CRYSTAL

GROWTH
Molecular Effects of Anionic Surfactants on Lysozyme & DESIGN
Precipitation and Crystallization
2005
O. D. Velev, Y. H. Pan, E. W. Kaler, and A. M. Lenhoff* VOL. 5, NO. 1
Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716 351-359
Received April 23, 2004; 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 ). 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 packing parameters of the crystals. Other questions at

Increasing the efficiency with which proteins can be
crystallized and improving the quality of the crystals
obtained are problems of major importance for struc-
tural 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 mol-
ecules, with hydrophilic groups that can either be
nonionic (e.g., beta-octyl glucoside and polyoxyethylene-
based surfactants) or ionic (e.g., decanoic and dodecanoic
acid, sodium dodecyl sulfate, and cetyltrimethylammo-
nium 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 pyrene-
based fluorescent surfactants. The originally nonfluo-
rescent protein crystals slowly become fluorescent due
to the uptake of the surfactant into the crystal lattice,
demonstrating that surfactants are adsorbed and ac-
cumulate 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:
lenhoff@che.udel.edu.
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.
10.1021/cg049852r CCC: $30.25 2005 American Chemical Society
Published on Web 09/10/2004
the molecular level include whether the surfactant
molecules are bound in specific locations and orienta-
tions 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 ap-
proximately 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 ra-
tios range from less than 1:1 to 10:1. Due to the low
concentrations used, formation of free surfactant mi-
celles is not expected in any of these systems, and most
352 Crystal Growth & Design, Vol. 5, No. 1, 2005
of the interactions involved are between pairs of inde-
pendent molecules.
The effects of surfactants on the protein-protein
interactions in solution were estimated via the osmotic
second virial coefficient measured by static light scat-
tering (SLS). This parameter captures well the repul-
sive-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 am-
phiphiles 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 crystal-
lized, 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 measure-
ments 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 deion-
ized 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 contain-
ers, 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 m collimation. The
rotating anode was operated at 50 kV and 80 mA and a 1.5
degree oscillation was used for each orientation. The low-
temperature data were collected on a Rigaku RU-300 (RAXIS-
IV) with a double focusing mirror system with 0.3 m
collimation. The rotating anode was operated at 50 kV and
Velev et al.
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 orienta-
tion. 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 re-
strained 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 2 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 head-
groups 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 crystalliza-
tion slot associated with conditions conducive to crys-
tallization.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 interac-
tions below the crystallization slot.14,15 As sodium do-
decyl 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
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 m.
Crystal Growth & Design, Vol. 5, No. 1, 2005 353
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 am-
phiphilic 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
354 Crystal Growth & Design, Vol. 5, No. 1, 2005 Velev et al.

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 m.

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 m, (b) ) 100 m.

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 pre-
cipitation 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 con-
sisting 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 shorter-
chain 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).
Anionic Surfactant Effects on Lysozyme Precipitation Crystal Growth & Design, Vol. 5, No. 1, 2005 355

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 m.

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 m, (b) ) 50 m.
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 pre-
sented 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 usu-
ally 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 ap-
pearing identical to those seen under the same condi-
tions 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 experi-
ments, 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 Figure 8. Schematic of the general scheme of the temporal
resolution of 1.4-2.0 . The rhomboidal and the layered evolution of the morphology of the lysozyme crystals obtained
chunky crystals were tetragonal, belonging to space in the presence of small amounts of anionic surfactants.
356 Crystal Growth & Design, Vol. 5, No. 1, 2005 Velev et al.

