beckman Coulter DU800) with scanning of the wavelengths from 230 to 350 nm. The TAA concentrations were 1, 0.5, and 0.25 mg/mL. The apparent molecular weight at each concentration was measured and then extrapolated to a zero protein concentration to determine the actual molecular weight. The procedure used to calculate the molecular weight is described elsewhere.23

3. Results and Discussion 3.1. Crystal Growth in PEG Solution. The TAA crystals were grown in the PEG-containing solutions using the hangingdrop vapor diffusion technique. The initial TAA drop contained 20 mg/mL of protein concentration and 12.5% PEG. It was held on a glass plate, and 25.0% PEG was used as the external solution to which the water in the drop diffused. After a few days, feather-like crystals (Figure 1a) were obtained. These crystals were broken into smaller ones for use as new seeds (microseeding technique). They were placed in 12.5% PEG solution and grew into polyhedral crystals (Figure 1b). Most of these crystals, however, were polycrystals. As described in the introduction, single crystals could be directly grown in a drop with the same composition under microgravity conditions. 3.2. Diffusion Coefficient and Its Dependence on TAA Concentration. Figure 2a shows autocorrelation function g(2)(q,t) versus lag time t at a scattering angle θ of 30°, 60°, and 90°. The concentration of TAA was 10 mg/mL, and that of

Onuma et al.

Figure 1. (a) Feather-like TAA crystals grown by the hanging-drop vapor diffusion technique. The initial drop contained 20 mg/mL of TAA and 12.5% PEG, and external solution was 25.0% PEG. (b) Polyhedral TAA crystal grown in 12.5% PEG solution using a part of (a) as a new seed (microseeding).

PEG was 1.0% (in 50 mM of NaAc buffer solution with 6.0 pH at 20 °C). A single-exponential function (solid lines), assuming a monomodal particle distribution, was able to fit all the data. The decay time τ distribution (Figure 2b) corresponding to 60°, as obtained by CONTIN analysis, showed a single peak. This single-peak distribution was also observed for a 3.0% PEG solution, but not for 6.0 and 12.5% PEG solutions. Figure 2c shows g(2)(q,t) vs t for a 12.5% PEG solution. The measured scattering angles and TAA concentration were the same as those used for the measurements shown in Figure 2a. In this case, single-exponential function (dotted lines) was unable to fit all the data, and a double-exponential function (solid lines) was needed. The τ distribution (Figure 2d) showed two peaks as expected, and the fast mode one corresponding to a small τ had fairly low intensity. We assume the fast mode corresponded to PEG molecules. To estimate whether the fast mode was PEG, the diffusion coefficient of PEG was measured in 50 mM of NaAc buffer solution. Figure 3a shows the concentration dependence of mutual diffusion coefficient D for the PEG molecules. Corresponding apparent hydrodynamic radius with each diffusion coefficient, which was calculated after the correction for viscosityη of the buffer solution (1.02 cp), was also plotted in the figure. There was a repulsive force between molecules, as shown by the positive slope of the correlation line between diffusion coefficient and PEG concentration. The translational diffusion coefficient at a zero PEG concentration, D0, was 77.8 × 10-12 m2/s, which corresponds to an actual hydrodynamic radius, rH, of 2.7 nm. Because this rH was large on the basis of molecular weight of PEG, we conducted molecular weight measurement using SLS for PEG solutions, and it was 7500

Effect of PEG on Intermolecular Interaction

Crystal Growth & Design, Vol. 9, No. 5, 2009 2519

Figure 2. (a) Autocorrelation functions at θ ) 30° (circles), 60° (triangles), and 90° (crosses) at 10 mg/mL concentration for 1.0% PEG solution. Solid lines are fitting lines assuming single-exponential function. Data points are reduced to 1/2 for easy to see. (b) Decay time distribution at θ ) 60° as analyzed using CONTIN method for data shown in (a). (c) Autocorrelation functions for 12.5% PEG solution. TAA concentration is the same as in (a). Single-exponential function (dotted lines) does not fit the data; double-exponential function (solid lines) is needed. (d) Decay time distribution at θ ) 60° for the data shown in (c). Two modes (fast and slow) were observed.

