Molecular Effects of Anionic Surfactants on Lysozyme Precipitation

Molecular Effects of Anionic Surfactants on Lysozyme Precipitation and Crystallization O. D.

Velev,†

Y. H.

Pan,‡

E. W. Kaler, and A. M. Lenhoff*

Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716 Received April 23, 2004;

CRYSTAL GROWTH & DESIGN 2005 VOL. 5, NO. 1 351-359

Revised Manuscript Received July 15, 2004

ABSTRACT: Surfactants are used as additives in some protein separations and crystallization procedures, but the mechanism of their action is still poorly understood. By measuring the osmotic second virial coefficients of lysozyme solutions by static light scattering, we show that small amounts of anionic surfactants of varying molecular weight always make the protein-protein interactions in solution more attractive and that both the charge of the headgroup and the length of the hydrophobic tail mediate the interactions. The same surfactants also modify lysozyme crystallization and promote formation of twinned phases with gross morphologies different from those seen in the absence of surfactant, some of which display remarkably structured patterns on a micrometer scale. The surfactant effects are, however, often only kinetic, as the phases obtained initially recrystallize slowly into large stable crystals. These crystals are of excellent X-ray diffraction quality and resolution (up to 1.4 Å). Their symmetry, unit cell dimensions, crystal contacts, and protein backbone conformation are the same as those commonly observed for lysozyme, but sometimes occur at atypical pH values. The data suggest new techniques for modification of protein crystallization. Introduction Increasing the efficiency with which proteins can be crystallized and improving the quality of the crystals obtained are problems of major importance for structural biology and for industrial separations. A large variety of chemical agents are commonly added during protein crystallization to effect crystal growth.1 Such additives often include amphiphilic surfactant molecules, with hydrophilic groups that can either be nonionic (e.g., beta-octyl glucoside and polyoxyethylenebased surfactants) or ionic (e.g., decanoic and dodecanoic acid, sodium dodecyl sulfate, and cetyltrimethylammonium bromide). Surfactants may be present during protein crystallization even if they are not purposely added because amphiphilic molecules such as detergents and lipids may persist as residual impurities after protein purification. The effects of surfactants on protein crystallization are, however, still poorly characterized and understood. A few studies have demonstrated that the addition of nonionic surfactants can improve the quality of protein crystals.1-5 However, little insight is available about the molecular mechanisms by which the surfactants modify the crystallization process. In an earlier experimental study,6 we characterized by quantitative fluorescence microscopy the infusion of lysozyme crystals with pyrenebased fluorescent surfactants. The originally nonfluorescent protein crystals slowly become fluorescent due to the uptake of the surfactant into the crystal lattice, demonstrating that surfactants are adsorbed and accumulate in the protein crystals. However, it has not been determined whether the surfactant affects the dynamics of nucleation and/or growth, or the protein * Author for correspondence. Phone: 302-831-8989. E-mail: [email protected]. † Present address: Department of Chemical Engineering, North Carolina State University, Raleigh, NC 27695-7905. ‡ Present address: Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716.

packing parameters of the crystals. Other questions at the molecular level include whether the surfactant molecules are bound in specific locations and orientations within the crystal structure, or whether they are merely amorphously adsorbed. The effect of surfactants on crystallization is based on the interaction of the surfactant with the protein. Protein-surfactant interactions in solution have been studied and characterized in detail previously in several systems utilizing ionic surfactants. Surfactant molecules bind to proteins in solution, with the energy of that interaction found to be on the order of 10 kT in two independent studies.7,8 Coprecipitation can be expected when the surfactant/protein molar ratio becomes approximately equal to the net charge of the protein of sign opposite to that of the surfactant.9-11 Larger quantities of strong detergents may solubilize the protein molecules and cause their denaturation by unfolding of the protein.12,13 This paper reports characterization of hen egg lysozyme interactions and crystallization in the presence of small amounts of amphiphilic molecules with an anionic headgroup, with the goal of understanding how the type and size of the surfactant hydrophobic and hydrophilic groups affect protein crystallization. The experiments were carried out with two homologous series of common amphiphiles, aliphatic sulfates and carboxylic acids of hydrocarbon chain length varying from C6 to C12. The lower molecular weight amphiphiles used are generally considered to be “mild” and do not cause protein precipitation from solution (notably, most of these molecules do not exhibit detergency and the term “surfactants” is used here in the broader definition of amphiphilic molecules that combine hydrophilic and hydrophobic groups). The surfactant/protein molar ratios range from less than 1:1 to 10:1. Due to the low concentrations used, formation of free surfactant micelles is not expected in any of these systems, and most

