Assessing Micellar Interaction and Growth in Detergent Solutions Used to Crystallize Integral Membrane Proteins Patrick J. Loll,† Carl Hitscherich, Jr.,‡ Vladimir Aseyev,‡,§ Margaret Allaman,‡ and John Wiencek*,‡
CRYSTAL GROWTH & DESIGN 2002 VOL. 2, NO. 6 533-539
Department of Biochemistry, Drexel University College of Medicine, 245 North 15th Street, Philadelphia, Pennsylvania 19102-1192, and Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City, Iowa 52242 Received August 1, 2002;
Revised Manuscript Received September 16, 2002
ABSTRACT: Integral membrane proteins are solubilized in aqueous solutions by the addition of detergent, creating protein-detergent complexes (PDCs), which can then be crystallized. Interactions between the detergent moieties of PDCs contribute significantly to their crystallization behavior. Interaction forces can be quantified using the second osmotic virial coefficient (B22). The B22 behavior of protein-free detergent micelles is a good predictor of the behavior of the corresponding PDCs under similar conditions, suggesting that detergent B22 measurements can be used as a screening tool when crystallizing PDCs. However, if the micelle size varies, B22 measurements will not accurately reflect micelle-micelle forces. We therefore examined micelle size in a model detergent system, using small-angle X-ray scattering and static and dynamic light scattering, assessing the effects of temperature, detergent concentration, and precipitant on B22 and micelle size. In the absence of poly(ethylene glycol) (PEG), decreases in B22 principally reflect increases in micelle-micelle attractive forces and do not reflect significant changes in micelle size. In the presence of PEG, the apparent hydrodynamic radius of detergent micelles shows a similar dependence upon micelle concentration as in the absence of PEG, suggesting that PEG does not effect significant changes in micelle size but rather acts by enhancing interaction forces between micelles. 1. Introduction Roughly one-quarter to one-third of all proteins are thought to be integral membrane proteins.1 These molecules are critically important to the functioning of living cells and are under intensive investigation as potential drug targets. However, relatively little is known about membrane protein structure: of the tens of thousands of proteins of known structure, fewer than 1% are membrane proteins. This is largely due to the difficulties associated with obtaining crystals suitable for X-ray diffraction analysis.2,3 Insights into the basic mechanisms controlling membrane protein crystal growth are required so that rational strategies may be devised to improve the success rate for crystallization. Membrane proteins have evolved to exist in the anisotropic, amphipathic environment of biological membranes. Hence, these proteins typically contain both regions that are embedded in the lipid bilayer and regions that are exposed to aqueous solution and are soluble in neither aqueous solutions nor organic solvents. However, the addition of detergents can mask the hydrophobic section of the protein, creating a water soluble protein-detergent complex (PDC). Crystals of membrane proteins for X-ray diffraction analysis are most frequently obtained by direct crystallization of PDCs. Because PDCs can contain as much as 50% detergent by weight, it is not surprising that * To whom correspondence should be addressed. Tel: (319)353-2377. Fax: (319)335-1415. E-mail:
[email protected]. † Drexel University College of Medicine. ‡ University of Iowa. § Permanent address: Institute of Macromolecular Compounds, Russian Academy of Science, Bolshoi Prospect 31, 199004, St. Petersburg, Russia.