(data not shown). Taking molecular weight distribution of PEG into account, this value is reasonable for PEG monomer. Note that rH of PEG is close to that of TAA monomers, as described below. For a 12.5% PEG solution, the apparent rH of PEG monomer was reduced to 1.5 nm due to repulsive intermolecular interaction, which almost corresponded to the τ of fast mode in Figure 2d, that is, the rH of fast mode was estimated as 1.4 nm after correction of the viscosity of the protein solution. Therefore, it is safe to conclude that the fast mode appearing in theτ distribution for a high PEG concentration corresponds to PEG molecules. The PEG molecule should be observable in the 1.0 and 3.0% PEG solutions. However, as the PEG concentration was reduced, the apparent rH increased, and the difference between it and that of the TAA monomers became smaller. This led to an anomaly in the D vs c relationship as described below. Figure 3b shows the concentration dependence of D for the TAA monomers for solutions with PEG concentrations of 0 to 12.5%. For the 0% PEG solution, the dependence showed the same tendency as that observed in the previous study (using 50 mM of Tris solution at pH of 7.5). There was a repulsive force between the TAA molecules, and interaction parameter (line slope) was estimated to be 5.65 after conversion of c to a dimensionless volume fraction. The smaller value of the interaction parameter than that for 50 mM of Tris solution, 6.07, was due to the lower pH of the solution. Because the isoelectric point of TAA monomers is approximately 4.30, the surface potential of the monomers in the present solution was smaller leading to lower repulsive electrostatic interaction. The D0 at a zero protein concentration was 66.1 × 10-12 m2/s, which gave

an rH of 3.2 nm. This value coincides with that for TAA monomers estimated in the previous study within the margin of error. The concentration dependence of D, however, showed anomalies when PEG was added to the solution. With 1.0% PEG the dependence had a positive slope for TAA concentrations of more than 2.5 mg/mL. Below 2.5 mg/mL, it had a negative slope, and the D quickly increased as the TAA concentration decreased. This positive to negative change in the slope was also observed with 3.0% PEG (Figure 3c). The positive slope changed to a negative one below a TAA concentration of 10 mg/mL and then took a steeper negative value below 2.5 mg/mL. These anomalies were not due to experimental error because each D was within ( 2%. They arose due to the closeness of rH between the PEG molecules and TAA monomers, the (relatively) low viscosity of the solution, and the intermolecular interaction of PEG. Because the apparent rH of PEG increased with a decrease in the PEG concentration, the difference between it and the rH of TAA monomers was smaller at low PEG concentrations. This effect was partly compensated for by the repulsive interaction between TAA monomers up to 3.0% PEG, which means a relative increase in the apparent rH of TAA monomers at low TAA concentrations. However, low viscosity in low-PEG solutions reduced the τ of the TAA monomers. Moreover, the contribution of a large number of PEG molecules as compared to that of TAA monomers to the total scattering intensity was more effective at low TAA concentrations. The number of PEG molecules was at least four times that of TAA monomers. The single peak in the τ distribution for 1.0 and 3.0% PEG solutions thus includes a contribution from PEG molecules, and their effect was distinct

2520

Crystal Growth & Design, Vol. 9, No. 5, 2009

Onuma et al.

Figure 4. (a) Solvent viscosity dependence on PEG concentration. Data at zero inclination was used as actual viscosity. (b) Change in hydrodynamic TAA monomer radius with PEG concentration.

Figure 3. (a) Concentration dependence of diffusion coefficient (closed cirlces) for PEG molecules. Open circles are apparent hydrodynamic radius corresponded to each diffusion coefficient. (b) Concentration dependence of diffusion coefficient for TAA monomers at various PEG concentrations. Note that relationship is the same between 6.0 and 12.5%. (c) Enlargement of (b) for less than 20 mg/mL TAA concentration for 1.0 and 3.0% PEG solutions.

at low TAA concentrations. We concluded that analysis of D vs c for TAA monomers is effective only for TAA concentrations of more than 2.5 mg/mL for a 1.0% PEG solution and 10 mg/mL for a 3.0% PEG solution. For a 6.0% PEG solution D vs c showed a neutral relationship. The effect of PEG molecules on D was not reflected in the results because the τ distribution peaks were completely separated between PEG and TAA. The difference in apparent rH between PEG and TAA monomer was large due to the high PEG concentration and small repulsive force between TAA monomers. Note that the intermolecular interaction of TAA monomers was almost the same for the 12.5% PEG solution as for the 6.0% one. More than doubling the PEG concentration had no effect on the slope of the relationship line. This indicates