10.1021/cg049852r CCC: $30.25 © 2005 American Chemical Society Published on Web 09/10/2004

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of the interactions involved are between pairs of independent molecules. The effects of surfactants on the protein-protein interactions in solution were estimated via the osmotic second virial coefficient measured by static light scattering (SLS). This parameter captures well the repulsive-attractive balance of the interactions between the protein molecules and can be used as a predictor for protein crystallization.14-18 The effects of the amphiphiles on the kinetics and morphology of crystal growth were followed, and the crystals obtained were characterized by X-ray diffraction to evaluate their quality and to resolve whether the surfactants become incorporated in the crystal lattice in an ordered fashion. Experimental Section Materials. Lysozyme was obtained from Sigma (6× crystallized, L-6876). The solutions for SLS were prepared with deionized water from a Millipore Milli-Q system. All solutions contained 0.3 M NaCl, 10 mg/L NaN3 (both from Sigma) and 3 × 10-4 M citrate buffer (citric acid from Fisher Scientific). The pH of the protein solutions was adjusted precisely to the experimental value, 6.5 or 10, by adding small amounts of 0.1 M HCl or NaOH while continuously stirring and measuring the pH. The pH of the batch electrolyte solutions used for diluting the protein samples was also adjusted in the same way and sustained by the small amount of buffer present. Sodium octanoate and sodium dodecyl sulfate were obtained from Sigma and all other surfactants from Aldrich. Special care was taken to minimize the presence of dust to obtain reliable SLS data, and to minimize the presence of external nuclei in the crystallization. All sample preparation procedures were carried out in a clean airflow bench (NuAir from Laminar Flow Products). The electrolyte solutions were filtered through 20 nm cutoff inorganic Anotop filters (Whatman). The protein solutions were prepared less than 2 h before the SLS measurements and were filtered through 100 nm Anotop filters and immediately diluted and sealed in ampules and vials. Methods. The SLS data were obtained using a Brookhaven BI9000 light-scattering apparatus, equipped with a laser of wavelength 488 nm. All measurements were performed at a scattering angle of 90° at 25 ( 0.1 °C. The absolute Rayleigh ratios of the samples were calculated by calibration with pure benzene before each experimental measurement. The details of the procedures used for data collection and processing are described in detail in ref 17. Lysozyme crystals were grown by a batch method in which the protein solutions were left for up to 12 months at 4 °C in plastic vials and glass ampules sealed with Teflon tape. The plastic vials were 1.5 mL polypropylene microcentrifuge tubes from Sigma, handled on the clean bench. The 2 mL glass ampules from Wheaton (NJ) were treated first with detergent, followed by Nochromix oxidizing solution, washed with deionized water, and dried in a clean environment before use. The morphology of the growing crystals was examined periodically by microscopy through the walls without opening the containers, to prevent contamination. The microscopy observations were carried out in regular and polarized transmitted light on a Nikon Optiphot-2. Water evaporation from the glass vials amounting to up to 50 vol % was observed over periods of several months. At the end of the experimental cycle, diffraction data were collected on some of the crystals at room temperature and -180 °C. The room temperature data were collected, for crystals with typical dimensions of 0.35-0.5 mm, on a Rigaku RU-200 (RAXIS-II) diffractometer with 0.3 µm collimation. The rotating anode was operated at 50 kV and 80 mA and a 1.5 degree oscillation was used for each orientation. The lowtemperature data were collected on a Rigaku RU-300 (RAXISIV) with a double focusing mirror system with 0.3 µm collimation. The rotating anode was operated at 50 kV and

Figure 1. A summary of the effect of small quantities of surfactants with increasing hydrocarbon tail length on the lysozyme virial coefficients in solution measured by SLS. All solutions are at a surfactant/protein molar ratio of 1:1, except for the last column (SDS). The error bar shown for one system is characteristic of all virial coefficient measurements reported here. 100 mA and one degree oscillation was used for each orientation. Most of the crystals selected for data collection were smaller than 0.3 mm to minimize mosaicity, and they were frozen in a cryoprotectant solution made up of 30% glycerol in mother liquor. Structural refinements of the data sets were carried out using the programs XPLOR 3.1 and CNS 0.9a. All reflections were used without amplitude or sigma cutoff. Refinements were carried out in several stages, starting from the lower resolution range and progressing to the high-resolution limit. A typical refinement cycle at higher resolution included energy minimization by the conjugate gradient method, and restrained individual b-factor refinement followed by simulated annealing at 3000 K. Visual inspection of molecular models was carried out on a SGI graphic workstation using Chain19 and O.20 Water molecules were added to the model at a later stage of refinement using both automatic water placement programs in XPLOR and by manual inspection. Waters with temperature factors greater than 50 Å2 were excluded from the model.