PDC crystallization is exquisitely sensitive to the properties of the detergent(s) solutions used. For example, crystallization of PDCs appears to be particularly favorable under conditions that lie near the solution’s cloud point.4-7 The cloud point is a phase transition that occurs in solutions containing detergent micelles. At the cloud point, micelles coalesce and separate from the aqueous phase, causing microscopic droplets of detergentrich phase to be dispersed throughout the solution and giving rise to a characteristic turbidity (hence the term “cloud point”). The attractive forces between detergent micelles that mediate micelle aggregation and subsequent phase separation are also expected to mediate attractions between the detergent moieties of PDCs. Thus, it seems reasonable that when solution conditions approach the cloud point, the interactions between PDCs can be sufficiently attractive to bring PDCs into close contact, allowing crystal lattice contacts to be formed.8 This hypothesis has been examined using the bacterial outer membrane protein OmpF porin, one of many proteins that crystallizes near the solution cloud point.9 Measurements of the second osmotic virial coefficient (B22) were used to quantify interparticle attractive forces in solutions containing micelles and OmpF PDCs.10 B22 values for PDCs were found to become more negative as the crystallization conditions are approached, corresponding to increasingly attractive forces between PDCs. Earlier work has prompted the suggestion that there exists a “crystallization slot” for soluble proteins, an optimum range of B22 values in which crystals are most likely to form.11-14 In an analogous fashion, the B22 values for pure detergent micelles become more negative as the solution cloud point is approached. In fact, B22
10.1021/cg025563w CCC: $22.00 © 2002 American Chemical Society Published on Web 10/09/2002
534
Crystal Growth & Design, Vol. 2, No. 6, 2002
values for protein-free micelles were found to be good predictors of the B22 values for PDCs under similar conditions, suggesting that micelle-micelle interactions could be used to identify solution conditions suitable for PDC crystal growth.15 B22 is commonly taken as a measure of the pairwise intermolecular forces between particles in solution;16 however, changes in the micelle aggregation state can confound B22 values estimated from experimental data. If the B22 values of protein-free micelles are to indeed prove useful as a screening tool for PDC crystallization, it will be important to understand precisely what solution properties are reflected in these measurements. For example, as solution conditions approach the cloud point and the crystallization slot for PDCs, B22 decreases. It is not clear whether this decrease reflects an increase in the attractive force between particles in solution, an increase in particle size, or some combination of the two. Static light scattering (SLS) alone cannot answer this question,10 and attempts to combine SLS with dynamic light scattering (DLS) are complicated by the presence of poly(ethylene glycol) (PEG) in crystallization buffers.17 In this paper, we begin to address this issue by first considering solutions of protein-free detergent micelles in solutions containing no PEG. Our results suggest that decreases in B22 principally reflect increases in micelle-micelle attractive forces and do not reflect significant changes in micelle size. We then consider solutions containing the precipitating agent, investigating the effects of PEG on hydrodynamic and thermodynamic intermicellar interactions, as well as upon micelle size and shape. Phase diagrams for solutions of various PEG concentrations are mapped, and the effects of temperature and detergent concentration on micelle physiochemical properties in the presence of PEG are investigated. The results are consistent with a model in which PEG does not effect significant changes in micelle size but rather acts by enhancing attractive forces between micelles. 2. Experimental Section 2.1. Materials. Buffer salts were obtained from EM Science and Fisher. n-Octyl-β-D-glucopyranoside (BOG; MW 292.4) was from Anatrace and n-octylpolyoxyethylene (C8En, n ) 2-9, approximate MW 350; abbreviated as octyl-POE9) was from Bachem; PEG-2000 was obtained from Fluka. The present study focuses on the properties of the mother liquor from which tetragonal crystals of OmpF porin were grown (space group P42)9. This solution (referred to hereafter as “crystallization buffer”) consisted of 0.5 M NaCl, 0.1 M sodium phosphate, pH 6.5, 1 mM sodium azide, 0.9% w/w BOG, and 0.09% w/w octyl-POE; the precipitant used is PEG2000. Crystals were observed to form between PEG concentrations of 12-15% w/w. Buffers were prepared by titration of 0.1 M monobasic and dibasic sodium phosphate solutions containing 0.5 M NaCl to yield a pH of 6.5. Sodium azide was added following titration, after which PEG was added to the desired concentration (ranging from 0 to 15% w/w). Detergentcontaining solutions were prepared with a constant weight ratio of octyl-POE to BOG of 1:10. The detergent concentration was defined as the total concentration of both detergents (cdet) and was varied from 0 to 200 mg/mL. The micellar concentration (cm) was assumed to be the total detergent concentration in excess of the critical micelle concentration or CMC (cm ) cdet - cCMC). All solutions were prepared using deionized water (Maxima, ELGA Inc.) and were filtered using 0.2 µm filters (Puradisk