that the effect of PEG is not explained by simple depletion interaction, which physically increases the attractive force between molecules by osmotic pressure against the repulsive electrostatic interaction. This anomaly was not observed for a high-molecular-weight protein.18 3.3. Viscosity of Solution and rH. Figure 4a shows the η of PEG-containing solutions against the inclination angle, θ. The η had a slight dependence on θ for the 12.5% PEG solution. The actual viscosity of each solution was determined by extrapolation of the line to θ ) 0. Figure 4b shows the rH of the TAA monomers calculated using the data plotted in Figures 3b and 4a. Note that the rH decreased as the PEG concentration was increased. The rH decreased from 3.2 to 2.0 nm for the 12.5% PEG solution, which is not explained by attractive depletion interaction in PEG-containing solutions, as described in the intermolecular interaction anomaly (Figure 3a). 3.4. Molecular Weight Measurement. To determine whether the unexpected change in rH is specific for DLS, we did molecular weight measurement for a 12.5% PEG solution using SLS. Figure 5 shows a Zimm plot for various TAA concentrations (5-30 mg/mL). The SLS measurement at each c was done for θ ) 25 to 150° with an angular resolution of 1°, as was done in the previous study.19 A 12.5% PEG solution without TAA was used as a reference in measuring the scattering intensity. The inverse excess Rayleigh ratio of the TAA solutions against the 12.5% PEG buffer, Kc/∆R(θ), showed no dependence on θ for all c; that is, no aggregates formed in the solutions. We extrapolated these data to the double limit of c ) 0 (closed triangle symbols) and θ ) 0 and calculated the Mw from the intersection with the Kc/∆R(θ) axis to be 31.0 kDa. This Mw is much smaller than that of TAA monomers measured using solutions without PEG, 49.0 kDa. The coincidence of the

Effect of PEG on Intermolecular Interaction

Crystal Growth & Design, Vol. 9, No. 5, 2009 2521

Figure 5. Zimm plots for calculated molecular weight of TAA in 12.5% PEG solution. TAA concentrations were 30 (open circles), 20 (open reverse triangles), 10 (open squares), and 5 (crosses) mg/mL. Solid reverse triangles represent data at each scattering angle for c ) 0 mg/ mL extrapolated from the data for c ) 30, 20, 10, and 5 mg/mL. The y-axis intercept is the inverse of molecular weight.

SLS data with the DLS data suggests that adding PEG may cause TAA monomers to shrink or fragment. Decrease in rH and Mw with an increase in the concentration of neutral-charged polymers, such as PEG, have been observed in previous studies.24–26 However, the behaviors we observed were different. The previous studies used large moelcular weight polymers relative to that of the proteins and polymer-protein complexs formed. The Mw of polymer is at least a few times larger than that of protein, and the rH of polymer is also larger. As the polymer concentration is increased, the number of proteins bound to polymer chain decreases, which leads to a decrease in the rH and Mw of the complex. In our study, the Mw of the PEG was much smaller than that of the TAA, and the rH values were comparable. We did not observe the formation of a TAA-PEG complex. 3.5. SDS-PAGE Dependence on PEG Concentration. We did SDS-PAGE analysis for several kinds of TAA solutions. Figure 6a shows the results obtained under the reduced condition. Lane 1 was for a TAA solution without PEG. Lanes 2-9 were for solutions containing various concentrations of PEG. The TAA monomer band was in the same position up to 1.0% PEG (lane 5); it then gradually moved to lower molecular weight as the PEG concentration was increased. This movement was especially noticeable when the PEG concentration was increased from 1.6 to 6.3%. At 6.3%, the monomer band reached the lowest position and stayed there even when the PEG concentration was increased to 12.5%. This finding is consistent with the DLS result. The intermolecular interaction parameter (see Figure 3b) reduced from 1.0 to 6.0% PEG; however, it remained constant despite the increase in PEG concentration to 12.5%. The molecular weight of the TAA estimated from Figure 6a was approximately 50 kDa for the solution without PEG, and it was 40 kDa with 12.5% PEG. The 40 kDa is larger than that calculated from the SLS measurements. The results for lane 10 will be explained below. A shift of the monomer band was observed independent of the SDS-PAGE conditions and the kind of fraction. Figure 6b shows the SDS-PAGE results for the reduced and nonreduced conditions for solutions made from different fractions (fractions 1 and 2). For the reduced conditions (lanes 1-4), the SDS-PAGE results for both fractions exhibited the same behavior: the band showed a smaller molecular weight for 12.5% PEG solution. For the nonreduced conditions (lanes 5-8), the results were qualitatively the same as those for the reduced