Results Static Light Scattering. The main purpose of the SLS measurements was to compare the effect on the virial coefficient of surfactants with two different headgroups and tails ranging from C8 to C12, all at a surfactant/protein molar ratio of 1:1. The experiments were performed at two different pH values, 6.5 and 10. As shown earlier,14-18 the virial coefficients of lysozyme solutions at both these pH values are negative over a range of ionic strengths and are within the “crystallization slot” associated with conditions conducive to crystallization.14,15 Our results, presented graphically in Figure 1, show that the surfactant-containing solutions always have lower virial coefficients than the pure protein solutions, indicating a shift to more attractive intermolecular potentials. At pH 10, for example, the alkyl sulfates gradually decrease the virial coefficient from -5.5 to -7.2 × 10-4 mol mL/g2 and then cause precipitation, as expected for highly attractive interactions below the crystallization slot.14,15 As sodium dodecyl sulfate is a strong detergent and causes immediate precipitation even at a molar ratio of 1:1, the data in the last column were collected at an SDS/lysozyme ratio

Anionic Surfactant Effects on Lysozyme Precipitation

Figure 2. Effect of the surfactant/protein molar ratio on the lysozyme virial coefficients measured for the case of (a) octanoic acid and (b) octyl sulfate. The virial coefficients are always in the more attractive region, but there is a specifically strong effect at certain low molecular ratios.

of 1:10, where no precipitation was observed. Dodecanoic acid has a very low solubility at pH 6.5, so no data could be collected under these conditions. The uniform decrease in the value of the virial coefficients can be attributed to the binding of the surfactant molecules and their consequent effects on protein-protein interactions. As a rule, the protein solutions at pH 10, where the protein has a smaller initial charge, have lower virial coefficients than those at pH 6.5, and since binding of the negatively charged surfactant would also offset the positive charge on the

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protein, the relative electrostatic contribution to the change in B22 is significant. Additional effects clearly play a role as well, though. First, comparison of the surfactants from the two homologous series, sulfates and carboxylic acids, shows that the systems with sulfates always have lower virial coefficients and start precipitating first. Second, the B22 values decrease monotonically with increasing hydrophobic tail length of the surfactants, until precipitation begins (Figure 1). The effect of surfactant concentration can be explored for the two “mildest” surfactants from the homologous series, which allow higher concentrations to be attained (Figure 2). As expected, in all systems the addition of surfactant leads to more attractive interactions, with especially strong effects below surfactant/protein molar ratios of 1:1 (sulfate) or 1:2 (carboxylate). The minima at intermediate ratios are larger than the characteristic errors in B22 (see error bar in Figure 1) and thus probably reflect real interactions. However, the origin of this effect is still unclear. It may arise, for instance, from formation of specific structures of two protein molecules bound by one or two intermediate surfactant molecules; this effect could be lost when more amphiphilic molecules become adsorbed on the protein and randomize the structure. All the data consistently demonstrate that, for these cases, protein-protein interactions in solution can be manipulated toward stronger attraction by adding selectively chosen surfactants. As shown below, this change in the interaction energy leads to significant changes in the crystallization pattern of lysozyme. Crystal Morphology and Its Evolution with Time. The slightly negative virial coefficients measured for the lysozyme solutions without additives are within the crystallization slot for proteins, and indeed in experiments without surfactants, crystals were observed after an initial nucleation period of a few weeks to a few months.17 The crystals grown at pH 6.5 were of the usual tetragonal symmetry (Figure 3a), but at pH 10 the crystal symmetry changed to orthorhombic (Figure 3b), which correlates with the significantly lower virial coefficients measured at that pH.17 No recrystallization or differences between different vials of a given sample were observed. In contrast, when surfactants were added to the samples a rich variety of crystal morphologies were

Figure 3. Microphotographs of lysozyme crystals without surfactant: (a) tetragonal crystals grown at pH 6.5; (b) orthorhombic crystals at pH 10. Scale bars ) 200 µm.