Loll et al. 25 TF, Whatman). Filters were washed with water before using; filtration was performed at 60-70 °C, and the integral intensity of scattered light was measured before and after filtration to ensure that no change in detergent and/or PEG concentration took place. 2.2. Instrumentation and Methods. 2.2.1. Light Scattering Measurements. SLS and DLS experiments were conducted essentially as previously described,10,17 using an ALV/SP-125 compact DLS/SLS goniometer and an ALV-5000/E multiple τ digital correlator. A diode-pumped, solid state laser (COHERENT, DPSS532-400) operating at 532 nm wavelength and a 35 mW He-Ne Uniphase laser operating at 632.8 nm were used as light sources. SLS data were measured at a scattering angle of 90° over a temperature range of 10-55 °C. In DLS experiments, the correlation function of the scattered light intensity G2(t) was measured in the self-beating mode18 at scattering angles between 30 and 150°. The temperature was varied from 10 to 55 °C. Correlation functions were obtained with a shortest sample time of 12.5 ns and a last delay time of 393.2 ns. Time correlation functions were analyzed using the regularized inverse Laplace transform technique in unweighted mode. Reported Rh values reflect peak values, with error estimates reflecting repeated measurements. The specific refractive index (dn/dc) was measured with a Bellingham and Stanley 60/ED Abbe refractometer and the laser light source described above, using the methods detailed in Hitscherich et al.10 2.2.2. CMC Determination. SLS was used to determine the CMC for the BOG/octyl-POE detergent system over the temperature range of 10-55 °C.10,19,20 The CMC was identified by the characteristic sharp break in the plot of Rayleigh ratio vs detergent concentration, which is indicative of micelle formation. 2.2.3. Small-Angle X-ray Scattering (SAXS). If the size of a scattering particle is comparable to the wavelength of the incident radiation source, the radius of gyration of the particle (Rg) can be determined from the angular dependence of the scattered radiation. Thus, detergent micelles (Rg ∼ 2 nm) require X-rays for proper Rg measurements. Apparent radii of gyration were determined using an X-ray apparatus consisting of a standard copper target Ro¨ntgen tube as the radiation source, a camera, a collimation system of the Kratky type, and a PSD 50M position sensitive linear detector (Hecus M. Braun, Graz). The vacuum camera body has an adjustable highprecision line collimation system, which allows one to study angles in the q range of 6-0.05 nm-1. A 1 mm diameter quartz capillary was used as a flow-trough sample cuvette. A nickel foil was used to filter the incident beam. Data collection was controlled by a software package provided by the manufacturer (ASA V2.4). All measurements were conducted at 22 °C. Rg was obtained from the low angle region of the Guinier plots.21 2.2.4. Cloud Point Determination. Detergent cloud points were determined by visually monitoring solution clouding while slowly decreasing the temperature of the solution.17 An optimal scanning rate of 6 °C per hour was determined by trial and error and was used for all estimates. 2.2.5. Viscosity and Density Measurements. The viscosities of all solutions were measured as a function of temperature using a Brookfield DVII+ viscometer. Density measurements were conducted at the same temperatures using an Anton PAAR DMA 5000 density meter.
3. Results and Discussion 3.1. PEG-Free Detergent Solutions: Effects of Temperature and Detergent Concentration. 3.1.1. SLS. For the BOG/octyl-POE system, the CMC was found to be independent of temperature with a constant value of 4.7 ( 0.5 mg/mL. The intensity of scattered light increased linearly with increasing cm from the CMC (cm ) 0) to approximately 40-50 mg/mL. Estimates of the micellar molar mass were obtained from a Debye plot extrapolated to infinite dilution (Figure 1 and
Micellar Interaction and Growth in Detergent Solutions
Figure 1. Representative Debye plots from BOG/octyl-POE micelles in crystallization buffer measured at 90° scattering angle and four different temperatures: 10 (open circles), 22 (filled diamonds), 30 (open diamonds), 40 (open squares), and 45 °C (filled triangles). The inset shows the cm range used in the Mw extrapolation. Table 1. Physical Parameters of BOG/Octyl-POE Micelles Dissolved in Crystallization Buffer as a Function of Temperature temp ((0.1 °C)a
Mw ((2000 g/mol)
B22 × 104 ((0.5 cm3 mol g-2)
Rh,0b ((0.05 nm)
10 22 30 40 45 55
31 000 30 000 32 000 30 000 28 000 31 000
-2.0 -3.0 -2.2 -2.3 -2.1 -2.7
2.39 2.47
Crystal Growth & Design, Vol. 2, No. 6, 2002 535
Figure 2. Plot of the apparent hydrodynamic radius (Rh) vs micelle concentration (cm) obtained for mixed BOG/octyl-POE micelles in crystallization buffer at 90° scattering angle: 10 (open circles), 22 (closed diamonds), 40 (open squares), and 55 °C (closed squares). The thick solid line represents values of the micellar overlap concentration (cmic*) estimated for the apparent Rh measured at 55 °C and the micellar molar mass at infinite dilution.
