Crystallographic characterization of N-oxide tripod amphiphiles

Article abstract of DOI:10.1021/ja9085148

Tripod amphiphiles are designed to promote the solubilization and stabilization of intrinsic membrane proteins in aqueous solution; facilitation of crystallization is a long-range goal. Membrane proteins are subjects of extensive interest because of their critical biological roles, but proteins of this type can be difficult to study because of their low solubility in water. The nonionic detergents that are typically used to achieve solubility can have the unintended effect of causing protein denaturation. Tripod amphiphiles differ from conventional detergents in that the lipophilic segment contains a branchpoint, and previous work has shown that this unusual amphiphilic architecture can be advantageous relative to traditional detergent structures. Here, we report the crystal structures of several tripod amphiphiles that contain an N-oxide hydrophilic group. The data suggest that tripods can adapt themselves to a nonpolar surface by altering the hydrophobic appendage that projects toward that surface and their overall orientation relative to that surface. Although it is not possible to draw firm conclusions regarding amphiphile association in solution from crystallographic data, trends observed among the packing patterns reported here suggest design strategies to be implemented in future studies.

Full text of DOI:10.1021/ja9085148

Published on Web 01/22/2010
Crystallographic Characterization of N-Oxide Tripod
Amphiphiles
Pil Seok Chae, Ilia A. Guzei, and Samuel H. Gellman*
Department of Chemistry, UniVersity of Wisconsin, Madison, Wisconsin 53706
Received October 6, 2009; E-mail: gellman@chem.wisc.edu
Abstract: Tripod amphiphiles are designed to promote the solubilization and stabilization of intrinsic
membrane proteins in aqueous solution; facilitation of crystallization is a long-range goal. Membrane proteins
are subjects of extensive interest because of their critical biological roles, but proteins of this type can be
difficult to study because of their low solubility in water. The nonionic detergents that are typically used to
achieve solubility can have the unintended effect of causing protein denaturation. Tripod amphiphiles differ
from conventional detergents in that the lipophilic segment contains a branchpoint, and previous work has
shown that this unusual amphiphilic architecture can be advantageous relative to traditional detergent
structures. Here, we report the crystal structures of several tripod amphiphiles that contain an N-oxide
hydrophilic group. The data suggest that tripods can adapt themselves to a nonpolar surface by altering
the hydrophobic appendage that projects toward that surface and their overall orientation relative to that
surface. Although it is not possible to draw firm conclusions regarding amphiphile association in solution
from crystallographic data, trends observed among the packing patterns reported here suggest design
strategies to be implemented in future studies.
which are usually incorporated into the resulting crystals.6 The
amphiphiles are intended to cover the large hydrophobic patches
Introduction
Membrane proteins constitute a substantial fraction of the
human proteome, and many members of this protein class are
known to play crucial biological roles (e.g., receptors, ion
channels).1 Despite the prevalence and importance of membrane
proteins, however, structural and functional analysis of these
biomacromolecules is far less developed than for soluble
proteins.2 The traditional approach to protein characterization
begins with purification from a biological source, followed by
studies of solutions with controlled composition that contain
the protein and as little else as possible. One common goal is
to obtain high-resolution structural information, via NMR or
via crystallization followed by X-ray diffraction; crystal growth
usually requires homogeneous solutions of pure protein.3
Extending the traditional approach to membrane proteins is
challenging because these molecules have evolved to reside in
a lipid bilayer, which is a highly asymmetric environment, rather
than in aqueous solution.4 Extraction of intrinsic proteins from
biological membranes requires the use of small amphiphilic
molecules, generally detergents, as do all subsequent manipula-
tions of the protein sample.5 Even the crystallization of these
proteins is conducted in the presence of amphiphilic additives,
found on the surfaces of membrane proteins in their native states,
surfaces that are normally embedded in the lipophilic core of
the membrane.7
It can be quite demanding to identify an amphiphile that
effectively solubilizes a membrane protein of interest in a native-
like state, because the qualities that enable amphiphiles to cover
exposed lipophilic side chains on the protein also enable the
amphiphiles to interact favorably with lipophilic side chains that
would be buried in the core of the folded protein. Thus,
detergents often induce protein unfolding.5a,b,8 Extensive empiri-
cal study has identified a few guidelines for selecting “mild”
detergents; the most general guideline is avoidance of ionic
detergents (e.g., SDS, CTAB). Membrane protein researchers
generally favor detergents with uncharged polar headgroups,5d,9
popular examples of which include octyl glucoside (OG),5a
dodecyl maltoside (DDM),10 lauryldimethylamine oxide
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J. AM. CHEM. SOC. 2010, 132, 1953–1959 1953