Figure 6. SDS-PAGE results for TAA solutions. (a) Under reduced conditions. Lanes 1-9 correspond to 0, 0.016, 0.063, 0.25, 1.0, 1.6, 3.1, 6.3, and 12.5% PEG solutions, respectively. Lane 10 corresponds to solution made from TAA crystals grown from 12.5% PEG solution and pure water. (b) Comparison of reduced condition data (lanes 1-4) with nonreduced ones (lanes 5 to 8). Lanes 1 and 5 are TAA solutions collected from fraction 1 without PEG. Lanes 2 and 6 are TAA solutions collected from fraction 2 without PEG. Lanes 3 and 7 are TAA solutions of fraction 1 with 12.5% PEG, and lanes 4 and 8 are solutions of fracion 2 with 12.5% PEG. (c) Under reduced condtions. Lanes 1 to 6 are (fraction 1 and 0% PEG), (fraction 2 and 0% PEG), (fraction 1 and 12.5% PEG), (fraction 2 and 12.5% PEG), (fraction 1 and 6.3% PEG), and (fraction 2 and 6.3% PEG), respectively. Lanes 7-10 are (fraction 1 and 1 M of AS), (fraction 2 and 1 M of AS), (fraction 1 and 0.5 M of AS), and (fraction 2 and 0.5 M AS), respectively.

conditions although the decrease in molecular weight for the 12.5% PEG solutions was slightly larger than that under the reduced conditions. These results indicate that a decrease in molecular weight for PEG-containing solution is a common feature independent of the TAA isomer and/or SDS-PAGE conditions.

2522

Crystal Growth & Design, Vol. 9, No. 5, 2009

Onuma et al.

Figure 7. CD spectrum for 0 (blue) and 12.5% (red) PEG solutions. TAA concentrations were fixed at 1.0 mg/mL. Spectra coincide, suggesting no change in secondary structure of TAA monomers with PEG concentration.

To determine whether this anomaly is seen for other crystallization-inducing salts, we conducted SDS-PAGE analysis using ammonium sulfate (AS). Figure 6c shows the results for the reduced conditions. The solutions made from fractions 1 and 2 were examined for different concentrations of AS (lanes 7-10). The results for PEG solutions (lanes 3-6) were added for comparison. The monomer band stayed at the same position as that of the original TAA monomers (lanes 1 and 2), and no decrease in the molecular weight was observed. This indicates that the decrease in the molecular weight seen in the SDS-PAGE analysis is a specific result of using PEG as the crystallizationinducing reagent. The SDS-PAGE results are consistent with those for light scattering, which suggests that TAA monomers in PEGcontaining solutions shrink and/or fragment. If part of a molecule is actually broken by PEG, TAA crystals grown in a PEG solution should include weak bonds within molecule although this fragmentation can be recovered in crystallization. The fragmentation would again appear when the grown crystals were dissolved. Lane 10 in Figure 6a shows the SDS-PAGE result for a solution made by dissolving grown TAA crystals in distilled water. We removed grown crystals from PEG solutions and dissolved them in the water after removing the surrounding PEG+TAA solution by gentle and successive washing. Note that the band corresponding to the solution made from TAA crystals was in the same position as that for the reference TAA solution (lane 1) without PEG. The result shown in lane 10 reveal no evident band at a small molecular weight where a band should appear if the TAA molecules were partly broken in the PEG solution. Instead, a slight band at a high molecular weight, ∼200 kDa, was observed. The reason for this is unclear. Anyway, the result shown in the lane 10 suggests that the fragmentation of TAA molecules is a difficult explanation to accept for the light scattering and SDS-PAGE results. Another possible explanation for the SDS-PAGE results is shrinkage of the TAA molecules in the PEG solutions. However, the DLS and SLS data refuse this. The differences observed in the results between the TAA solution without PEG and that with 12.5% PEG are not explained by molecule shrinkage (see Figures 4b and 5). This means that the only explanation for each data anomaly is that the PEG modified the dynamics and scattering intensity of TAA molecules, and this results in an “apparent decrease” in rH and Mw of TAA monomers.