2.54 2.68
a
Typical standard errors for these measurements are given. CMC and dn/dc values remained constant over the range studies, with values of 4.7 ( 0.5 mg/mL and 0.144 ( 0.002 mL/g, respectively. b Index 0 denotes values taken at infinite dilution.
Table 1). The micellar molar mass was found to be independent of temperature within the error of the measurement. Likewise, dn/dc values were also found to be independent of temperature with a value of 0.144 mL/g. 3.1.2. DLS. The apparent hydrodynamic radius (Rh) was measured at scattering angles 30, 90, and 150° for selected samples at temperatures ranging from 10 to 55 °C. Because no angular dependence of Rh was observed, a scattering angle of 90° was chosen for subsequent investigation. The viscosity (η) and refractive index (n) were measured at each temperature and used in the calculation of Rh. At detergent concentrations up to 50 mg/mL, the increase in apparent Rh is practically temperature-independent (Figure 2). Above this concentration, the measured Rh value shows a small positive correlation with temperature. The apparent Rh passes through a maximum at cdet ∼100 mg/mL. 3.1.3. SAXS. The intensity of scattered laser light from BOG/octyl-POE mixed micellar solutions has no angular dependence, making it impossible to measure the radius of gyration (Rg) by SLS. Accordingly, SAXS measurements were used to determine the Rg. The dependence of apparent Rg upon cm is similar to that of apparent Rh (Figure 3). However, Rg is smaller than Rh and reaches its maximum at a lower concentration than Rhsabout 30-40 mg/mL. The parameter Rg/Rh can be used to describe the shape of noninteracting particles.22 The value of Rg/Rh for the BOG/octyl-POE micelles at 22° has a value of
Figure 3. Plots of hydrodynamic radius (Rh), radius of gyration (Rg), and their ratio as a function of micellar concentration (cm). Data correspond to mixed BOG/octyl-POE micelles in crystallization buffer at 22 °C. The long exposure times used preclude the use of replicates; however, experience suggests that Rg values are reproducible to within 0.01 nm. Error propagation would then yield estimates for the error in Rg/Rh of approximately 0.018.
0.89 at infinite dilution. For comparison, the lowest possible value that Rg/Rh can assume for noninteracting particles is 0.78, which corresponds to a perfect sphere. The infinite dilution result obtained for the BOG/octylPOE system therefore suggests that the micelles are somewhat elongated. Equations exist that relate the Rg and Rh values for ellipsoids to the major and minor axis lengths.23,24 Because both Rg and Rh are experimentally measured at infinite dilution for the 22 °C data shown in Table 1, the major and minor axis size can be estimated by solving these equations simultaneously. All axis lengths discussed here are from center to edge (i.e., radii, as opposed to diameters). If an oblate ellipsoid is assumed, the major and minor axis lengths obtained are 3.42 and 0.74 nm, respectively. The minor axis should be indicative of the detergent molecule’s length within the micelle, which seems unrealistically small in this case. Molecular dynamics simulations suggest that the detergent’s extended length within the micelle
536
Crystal Growth & Design, Vol. 2, No. 6, 2002
should be 1.52 nm.25 However, assuming a prolate ellipsoid geometry yields 4.30 and 1.66 nm major and minor axes, respectively. These results are in reasonable agreement with the molecular dynamics simulation, as well as the results of Kameya and Takagi,20 who measured the properties of pure BOG detergent micelles in water and surmised that the micelle was a prolate ellipsoid having a major axis of 4.6 nm and a minor axis of 1.4 nm. Marone et al.26 also found BOG to be a prolate ellipsoid in a 0.28 M NaCl solution with a major axis of 5.00 nm and a minor axis of 1.06 nm. Other published results suggest BOG micelles to be prolate ellipsoid or short cylinders with comparable dimensions27,28 (note that a short cylinder is well-modeled by a prolate ellipsoid). The modest difference in the various measurements can be ascribed to differences in the measurement methods (SANS, DLS, and SAXS), the solvent composition, and the detergent mixture (pure BOG vs the mixed detergent system studied here). Certainly, our results support the prolate ellipsoid geometry. If growth of the micelles occurs, the relative ratio of Rg/Rh will only increase (assuming constant ellipsoid shape and unchanged micelle hydration). In Figure 3, the ratio of Rg/Rh is seen to decrease with increasing cm, dropping below 0.78 at concentrations in excess of 20 mg/mL. Because this value is impossibly low, the Rh and/or Rg measurements must be biased by intermicellar interactions, resulting in the measurement of apparent radii, which differ significantly from the true radii. These results indicate that the negative slopes observed in the Debye plots represent attractive interactions and not micellar growth. Thus, BOG/octyl-POE mixed micelles exist as prolate ellipsoids that do not grow significantly with increasing micellar concentration but do exhibit increasingly attractive micellemicelle interactions as their concentration rises. 