Table 1. Solution Conditions and Crystal Collection and Refinement Statistics

Orthorhombic, Space Group P212121, Z ) 4
surfactanta none C8 C8 C10S
surfactant/protein 1:1 10:1 1:1
pH 10 10 10 10
Data Collection
a () 30.5b 30.8 30.8 30.4b
30.4b
b () 57.9b 58.8 59.1 57.7b
57.7b
c () 68.1b 68.2 68.3 67.5b
67.7b
diffraction limitc () 1.60b 2.00 2.00 1.40b
1.40b
measured reflections 110955b 46043 42638 471765b
155404b
unique reflections 16379b 8388 8435 20153b
22219b
Rmerged (%) 3.9b 6.9 10.3 3.5b
3.4b
overall completeness (%) 99.9b 95.6 96.2 92.7b
91.8b
completeness (%, last shell) 99.8b 73.0 82.8 79.1b
37.1b
R value (last shell) 0.187b 0.144 0.288 0.269b
0.274b
Refinement
resolution limit () 1.6 2.0 1.5 1.5
R value 0.196 0.198 0.206 0.178
Rfree value 0.246 0.256 0.266 0.267
reflections used 15700 7855 19106 18211
waters 168 82 168 150
Tetragonal, Space Group P43212, Z ) 8
surfactanta none C8 C8 C8S C10 C10S C10S
surfactant/protein 1:1 10:1 1:1 1:1 1:1 1:1
pH 6.5 6.5 6.5 6.5 10 6.5 10
Data Collection
a ) b () 78.1b 78.9 78.9 77.0 77.3b 78.9 78.8
77.8b 77.2b
c () 37.2b 38.2 38.1 38.5 37.3b 38.0 38.0
37.1b 37.7b
resolution limitsc () 1.70b 1.90 1.90 1.55b 1.50b 2.00 2.07
1.75b 1.40b
measured reflections 154088b 61036 69496 305031b 205837b 49251 62485
209845b 224083b
unique reflections 12998b 9356 9566 18179b 19587b 8406 8364
13052b 18726b
Rmerged (%) 4.7b 3.5 6.3 3.7b 3.6b 6.4 9.7
4.9b 4.3b
overall completeness (%) 98.8b 96.4 98.6 97.7b 95.0b 98.1 97.6
99.6b 81.2b
completeness (%, last shell) 99.9b 77.0 92.2 92.7b 62.0b 88.7 95.0
100b 31.9b
R value (last shell) 0.234b 0.130 0.134 0.248b 0.248b 0.203 0.277
0.279b 0.219b
Refinement
resolution limit () 1.6 2.0 1.55 1.6 1.4 2.0 1.5
R value 0.202 0.203 0.201 0.196 0.198 0.180 0.178
Rfree value 0.270 0.256 0.255 0.280 0.251 0.268 0.267
reflections used 14590 8040 15037 14762 19468 7188 18211
waters 180 69 139 152 194 89 150
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/(I) > 2, R value < 0.300 in the last shell. d Rmerge ) (|I - I|)/I.
group P43212, while the needlelike crystals were ortho-
rhombic 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 , respectively. When compared with the
structures crystallized without surfactant, the RMS
distances in all atoms were no greater than 1.4 in
P43212 symmetry and 0.9 in P212121 symmetry.
Difference Fourier maps, calculated using the structure
factors obtained with and without the presence of the
surfactant, i.e., Fowith surfactant - Fowithout sur-
factant, revealed no distinctive features that can be
Anionic Surfactant Effects on Lysozyme Precipitation
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 mol-
ecules 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 sur-
factant-containing crystals. The resolution limit of 1.4
is better than the limit of 1.6-1.7 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 oth-
ers,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 ran-
domly observed change, despite the absence of surfac-
tant 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 pro-
cess.
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 crystal-
lization 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 crystal-
lization 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 crystal-
lization. However, our results also demonstrate bene-
ficial 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 interac-
tions toward greater attraction. As the amphiphiles are
oppositely charged to the net charge on the protein, one
likely mechanism for the increased attraction is elec-
trostatic, i.e., surfactant binding decreases the charge-
charge 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
Crystal Growth & Design, Vol. 5, No. 1, 2005 357
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 interac-
tions. One possible mechanism for this is hydrophobic
anchoring of the surfactants on the protein, allowing
better neutralization of the surface charges. An alterna-
tive 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 unfavor-
able 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 interac-
tions 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 sig-
nature develops can provide interesting leads to explain
and inhibit twinning in practical crystallization pro-
cesses.
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, cor-
respond 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 surfac-
tant 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 ori-
entation 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 con-
figurations found in the most extensive crystal contacts.
As surfactant adsorption on the protein may dampen
some of the unfavorable configurations, it could in
358 Crystal Growth & Design, Vol. 5, No. 1, 2005
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 protein-
protein 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,
Velev et al.
adsorb strongly and form precipitates with long-term
kinetic stability by profoundly modifying the protein-
protein 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 low-
quality 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 surfactant-
protein interactions on the molecular scale may help to
improve protein separation processes of practical im-
portance. Characterizing the correlation between sur-
factant chemistry and its relation to a specific type
Anionic Surfactant Effects on Lysozyme Precipitation
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 recrystal-
lization. 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 propen-
sity to precipitate. The surfactants mediate the attrac-
tive interactions between the proteins via both electro-
static and hydrophobic contributions to the interaction
energy.
The results reported here may be relevant to the
widely observed and generally undesirable crystal twin-
ning. 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 uncon-
trolled 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 sur-
factants have already been successfully used as addi-
tives under empirically determined experimental con-
ditions.
Crystal Growth & Design, Vol. 5, No. 1, 2005 359
Acknowledgment. This study was supported by
grants from NASA (NAG8-1346) and NSF (BES-
9510420 and BES-0078844).
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