Figure 8. Ultracentrifuge calculated molecular weight of TAA monomers for 12.5% PEG solutions. (a) Absorbance at different wavelengths. Red, blue, and black correspond to TAA solutions with concentrations of 1.0, 0.5, and 0.25 mg/mL. (b) Concentration gradient (absorbance) vs position in 0.5 mg/mL solution measured at 12 000 rpm. Solid curve is fitting assumimg monocomponent particle in solution. (c) Relationship between apparent molecular weight and absorbance. Open symbols are for 20 000 rpm; closed ones are for 12 000 rpm. Synbol colors corresponds to the TAA concentrations in (a). Actual molecular weight is extrapolation of line to zero limit of absorbance.

3.6. CD Spectrum and Analytical Ultracentrifuge Results. If the anomalies observed for the light scattering and SDS-PAGE data are apparent, the molecular structure of the TAA monomers should retain a high-order structure independent of the PEG concentration. Figure 7a shows CD spectra for solutions without PEG (blue line) and 12.5% PEG (red line). The far-UV spectra coincided completely around 200-250 nm, indicating that the PEG did not affect the secondary structure of the TAA monomers. Figure 8 shows the analytical ultracentrifuge data. The absorbance for each TAA solutions (0.25, 0.5, and 1.0 mg/mL) with 12.5% PEG showed a clear peak at around 280 nm (Figure 8a). The absence of a peak at 350 nm indicates the absence of aggregates in the solutions, which is consistent with the DLS results. Figure 8b shows an example absorbance distribution (concentration distribution) at each position in the solution from

Effect of PEG on Intermolecular Interaction

Figure 9. TR-SLS data for 20% PEG and 25 mg/mL of TAA solution. (a) Scattering intensity vs scattering angles for TAA solution at time zero. Inset shows that for 20% PEG solution without TAA. (b) Change in Mw (open circles) and Rg (open reverse triangles) over time. (c) Change in df over time.

the rotation center for a 0.5 mg/mL solution measured at 12 000 rpm. This data was fitted using a concentration-gradient equation assuming a monocomponent, and the apparent molecular weight was calculated to be 50.8 kDa. Figure 8c shows the change in apparent molecular weight with absorbance. The zero limit of absorbance, that is, the zero limit of the protein concentration, shows the actual molecular weight of the TAA monomers. It was 51.0 kDa for both 12 000 and 20 000 rpm. The difference evident between the molecular weight vs absorbance relationships for 12 000 and 20 000 rpm was negligibly small. This indicates that the intermolecular interaction between TAA monomers in 12.5% PEG solution was neutral, which is consistent with the results plotted in Figure 3b. Because the measurement error associated with the analytical centrifuge technique is approximately 5%, the calculated molecular weight of 51.0 kDa was virtually the same as for the 0% PEG solutions using SLS (49.0 kDa). The CD and analytical centrifuge data