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Figure 4. Crystals with starlike morphology grown in the presence of alkyl sulfates: (a) bright field illumination of crystals grown at pH 6.5 in the presence of octyl sulfate; (b) microphotograph of the same sample in crossed polarizers shows that the structures, albeit twinned, are crystalline. Scale bars ) 200 µm.

Figure 5. Two modifications of the twinned crystals obtained in the presence of fatty acids at pH 10. (a) Single small crystals and ball-like twinned formations; (b) shapeless layered chunk from crystalline protein obtained with octanoic acid. Scale bar (a) ) 200 µm, (b) ) 100 µm.

observed. These formed relatively quickly and exhibited subsequent metamorphoses and recrystallizations. The first solid phase seen in many of the samples was a fine and visibly amorphous precipitate. Although no precipitation was observed during the initial hours in the course of the light-scattering experiments at 25 °C, small amounts of precipitate typically formed after the protein solutions were refrigerated overnight. Virtually no precipitate was observed in the samples at pH 6.5, except for a single case with the highest amount (10:1) of octanoic acid. Some degree of precipitation was observed in most of the samples at pH 10, with the largest amounts in the solutions with sulfates. A variety of crystals with different morphologies atypical for solutions without surfactants were observed in the subsequent few weeks. In most cases, these could be recognized as morphologically twinned crystals consisting of ingrown clusters of different orientation. The samples at pH 6.5 usually grew rhomboidal-shaped crystals such as the ones of tetragonal symmetry normally seen at this pH (Figure 3a), but in the samples with the highest concentration of octyl sulfate (3:1) the rhomboidal crystals were accompanied by a specific “starlike” twinned modification shown in Figure 4. A much larger variety of crystalline morphologies grew from the protein solutions originally precipitated at pH 10. Within two weeks, the precipitate recrystal-

lized into twinned crystals, the morphology of which depended on the chemical nature of the surfactant. Starlike twinned crystals similar in appearance to those in Figure 4 were exclusively observed in solutions of sulfates. Samples with higher molecular weight alkanoic acids demonstrated another specific “signature” in the growth of twinned phases: ball-like formations of large, clearly distinguishable, presumably tetragonal crystals (Figure 5a). These crystals then often grew in one specific direction, forming pillared stacks of crystals in the form of cylinders (Figure 6). The common morphology observed with the shorterchain octanoic and decanoic acids was large crystal-like faceted chunks, which usually did not display any specific gross shape or symmetry (Figure 5b). The protein phase in the chunks was, however, crystalline, as proven by the rotation of polarized light. A common feature of many of these chunks was the presence of lines of different optical density when viewed along one direction of observation, suggesting that the crystals represent a specific twinned morphology. These stripes suggest that the chunks are probably twinned in the form of stacks of crystalline layers and may be a product of a step bunching growth mechanism. In some samples of lower protein concentration, the layered chunks grew as remarkably symmetric crystals with fascinating periodically layered structures on a micrometer scale (Figure 7).



Figure 6. Bright-field (a) and dark-field (b) images of twinned crystals with “pillared” appearance obtained at pH 10 in the presence of dodecanoic acid. Scale bars ) 200 µm.

Figure 7. Highly symmetric structures slowly grown in the presence of octanoic acid at pH 10. (a) Large layered crystal; (b) close-up of the structure showing the regular striped pattern. Scale bar (a) ) 200 µm, (b) ) 50 µm.

Additional transformations were observed after the solutions were left unperturbed for periods ranging from a few weeks to a few months. A summary of the general path followed in the recrystallizing solutions is presented in Figure 8; it should be emphasized, however, that the course of recrystallization in any given sample appeared to follow a random path, with several different twinned phases frequently coexisting with the separated crystals. The starlike plates formed with sulfates usually recrystallized slowly into large chunky crystals similar to those observed with carboxylates (Figure 5b). More importantly, all of the samples evolved within 4-8 months into rhomboid- or needle-shaped crystals appearing identical to those seen under the same conditions in the absence of surfactants (Figure 3). The ultimate formation of either rhomboid or needlelike crystals at the final stage was invariant; although both crystal morphologies were observed, the plastic vials appeared to favor the needlelike modifications. In many samples, these highly symmetric crystals grew to sizes of a few millimeters. X-ray Characterization. Crystallographic data were collected from 14 crystals from different batch experiments, selected to sample a wide range of surfactant types, protein concentrations, and solution pH. The solution conditions represented are listed in Table 1, along with data collection and refinement statistics for these crystals. All the data sets were collected to a resolution of 1.4-2.0 Å. The rhomboidal and the layered chunky crystals were tetragonal, belonging to space