A R T I C L E S
Chae et al.
Figure 1. Chemical structures of widely used detergents for membrane protein studies.
(LDAO),11 Triton X-100,12 and CHAPS13 (Figure 1). A
detergent that is effective for extraction of an intrinsic protein
from its membrane frequently turns out not to be ideal for
subsequent characterization; in these cases, a detergent exchange
procedure is employed after the initial isolation.14 Thus, for
example, Triton X-100 is often effective for disruption of a
biological membrane and concomitant “capture” of embedded
proteins in a native-like state, but this detergent has generally
not been amenable to crystallization.15 Therefore, a sample
solubilized with Triton X-100 can have the detergent component
exchanged for more crystallization-prone examples such as
DDM, OG, or LDAO, which may not have been capable of
extracting the protein from its native membrane.16
few efforts of this type have been reported.17 Such research
efforts can be difficult to pursue because they require both
expertise in the synthesis and characterization of organic
molecules and expertise in the biochemistry of membrane
proteins, skill sets that generally do not overlap.
Our interest in the design of new amphiphiles for solubiliza-
tion and ultimately crystallization of membrane proteins has
focused on a simple strategy for modulating conformational
mobility.18 The conventional detergents most commonly em-
ployed in successful membrane protein crystallization efforts,
DDM, OG, and LDAO, all contain linear alkyl groups, which
are highly flexible.15 Because membrane protein crystallization
remains notoriously difficult, even when these detergents are
used, we wondered whether the inherent flexibility of linear alkyl
groups works against formation of a crystalline lattice.18 The
rarity of membrane protein crystals grown in the presence of
CHAPS or other rigid amphiphiles suggests that some degree
of flexibility may be important in the amphiphile structure.
Therefore, we have designed, synthesized, and evaluated new
amphiphiles that contain at least one branchpoint.18 This feature,
a carbon atom bearing three or four non-hydrogen bonding
partners, limits conformational mobility about nearby single
Most biochemical detergents share a simple architecture: a
polar “headgroup” is placed at one end of a long alkyl “tail”
(as illustrated by DDM, OG, and LDAO). A few common
examples, such as Triton X-100 and CHAPS, deviate from this
structural pattern, and the widespread use of these atypical
detergents suggests that it might be fruitful to explore a broader
range of variations in amphiphilic architecture. To date, only a
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N-Oxide Tripod Amphiphiles
A R T I C L E S
Figure 2. Chemical structures of amphiphiles.
bonds19 but does not achieve the level of rigidity induced by a
multicyclic skeleton such as that found in CHAPS. Our approach
is illustrated by structure 1, which contains a quaternary carbon.
Because the quaternary carbon bears three hydrophobic ap-
pendages, along with a polar appendage, we refer to molecules
of this type as “tripod amphiphiles”. This design is modular;
the size and conformational features of each hydrophobic
appendage and of the hydrophilic appendage can be individually
varied to tune amphiphile behavior. Tripod amphiphiles can be
viewed as species that combine features of both conventional,
highly flexible detergents and “facial amphiphiles”, as identified
by Kahne et al.20
of 1 toward bR and Rho was shown to be superior to that of a
related conventional detergent (LDAO) and to that of a handful
of other tripod N-oxides. Tripod amphiphile 1 is now com-
mercially available under the name “Tripao”.
It would be valuable to have molecular-level understanding
regarding the manner in which detergents or other amphiphiles,
such as 1, associate with one another in solution and with
lipophilic surfaces of membrane proteins. Unfortunately, this