Crystal Growth & Design, Vol. 9, No. 5, 2009 2523

indicate that the decreases in rH and Mw for the TAA monomers observed by light-scattering and SDS-PAGE measurements in the high PEG concentration solutions were the apparent ones. The reasons for anomalies in the light scattering and SDS-PAGE data would be caused by some kind of solution structure in PEG-containing solution. Polymers such as PEG are known to be able to form a network in solution by entangling the molecular chains. This network becomes more distinct as the concentration and/or molecular weight of the polymer are increased. When the molecular weight of PEG is small, that is, the molecule size is small as compared to that of a protein molecule, PEG-containing buffer acts as a uniform solvent for proteins. However, as the PEG molecular weight is increased and the size of the PEG molecules becomes comparable to that of the protein molecules, as in the present case, the solutions are not a binary system (protein-PEG solvent), but are a ternary system (protein-PEG molecule-buffer solvent). The PEGformed network affects the dynamics of the protein molecules, and the principle of free protein molecule movement in a uniform solvent no longer holds. It has been shown that a network consisting of high molecular weight polymers causes the high-speed fluctuation in solution. This would accelerate the diffusion of TAA molecules and thereby decreases the rH of TAA monomers. The fluctuation induced by a PEG network could also affect the electrophoresis of TAA molecules in SDS-PAGE. The absolute scattering intensity from TAA molecules would decrease because a PEG-inducing network acts as a shell for the protein molecules. The critical PEG concentration, c*, above which the molecular chain entangling starts was estimated using the equations, c* ) MPEG/(NARPEG3)27 and c* ) MPEG/{NA(2RPEG3)},28 where MPEG, NA, and RPEG are the molecular weight of PEG, Avogadro’s number, and the gyration radius of a PEG monomer.27 Although RPEG was not directly measured, it could be estimated using the rH of PEG. It was approximately 2.0-2.5 nm assuming PEG molecules have a sphere or random coil shape. The c* was ∼100% by using the former equation, which far exceeds the actual concentration as pointed out in ref 29. The latter equation gave c* ∼ 13%, which is close to the concentration, 12.5%. However, we should point out that the anomalies already appeared at lower PEG concentration (∼1%) as seen in the decrease in rH. This suggests that there should be a strong inhomogeneous concentration distribution of PEG molecules in the solution. The locally concentrated PEG induced network formation, and this affected the scattering intensity of the TAA monomers. Effect of polymer network to protein crystallization has been investigated using the agarose gel as a medium. Although several discrepancies concerning the promotion and inhibition effect of gel to protein nucleation have been reported,30–33 conclusive information was recently obtained from dynamic surface-tension measurements.34 The surface tension of lysozyme solution containing agarose gel was measured, and it was concluded that the gel network inhibits the protein nucleation by increasing the repulsive force between protein molecules and the structural mismatch between crystal-protein molecule interface. This effect would result the formation of submicron-sized protein clusters in a solution, as we will show in the following section. The discussion in ref 34 supports our results (especially for the effect of polymer network to intermolecular interaction in Figure 3b) and conclusions. 3.7. TR-SLS Measurements. If we assume a PEG-inducing network, interesting behavior is predicted in the association of TAA. The PEG network would act as a barrier for association,

2524

Crystal Growth & Design, Vol. 9, No. 5, 2009

which suggests the formation of a “stable TAA aggregate.” In this solution, further association of the aggregate would not proceed. Figure 9 shows the results of TR-SLS measurements of the TAA aggregates for a 20.0% PEG solution: (a) scattering intensity vs scattering angle at time zero, (b) change in apparent molecular weight Mw and gyration radius Rg, and (c) change in fractal dimension df. The concentration of TAA was 25 mg/ mL. Up to ∼19.0% PEG, no aggregation of TAA was observed. At 20.0% PEG, aggregates larger than 100 nm in radius suddenly appeared as soon as the solution was prepared. Such aggregates are not induced by the PEG itself, as revealed by the scattering intensity data, which showed no dependence on the scattering angle for 20.0% PEG buffer without TAA (see inset in Figure 9a). These aggregates increased Mw and Rg over time; both reached a maximum after ∼250 min (Figure 9b). The Rg increased from 170 to 380 nm over time and then showed no further increase. The association stopped, and the aggregates stably remained in this state for more than a few weeks. No precipitation was observed although this aggregate size far exceeded the critical radius for nucleation. The change in df showed an interesting feature. It quickly increased from 1.40 to 1.70 within the first 100 min, and there was no remarkable increase thereafter. It eventually reached an equilibrium value of ∼1.85. This value is typical for amorphous or polycrystal aggregates and means that submicron-sized aggregates whose inner structure did not change to single-crystalline state stably exist in the solution. We assume that this phenomena well coincides with the formation of TAA polycrystals in PEG solutions. Crystals would grow not only by monomer association but also by aggregate association, which would result in lowquality crystals with a mosaic structure. The effect of microgravity might hinder the formation of stable low-crystalline aggregates by breaking the PEG-inducing solution network. The microgravity would eliminate the inhomogeneous distribution of PEG molecules and reduce the PEG concentration than c* in all position of solution.

References (1) Bodo, G. Nature 1955, 176, 829–830. (2) Swanson, R.; Trus, B.; Mandel, N.; Mandel, G.; Kallai, O. B.; Dickerson, R. E. J. Biol. Chem. 1977, 252, 759–775. (3) Bushnell, G. W.; Louie, G. V.; Brayer, G. D. J. Mol. Biol. 1990, 214, 585–595. (4) Georgalis, Y.; Zouni, A.; Eberstein, W.; Saenger, W. J. Cryst. Growth 1993, 126, 245–260. (5) Boyer, M.; Roy, M.-O.; Jullien, M. J. Cryst. Growth 1996, 167, 212– 220.