3.2. Phase Diagram: Effect of PEG. Pure aqueous solutions of BOG do not phase separate between 0 and 100 °C;4,29 however, the addition of PEG can induce phase separation in BOG solutions at temperatures relevant to membrane protein crystallization. In the presence of PEG, mixed BOG/octyl-POE micelles exhibit phase separation similar to that seen with pure BOG micelles.17 The precise location of the detergent cloud point was mapped for BOG/octyl-POE mixed micelle solutions containing various PEG concentrations at temperatures between 0 and 40 °C (Figure 4). Solutions with a cPEG of 6% w/w or less were not observed to cloud at temperatures between 0 and 40 °C, while at PEG concentrations above 6% w/w, solutions could be induced to separate into two immiscible phases upon cooling. Figure 4 depicts the cloud point dependence upon temperature, using fixed PEG concentrations. With increasing cPEG, the phase separation occurs at higher temperatures and lower detergent concentrations. The overlap concentration for PEG-2000 is 4.2% w/w.17 Above this concentration, PEG solutions are semidilute, and PEG molecules begin to become entangled and to form extended networks. It is interesting to note that BOG/octyl-POE solutions will only cloud at PEG concentrations in excess of the overlap concentration, suggesting that the formation of a network of
Loll et al.
Figure 4. Position of the cloud point boundary as a function of detergent concentration and temperature for solutions containing 10 (filled squares), 12 (filled triangles), and 15% (open circles) w/w PEG.
Figure 5. Representative Debye plots for BOG/octyl-POE micelles in various precipitant compositions: 0% PEG (open circles), 6% PEG (filled triangles), 10% PEG (filled squares), 12% PEG (open inverted triangles), and 14% PEG (open squares). The inset shows the range of cm used for calculation of B22 (only 0, 10, and 14% PEG are shown in the inset for clarity).
entangled PEG molecules may be required to promote detergent phase separation. 3.3. Micellar Properties: Effects of PEG Concentration and Micellar Concentration. 3.3.1. SLS. Debye plots obtained at 22 °C are linear at low detergent concentrations but show a pronounced PEG-dependent curvature at micelle concentrations above 20 mg/mL (Figure 5). Estimates of the micellar weight-averaged molar mass (Mw) and B22 were obtained utilizing a linear fit extrapolated to infinite dilution, using cm < 20 mg/mL (Table 2). The estimated Mw values were found to be independent of cPEG within the error of the measurement. B22 was found to decrease significantly when cPEG approached the concentrations required to induce OmpF crystallization.10 Similar cPEG values are required to drive the micellar system toward the cloud point. 3.3.2. DLS. The apparent hydrodynamic radius (Rh) was measured at scattering angles 30, 90, and 150° for selected solutions at cPEG ranging from 0 to 12% w/w. As was the case with PEG-free solutions, no angular dependence of Rh was observed, and accordingly, a scattering angle of 90° was chosen for subsequent
Micellar Interaction and Growth in Detergent Solutions
Crystal Growth & Design, Vol. 2, No. 6, 2002 537
Table 2. Physical Parameters of Detergent Solutions with Various Precipitant Compositions cPEG (w/w %)
Mw ((2000 g/mol)
B22 × 10-4a,b ((1.0 cm3 mol g-2)
CMC ((1.0 mg/mL)
dn/dc ((0.002 mL/g)
Rh ((0.05 nm)
η ((0.005 mPa s)
n ((0.0005)
0 2c 4c 6 8c 10 12 14c 15
30 000 35 000 31 000 30 000 28 000 30 000 27 000 25 000 39 000
-3.0 -2.1 -1.5 -3.0 -2.8 -2.4 -3.3 -4.7 -4.6
4.7 4.7 4.7 5.5 6.0 5.0 7.0 8.0 6.5
0.144 0.126 0.132 0.122 0.129 0.133 0.123 0.113 0.113
2.47
1.04 1.25 1.44 1.73 2.05 2.41 2.90 3.43 3.67
1.339 1.341 1.344 1.350 1.350 1.356 1.359 1.358 1.363
2.41 2.48 2.35
a Data obtained at 22 °C. b SLS and DLS measurements were taken at a 90° scattering angle. c Measurements were taken using a 632.8 nm laser source.