Figure 8. Schematic of the general scheme of the temporal evolution of the morphology of the lysozyme crystals obtained in the presence of small amounts of anionic surfactants.

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Table 1. Solution Conditions and Crystal Collection and Refinement Statistics Orthorhombic, Space Group P212121, Z ) 4 none C8 1:1 10 10

surfactanta surfactant/protein pH a (Å)

30.5b

Data Collection 30.8

b (Å)

57.9b

58.8

c (Å)

68.1b

68.2

diffraction limitc (Å)

1.60b

2.00

measured reflections

110955b

46043

unique reflections

16379b

8388

Rmerged (%)

3.9b

6.9

overall completeness (%)

99.9b

95.6

completeness (%, last shell)

99.8b

73.0

R value (last shell)

0.187b

0.144

resolution limit (Å) R value Rfree value reflections used waters

1.6 0.196 0.246 15700 168

C8 10:1 10

C10S 1:1 10

30.8 30.4b 59.1 57.7b 68.3 67.7b 2.00 1.40b 42638 155404b 8435 22219b 10.3 3.4b 96.2 91.8b 82.8 37.1b 0.288 0.274b

30.4b 57.7b 67.5b 1.40b 471765b 20153b 3.5b 92.7b 79.1b 0.269b

Refinement

surfactanta surfactant/protein pH

none

a ) b (Å)

78.1b

c (Å)

37.2b

resolution limitsc (Å)

1.70b

measured reflections

154088b

unique reflections

12998b

6.5

Rmerged (%)

4.7b

overall completeness (%)

98.8b

completeness (%, last shell)

99.9b

R value (last shell)

0.234b

resolution limit (Å) R value Rfree value reflections used waters

1.6 0.202 0.270 14590 180

2.0 0.198 0.256 7855 82

1.5 0.206 0.266 19106 168

Tetragonal, Space Group P43212, Z ) 8 C8 C8 C8S 1:1 10:1 1:1 6.5 6.5 6.5 Data Collection 78.9 77.8b 38.2 38.1 37.1b 1.90 1.90 1.75b 61036 69496 209845b 9356 9566 13052b 3.5 6.3 4.9b 96.4 98.6 99.6b 77.0 92.2 100b 0.130 0.134 0.279b 78.9

2.0 0.203 0.256 8040 69

Refinement 1.55 0.201 0.255 15037 139

1.5 0.178 0.267 18211 150

C10 1:1 10

C10S 1:1 6.5

C10S 1:1 10

77.0

77.3b

78.9

38.5

37.3b

38.0

1.55b

1.50b

2.00

305031b

205837b

49251

18179b

19587b

8406

3.7b

3.6b

6.4

97.7b

95.0b

98.1

92.7b

62.0b

88.7

0.248b

0.248b

0.203

78.8 77.2b 38.0 37.7b 2.07 1.40b 62485 224083b 8364 18726b 9.7 4.3b 97.6 81.2b 95.0 31.9b 0.277 0.219b

1.6 0.196 0.280 14762 152

1.4 0.198 0.251 19468 194

2.0 0.180 0.268 7188 89

1.5 0.178 0.267 18211 150

a C8, octanoic acid; C8S, octyl sulfate; C10, decanoic acid; C10S, decyl sulfate. b Data were collected at low temperature. c Resolution limit was determined so I/σ(I) > 2, R value < 0.300 in the last shell. d Rmerge ) ∑(|I - 〈I〉|)/∑I.