Onuma et al. (6) Muschol, M.; Rosenberger, F. J. Chem. Phys. 1997, 107, 1953–1962. (7) Lafont, S.; Veesler, S.; Astier, J. P.; Boistelle, R. J. Cryst. Growth 1997, 173, 132–140. (8) Ries-Kautt, M. M.; Ducruix, A. J. Biol. Chem. 1989, 264, 745–748. (9) Vaney, M. C.; Broutin, I.; Retailleau, P.; Douangamath, A.; Lafont, S.; Hamiaux, C.; Prange, T.; Ducruix, A.; Ries-Kautt, M. Acta Crystallogr. Sect. D, Biol. Crystallogr. 2001, 54, 929–940. (10) Lekkerkerker, H. N. W. Physica A 1997, 244, 227–237. (11) Budayova, M.; Bonnete, F.; Tardieu, A.; Vachette, P. J. Cryst. Growth 1999, 196, 210–219. (12) McPherson, A. In Crystallization of Biological Macromolecules; Cold Spring Harbor Laboratory Press: New York, 1999. (13) Kulkarni, A. M.; Chatterjee, A. P.; Schweizer, K. S.; Zukoski, C. F. J. Chem. Phys. 2000, 113, 9863–9873. (14) Finet, S.; Tardieu, A. J. Cryst. Growth 2001, 232, 40–49. (15) Bonnete, F.; Vivares, D.; Robbert, C.; Colloch, N. J. Cryst. Growth 2001, 232, 330–339. (16) Tanaka, S.; Ataka, M. J. Chem. Phys. 2002, 117, 3504–3510. (17) Vivares, D.; Bonnete, F. Acta Crystallogr. Sect. D, Biol. Crystallogr 2002, 58, 472–479. (18) Tanaka, S.; Ataka, M.; Onuma, K.; Kubota, T. Biophys. J. 2003, 84, 3299–3306. (19) Onuma, K.; Inaka, K.; Furubayashi, N.; Shibata, F.; Kobayashi, Y.; Ohnhishi, Y.; Sato, M. Recent. Res. DeVel. Phys. Chem. 2007, 9, 173– 191. (20) Higashiura, A.; Tanaka, H.; Inaka, K.; Sato, M.; Sugiyama, S.; Takahashi, S.; Yamanaka, M.; Motohara, M.; Sano, S.; Kobayashi, T.; Suzuki, M.; Tanaka, T.; Nakagawa, A. Acta Crystallogr. 2006, A62, s123. (21) Kanzaki, N.; Ueyda, T. Q. P.; Onuma, K. J. Phys. Chem. B 2006, 110, 2881–2887. (22) Onuma, K.; Watanabe, A.; Kanzaki, N.; Kubota, T. J. Phys. Chem. B 2006, 110, 24876–24883. (23) Tanford, C.; Reynolds, J. A. Biochim. Biophys. Acta 1976, 457, 133– 170. (24) Azegami, S.; Tsuboi, A.; Izumi, T.; Hirata, M.; Dubin, P. L.; Wang, B.; Kokufuta, E. Langmuir 1999, 15, 940–947. (25) Matsudo, T.; Ogawa, K.; Kokufuta, E. Biomacromolecules 2003, 4, 728–735. (26) Matsudo, T.; Ogawa, K.; Kokufuta, E. Biomacromolecules 2003, 4, 1794–1799. (27) Cotton, J. P.; Nierlich, M.; Boue, F.; Daoud, M.; Farnoux, B.; Jannink, G.; Duplessix, R.; Picot, C. J. Chem. Phys. 1976, 65, 1101–1108. (28) Graessley, W. W. Polymer 1980, 21, 258–262. (29) Ying, Q.; Chu, B. Macromolecules 1987, 20, 362–366. (30) Thiessen, K. J. Acta Crystallogr. D Biol. Crystallogr. 1994, 50, 491– 495. (31) Robert, M. C.; Bernard, Y.; Lefaucheux, F. Acta Crystallogr. Sect. D, Biol. Crystallogr. 1994, 50, 496–503. (32) Finet, S.; Bonnete, F.; Frouin, J.; Provost, K.; Tardieu, A. Eur. Biophys. J. 1998, 27, 263–271. (33) Vidal, O.; Robert, M. C.; Boue, F. J. Cryst. Growth 1998, 192, 257– 270. (34) Wang, L.; Liu, X. Y. Biophys. J. 2008, 95, 5931–5940.

CG900019E