Figure 6. Plot of the apparent hydrodynamic radius (Rh) vs micellar concentration (cm) for mixed BOG/octyl-POE micelles in various PEG solutions: 0% PEG (open circles), 6% PEG (filled triangles), 10% PEG (filled squares), and 12% PEG (open inverted triangles).
investigations. The refractive index and viscosity of the solvent (PEG solution without detergent) were measured at each temperature and used in the Rh calculation. Apparent Rh values increase with micelle concentration (Figure 6). Apparent Rh is independent of cPEG up to a cm of 20 mg/mL but shows a marked dependence upon cPEG at higher detergent concentrations. 3.4. Micellar Properties: Effects of Temperature and Detergent Concentration. Estimates of the micellar Mw were obtained at different temperatures and a fixed PEG concentration of 10% w/w, using Debye plots extrapolated to infinite dilution (Figure 7 and Table 3). In contrast to what is seen in the absence of PEG, apparent micelle Mw increases with decreasing temperature. In the presence of PEG, the apparent Rh values measured by DLS increase with increasing cm. The rate of this increase is temperature-dependent, with Rh increasing more rapidly at lower temperatures (Figure 8). Discussion Previous SLS experiments using the detergent-solubilized integral membrane protein OmpF porin have revealed that B22 values for PDCs fall rapidly as solution conditions are allowed to approach crystallization conditions. B22 values for protein-free detergent micelles displayed a similar trend, raising the intriguing pos-
Figure 7. Representative Debye plots from BOG/octyl-POE micelles in crystallization buffer containing 10% PEG, measured at five different temperatures: 10 (open circles), 22 (filled diamonds), 35 (open squares), 45 (filled triangles), and 55 °C (filled squares).
sibility that physical measurements of detergent properties in the absence of protein might be useful in predicting conditions that would be of general utility in crystallizing new membrane proteins. However, the mechanistic basis for the observed B22 behavior has not been clear. The measured drop in B22 near the crystallization boundary might represent an increase in micelle-micelle attractive forces, micelle growth/aggregation, or some combination of the two. Indeed, either effect can be rationalized as promoting crystal formation.17 To achieve a detailed mechanistic understanding of membrane protein crystal growth, we have attempted to distinguish the relative importance of these two effects. SLS alone is not able to address the issue of whether micelles are varying in size, since the micelle radius is significantly smaller than the wavelength of visible light. DLS reveals a change in apparent micelle hydrodynamic radius, but these measurements can be related to actual particle size only after careful correction for hydrodynamic and thermodynamic interactions between the micelles. To distinguish between attractive interactions and growth, SAXS was utilized to independently measure Rg. Initial SAXS experiments focused on detergent solutions containing no PEG. While such solutions do not display cloud point behavior, they do show significant changes in apparent Rh when detergent concentration is increased. When Rh values obtained from DLS were compared to Rg values obtained from SAXS, it became clear that the apparent changes in radius did not reflect
538
Crystal Growth & Design, Vol. 2, No. 6, 2002
Loll et al.