group P43212, while the needlelike crystals were orthorhombic with a space group of P212121. The structures were solved by molecular replacement using the atomic coordinates of hen egg lysozyme files 6LYT and 193L from the Protein Data Bank as the starting models for the tetragonal and orthorhombic form, respectively. All structural refinements of the data sets were carried to R values of about 20% with good geometry. The refined molecular structure of the lysozyme in all crystals was very similar to that of the initial PDB

models (193L or 6LYT); the RMS differences in all atoms and in the main chain atoms were no greater than 1.6 and 1.2 Å, respectively. When compared with the structures crystallized without surfactant, the RMS distances in all atoms were no greater than 1.4 Å in P43212 symmetry and 0.9 Å in P212121 symmetry. Difference Fourier maps, calculated using the structure factors obtained with and without the presence of the surfactant, i.e., Fo〈with surfactant〉 - Fo〈without surfactant〉, revealed no distinctive features that can be


attributed to ordered amphiphilic or solvent molecules. In summary, the lysozyme structures, as well as the water shell surrounding the protein, are very similar within each symmetry group for crystals obtained with or without surfactant, and no ordered surfactant molecules were found in any of the refined structures. There are, however, two notable features of the data regarding the effect of the surfactant. The first is the fairly high-resolution achieved with many of the surfactant-containing crystals. The resolution limit of 1.4 Å is better than the limit of 1.6-1.7 Å achieved in the absence of surfactant in this and in a previous study17 under the same procedures of batch crystallization. These high resolution data are comparable to the best results available in the PDB (most of which were obtained on crystals grown in space). Thus the presence of surfactant does not adversely impact the protein crystal quality measurably, and may actually enhance it, similarly to crystallization data obtained by others,1,4,5 although more carefully controlled experiments would be needed to confirm this. The second effect, which can be specifically attributed to the presence of the additives, is the perturbation of many of the samples at pH 10 to the tetragonal crystal form, contrary to previous experiments without surfactant, where only orthorhombic crystals formed at this pH.17 This randomly observed change, despite the absence of surfactant effects on the respective crystal structures, suggests that the two crystal forms are very similar in free energy, and could be inferred to be a direct result of surfactant interference with crystal nucleation and growth during the spontaneous recrystallization process. Discussion Due to the difference in the molecular weight of the protein and the surfactants used, the physical amount of surfactant in the solutions for the SLS and crystallization experiments is small, typically ranging from ca. 0.01 to 0.1 wt %; this may approach the level of amphiphilic impurities in the protein or in the crystallization media. The light scattering and crystallization data demonstrate that even this relatively small amount of surfactant can substantially change protein-protein interactions and may be responsible for changes in the protein crystallization pattern. Thus, the presence of surfactants or lipids in the crystallization media may be responsible in part for the crystal twinning and irregular growth sometimes observed in protein crystallization. However, our results also demonstrate beneficial effects of the surfactants, which can be used to control the crystallization process and enhance crystal quality. The virial coefficient data show that the anionic surfactants effectively shift the protein-protein interactions toward greater attraction. As the amphiphiles are oppositely charged to the net charge on the protein, one likely mechanism for the increased attraction is electrostatic, i.e., surfactant binding decreases the chargecharge repulsion among the protein molecules. The difference in the strength of action of the sulfates and carboxylates exemplifies the importance of the type of charged group. Similar effects of tighter electrostatic binding of protein to sulfates compared to carboxylates


have been observed previously in protein adsorption on chromatographic media with different surface groups21 and have been explained on the basis of hydration effects.22 The data also show, however, that the hydrophobic protein-surfactant interactions are also of primary importance in mediating the protein-protein interactions. One possible mechanism for this is hydrophobic anchoring of the surfactants on the protein, allowing better neutralization of the surface charges. An alternative explanation is that an amphiphile can bridge adjacent protein molecules by electrostatic binding to one of those molecules and hydrophobic adsorption on the other one. This could dampen energetically unfavorable conformations, such as when a strongly charged hydrophilic group on the surface of one of the proteins approaches a hydrophobic patch on the surface of the other one. Alternatively, hydrophobic interactions may occur between amphiphiles electrostatically anchored on different protein molecules. The additional attractive component induced via surfactant perturbation of the protein-protein interactions is the obvious reason for the protein precipitation observed and the wide variety of morphologies seen upon subsequent recrystallization. Our earlier data on the energy of ionic surfactant adsorption in protein crystals gave an estimate of 10-12 kT per surfactant molecule;6 this value is similar to literature values for adsorption of alkyl sulfates on proteins in solution.7,8 Such adsorption is strong enough that it could well interfere with protein crystal nucleation and growth. An intriguing aspect of the crystallization abnormalities observed here is that the morphologies of the twinned phases depend specifically on the surfactant type. Understanding the way in which this surfactant “signature” develops can provide interesting leads to explain and inhibit twinning in practical crystallization processes. The long-term observations of the metamorphoses of the protein phase reveal that the twinning effects observed are kinetic in origin and ultimately transient. The most favorable crystalline symmetry and basic cell dimensions, which the crystals ultimately adopt, correspond to those obtained without surfactant. The only effect of the surfactant at these long times is seen in the perturbation of the crystal form registered at pH 10, i.e., the random formation of both tetragonal and orthorhombic crystals. The fact that no ordered surfactant molecules are found in the crystal structures shows that the surfactant executes its kinetic effects via subtle interference with the pattern of the protein-protein interactions. A simple model can qualitatively explain the behavior observed in both light scattering and crystallization experiments. It has been well recognized23 that the discrete protein-protein interaction profile along a certain configuration coordinate (e.g., the angular orientation of the molecules) exhibits a series of minima and maxima corresponding to more attractive and repulsive configurations, respectively (Figure 9). Some of the deepest global minima may correspond to configurations found in the most extensive crystal contacts. As surfactant adsorption on the protein may dampen some of the unfavorable configurations, it could in

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Figure 9. Schematics of the concept of how surfactant binding changes the energy of protein interactions, leading to the formation of “mutated” crystals or precipitates.

general lead to more energetically favorable proteinprotein interactions (Figure 9), which is witnessed in the measured lower virial coefficient. On the other hand, it is very unlikely that adsorbed surfactant molecules will disrupt the crystal contacts, which involve highly complementary configurations of pairs of oppositely charged groups and/or hydrophobic patches. The energy gained upon formation of some of these contacts in the crystallization of lysozyme has been estimated as >20 kT.24 The adsorption of a surfactant molecule within these contacts will not be energetically favorable, as it will lead to an energy gain of only 10-12 kT as opposed to the loss of >20 kT. Thus, the long-term crystalline structure will not be influenced by the surfactant adsorption, although the discrete crystallization and recrystallization routes by which it will be reached can be modified by the additives. The small changes in the energy vs configuration pattern caused by surfactant adsorption are the possible reason for formation of various twinned phases observed at pH 10. This, we believe, is the mechanism of the “mild” surfactant action studied in this paper. On the other hand, “hard” surfactants, such as larger quantities of sodium dodecyl sulfate and other strong detergents,

adsorb strongly and form precipitates with long-term kinetic stability by profoundly modifying the proteinprotein interaction pattern and possibly the protein structure and eventually forming hemi-micelles (Figure 9, bottom panel). We cannot conclude at this stage that the surfactants per se enhance the growth of high-quality protein crystals. Instead, one of the most likely reasons for the formation of the large and high-resolution crystals appears to be the ability of the surfactants to promote recrystallization of the phases formed. The surfactant causes the rapid appearance of precipitates and lowquality twinned crystals. This decreases the protein concentration in solution, so the next generation of crystals will subsequently grow slowly under conditions of low and constant supersaturation, which is known to be the best route for the formation of highly ordered crystals.23 This hypothesis again points to the kinetic nature of the surfactant effects in protein crystallization. Understanding the mechanism of the surfactantprotein interactions on the molecular scale may help to improve protein separation processes of practical importance. Characterizing the correlation between surfactant chemistry and its relation to a specific type


of crystal twinning may assist in identifying undesirable contaminants during protein crystallization. On the other hand, surfactants may help in processes where rapid crystallization is required, or when time permits waiting until high quality crystals grow via recrystallization. Use of surfactants in protein separations may expand well beyond crystallization. Protein mixtures can be controllably separated by selective precipitation with surfactants (manuscript in preparation), and the high symmetry and the spontaneous organization of the twinned structures into micrometer-sized layers (Figure 7) can potentially also be of use in new materials synthesis. Conclusions The surfactants studied strongly affect the pattern of protein interactions and crystallization. The virial coefficient data show that they make the proteins more “sticky”, leading to additional attraction and a propensity to precipitate. The surfactants mediate the attractive interactions between the proteins via both electrostatic and hydrophobic contributions to the interaction energy. The results reported here may be relevant to the widely observed and generally undesirable crystal twinning. Twinning may originate from adsorption on the protein of surfactant molecules that disrupt growth of the lattice. An important conclusion of this study is that the effect of the surfactant on the protein crystallization process is kinetic and not thermodynamic. The initial precipitation and twinning are highly disruptive and undesirable effects of the surfactant, but the crystals ultimately grown are essentially indistinguishable in their lattice parameters and molecular conformation from ones obtained without additives. Moreover, crystals grown in the presence of surfactant were large and diffracted to high resolution. Thus, while the uncontrolled presence of detergents and amphiphiles in protein crystallization solutions should be avoided, the addition of some “mild” anionic surfactants may aid crystallization without impairing, and possibly even improving, crystal quality. Not surprisingly, some surfactants have already been successfully used as additives under empirically determined experimental conditions.