Table 3. Physical Parameters of BOG-Octyl POE Micelles in Crystallization Buffer Containing 10% PEG as a Function of Temperature temp (°C)
Mw ((2000 g/mol)
B22 × 10-4 ((1.0 cm3 mol g-2)
CMC ((1.0 mg/mL)
dn/dc ((0.002 mL/g)
Rh,0a ((0.05 nm)
η ((0.005 mPa s)
10 22 35 45 55
37 000 30 000 20 000 18 000 18 000
-2.9 -2.4 -1.7 -1.3 -1.5
5.7 5.0 4.5 4.7 3.4
0.133 0.133 0.133 0.133 0.133
2.40 2.47 2.43 2.49 2.51
3.45 2.41 1.78 1.44 1.15
a
Index 0 denotes values taken at infinite dilution.
Figure 8. Plot of the apparent hydrodynamic radius (Rh) vs micellar concentration (cm) obtained for mixed BOG/octyl-POE micelles in crystallization buffer containing 10% PEG. The temperatures are the same as in Figure 7: 10 (open circles), 22 (filled diamonds), 35 (open squares), 45 (filled triangles), and 55 °C (filled squares).
any actual significant change in micelle size and therefore reflected thermodynamic interactions. This implies that the measured B22 values are true reflections of micelle-micelle interaction forces and are not confounded by changes in micelle geometry. We then turned to solutions containing the precipitating agent PEG. PEG is required to grow crystals of OmpF and is also required to induce phase separation in the BOG/octyl-POE system; hence, the behavior of micelles in the presence of PEG is of interest when considering mechanisms by which PDCs can be crystallized. However, the presence of PEG molecules, which are roughly comparable to micelles in size, significantly complicates the analysis of the light scattering data.17 B22 values for BOG/octyl-POE micelles reflect mildly attractive micelle-micelle forces at low PEG concentrations; however, when the PEG concentration approaches the levels required to induce PDC crystallization or detergent phase separation, B22 values decrease significantly, indicating an increase in micelle-micelle attractive forces, as has previously been described.10 In the presence of PEG, apparent Rh for detergent micelles tends to increase with increasing detergent concentration, as is seen in the absence of PEG; however, this increase is exaggerated when PEG is present in the solution. Because this apparent change in Rh has been shown to be entirely due thermodynamic interactions in the absence of PEG and not to any change in size, we suggest that the same holds true in the presence of PEG and that the observed B22 values are accurate reflections of micelle-micelle interaction forces. However, the caveat must be borne in mind that conclusions about micelle size are obtained from comparison of Rh and Rg measurements at low micelle concentration; it
is not possible to use such an approach to measure actual micelle size at or near the cloud point, under conditions where PDCs actually form crystals. The BOG/octyl-POE system displays a lower consolute boundary in the presence of PEG, i.e., lowering the temperature can induce phase separation, while raising the temperature drives the system away from the cloud point. In the presence of PEG, micelle molecular weight decreases with increasing temperature, an effect which is not observed without PEG. Examination of micelle B22 reveals that it is essentially temperature-independent in the absence of PEG; however, when PEG is present, increasing temperature leads to an increase in micelle B22 values to levels significantly higher than that seen in pure detergent solutions. These increased B22 values would correspond to a decrease in micellemicelle attractive forces. This can be understood in terms of the free energy of the system. As temperature increases, the enthalpic contribution to the free energy from micelle-micelle interactions is reduced. To compensate, the entropic contribution increases, which is achieved by increasing the number of micelles in solution (thereby decreasing the average micelle size). Conclusions We have used SAXS in conjunction with SLS and DLS to characterize the detergent system used in the crystallization of the integral membrane protein OmpF porin. In the absence of the precipitating agent PEG, we have determined that detergent micelles do not undergo large changes in size as micelle concentration increases. Increases in the apparent hydrodynamic radius observed with increasing concentration may therefore be ascribed to micelle-micelle attractive forces, which cause the diffusion rate for micelles to be underestimated. A similar dependence of apparent hydrodynamic radius upon micelle concentration is observed in the presence of PEG, and we argue that this also reflects intermicellar attractions and is not likely to be due to changes in micelle size. A consequence of this conclusion is that B22 values measured by SLS accurately reflect micelle-micelle interaction forces and are not biased by changes in micelle size. These detergent B22 measurements may now be tested as a screening tool in the crystallization of integral membrane proteins. Acknowledgment. This work was supported by grants to P.J.L. and J.W. from NASA (NAG8-1350 and NAG8-1840). References (1) Wallin, E.; von Heijne, G. Protein Sci. 1998, 7, 1029-1038.