Acknowledgment. This study was supported by grants from NASA (NAG8-1346) and NSF (BES9510420 and BES-0078844). References (1) Sauter, C.; Ng, J. D.; Lorber, B.; Keith, G.; Brion, P.; Hosseini, M. W.; Lehn, J. M.; Giege, R. J. Cryst. Growth 1999, 196, 365-376. (2) McPherson, A.; Koszelak, S.; Axelrod, H.; Day, J.; Robinson, L.; McGrath, M.; Williams, R.; Cascio, D. J. Biol. Chem. 1986, 261, 1969-1975. (3) McPherson, A.; Koszelak, S.; Axelrod, H.; Day, J.; Robinson, L.; McGrath, M.; Williams, R.; Cascio, D. J. Cryst. Growth 1986, 76, 547-553. (4) Mustafa, A. O.; Derrick, J. P.; Tiddy, G. J. T.; Ford, R. C. Acta Crystallogr. 1998, D54, 154-158. (5) Guan, R. J.; Wang, M.; Liu, X. Q.; Wang, D. C. J. Cryst. Growth 2001, 231, 273-279. (6) Velev, O. D.; Kaler, E. W.; Lenhoff, A. M.; J. Phys. Chem. B. 2000, 104, 9267-9275. (7) Jones, M. N.; Manley, P., J. Chem. Soc., Faraday Trans., I 1979, 75, 1736-1744. (8) Jones, M. N.; Manley, P., J. Chem. Soc., Faraday Trans., I 1980, 76, 654-664. (9) Fukushima, K., Murata, Y., Nishikido, N., Sugihara, G.; Tanaka M. Bull. Chem. Soc. Jpn. 1981, 54, 3122-3127. (10) Moren A. K.; Khan A. Langmuir 1995, 11, 3636-3643. (11) Moren A. K.; Khan A. Langmuir 1998, 14, 6818-6826. (12) Turro, N. J.; Lei, X.-G.; Ananthapadmanabhan, K. P.; Aronson, M. Langmuir 1995, 11, 2525-2533. (13) Valstar, A.; Almgren, M.; Brown, W.; Vasilescu, M. Langmuir 2000, 16, 922-927. (14) George, A.; Wilson, W. W. Acta Crystallogr. 1994, D50, 361365. (15) George, A.; Chiang, Y.; Guo, B.; Arabshahi, A.; Cai, Z.; Wilson, W. W. Methods Enzymol. 1997, 276, 100-110. (16) Muschol, M.; Rosenberger. F. J. Chem. Phys. 1995, 107, 1953-1962. (17) Velev, O. D.; Kaler, E. W.; Lenhoff, A. M. Biophys. J. 1998, 75, 2682-2697. (18) Neal, B. L.; Asthagiri, D.; Velev, O. D.; Lenhoff, A. M.; Kaler, E. W. J. Cryst. Growth 1998, 196, 377-387. (19) Sack, J. S. J. Mol. Graphics 1988 6, 224-225. (20) Jones, T. A.; Zou, J. Y.; Cowan, S. W.; Kjeldgaard, M. Acta Crystallogr. 1991, A47, 110-119. (21) DePhillips, P.; Lenhoff, A. M. J. Chromatogr. A 2001, 933, 57-72. (22) Asthagiri, D.; Schure, M. R.; Lenhoff, A. M. J. Phys. Chem. B 2000, 104, 8753-8761. (23) Durbin, S. D.; Feher. G. Annu. Rev. Phys. Chem. 1996, 47, 171-204. (24) Asthagiri, D.; Neal, B. L.; Lenhoff, A. M. Biophys. Chem. 1999, 78, 219-231.

CG049852R

Molecular Effects of Anionic Surfactants on Lysozyme Precipitation

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