Micellar Interaction and Growth in Detergent Solutions (2) Tsukihara, T.; Aoyama, H. Curr. Opin. Struct. Biol. 2000, 10, 208-212. (3) Ostermeier, C.; Michel, H. Curr. Opin. Struct. Biol. 1997, 7, 697-701. (4) Zulauf, M. In Crystallization of Membrane Proteins; Michel, H., Ed.; CRC Press: Boca Raton, 1991; pp 53-72. (5) Zulauf, M.; Rosenbusch, J. P. J. Phys. Chem. 1983, 87, 856862. (6) Rosenbusch, J. P. J. Struct. Biol. 1990, 104, 134-138. (7) Garavito, R. M.; Picot, D. Methods: A Companion to Methods in Enzymology 1990, 1, 57-69. (8) Song, L.; Gouaux, J. E. Methods Enzymol. 1997, 276, 6074. (9) Garavito, R. M.; Rosenbusch, J. P. Methods Enzymol. 1986, 125, 309-328. (10) Hitscherich, C. J.; Kaplan, J.; Allaman, M.; Wiencek, J.; Loll, P. J. Protein Sci. 2000, 9, 1559-1566. (11) Finet, S.; Bonnete, F.; Frouin, J.; Provost, K.; Tardieu, A. Eur. Biophys. J. 1998, 27, 263-271. (12) Ruppert, S.; Sandler, S. I.; Lenhoff, A. M. Biotechnol. Prog. 2000, 17, 182-187. (13) Vivares, D.; Bonnete, F. Acta Crystallogr. 2002, D58, 472479. (14) George, A.; Chiang, Y.; Guo, B.; Arabshahi, A.; Cai, Z.; Wilson, W. W. Methods Enzymol. 1997, 276, 100-110. (15) Loll, P. J.; Allaman, M.; Wiencek, J. J. Cryst. Growth 2001, 232, 432-438. (16) Stigter, D.; Hill, T. L. J. Phys. Chem. 1959, 63, 551-556. (17) Hitscherich, C. J.; Aseyev, V.; Wiencek, J.; Loll, P. J. Acta Crystallogr. 2001, D57, 1020-1029.
Crystal Growth & Design, Vol. 2, No. 6, 2002 539 (18) Chu, B. Laser Light Scattering: Basic Principles and Practice; Academic Press: San Diego, 1991. (19) Shtan’ko, S. P.; Volkov, V. A.; Shakhparanov, M. I. Russ. J. Phys. Chem. 1987, 61, 1470-1471. (20) Kameyama, K.; Takagi, T. J. Colloid Interface Sci. 1990, 137, 1-10. (21) Kratky, O.; Laggner, P. Encyclopedia of Physical Science and Technology; Academic Press: San Diego, 1992; Vol. 17. (22) Burchard, W. In Laser Light Scattering in Biochemistry; Harding, S. E., Sattelle, D. B., Bloomfield, V. A., Eds.; Royal Society of Chemistry: Cambridge, 1992; pp 3-22. (23) Young, C. Y.; Missel, P. J.; Mazer, N. A.; Benedek, G. B. J. Phys. Chem. 1978, 82, 1375-1378. (24) Thomas, H. G.; Lomakin, A.; Blankschtein, D.; Benedek, G. B. Langmuir 1997, 13, 209-218. (25) Bogusz, S.; Venable, R. M.; Pastor, R. W. J. Phys. Chem. 2000, B104, 5462-5470. (26) Marone, P. A.; Thiyagarajan, P.; Wagner, A. M.; Tiede, D. M. J. Cryst. Growth 1999, 207, 214-225. (27) Nilsson, F.; Soderman, O.; Johansson, I. Langmuir 1996, 12, 902-908. (28) Zhang, R.; Marone, P. A.; Thiyagarajan, P.; Tiede, D. M. Langmuir 1999, 15, 7510-7519. (29) Zulauf, M. In Physics of Amphiphiles: Micelles, Vesicles, and Macromolecules; Degiorgio, V., Corti, M., Ed.; Elsevier: Amsterdam, 1985; pp 663-673.
CG025563W