type of information is very difficult to acquire, given the
evanescent nature of micellar structures.22 Here, we report
crystal structures for 1, crystallized both from aqueous solution
and from organic solvents, and crystal structures of several other
tripod amphiphiles (2-6) as well as related dipod amphiphiles
(7,8) and an N-oxide that bears the hydrophobic segment of
Triton X-100 (which we designate “T-N-oxide”) (Figure 2).
Collectively, these structures offer insight on the ways in which
hydrophobic tripods prefer to pack against one another and a
basis for comparing tripod self-association with self-association
of more traditional hydrophobic moieties found in conventional
detergents. These structural data, necessarily acquired in the
highly ordered environment of a crystal, may not be directly
relevant to the loose associations that occur in aqueous solution;
nevertheless, trends in the packing data are suggestive with
regard to the ways in which tripod amphiphiles might interact
We have previously shown that 1 can extract bacteriorho-
dopsin (bR) from the “purple membrane” of Halobacterium
salinarum, and rhodopsin (Rho) from membrane preparations
derived from bovine retinas.18a,b The solubilized states of both
proteins are stable for several weeks.18b Both bR and a
potassium channel from Streptomyces liVidans have been
crystallized from solutions in which the protein is solubilized
by 1, although structures were not determined.18a,21 The behavior
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R.; Oesterhelt, D. Nature 1989, 340, 659–661. (b) Roth, M.; Arnoux,
B.; Ducruix, A.; Reiss-Husson, F. Biochemistry 1991, 30, 9403–9413.
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M.; Timmins, P. A. Structure 1995, 3, 1051–1059. (d) Penel, S.; Pebay-
Peroula, E.; Rosenbusch, J.; Rummel, G.; Schirmer, T.; Timmins, P. A.
Biochimie (Paris) 1998, 80, 543–551. (e) Belrhali, H.; Nollert, P.;
Royant, A.; Menzel, C.; Rosenbusch, J.; Landau, E. M.; Pebay-
Peyroula, E. Structure 1999, 7, 909–917. (f) Snijder, H. J.; Timmins,
P. A.; Kalk, K. H.; Dijkstra, B. W. J. Struct. Biol. 2003, 141, 122–
131. (g) Liu, Z.; Yan, H.; Wang, K.; Kuang, T.; Zhang, J.; Gui, L.;
An, X.; Chang, W. Nature 2004, 428, 287–292.
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1990, 31, 6717–6720. (b) Hoffmann, R. W. Angew. Chem., Int. Ed.
Engl. 1992, 31, 1124–1134. (c) Hoffmann, R. W.; Stahl, M.; Schopfer,
U.; Frenking, G. Chem.-Eur. J. 1998, 4, 559–566.
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Chem. Soc. 1992, 114, 7319–7320.
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Table 1. Thickness of the Nonpolar and Polar Layers in the
Crystal Structures of Amphiphiles
compound
nonpolar layer (Å)a
polar layer (Å)a
solventb
c
c
TDAO· 2H2O
water
18.12
4.66
1 · 2.5H2O
12.13
9.25
11.70
5.94
1.32
3.39
2.82
1.15
12.75
3.86
3.94
3.96
water
1 · 0.5H2O
DCM/Et2O
DCM/Et2O
DCM/Et2O
DCM/Et2O
DCM/Et2O
DCM/Et2O
DCM/Et2O
DCM/Et2O
DCM/Et2O
2
3
4
5
10.38
13.53
12.15
10.20
10.00
12.65
9.59
6 · 3H2O
7
8
T-N-oxide
17.7
a Determined with carbonyl carbon atom, except for TDAO. b Solvent
system used for crystal growth. c Determined with the nitrogen atom.
Figure 3. Packing patterns of TDAO (top; CSD code WURTIM) and T-N-
oxide (bottom) in the crystalline state. The parallelepipeds represent the
unit cells. The red lines represent the borders between polar and nonpolar
layers.
with hydrophobic surfaces in aqueous solution and may therefore
provide a useful basis for future designs.
Results and Discussion
The crystal structure of a conventional detergent bearing the
N-oxide headgroup, tetradecylamine oxide (TDAO), is available
in the Cambridge Structure Database23 (CSD code WURTIM)
and provides a useful point of reference for consideration of
the tripod amphiphile structures reported here. TDAO is a
slightly larger homologue of LDAO. The molecular packing in
this crystal (Figure 3, upper part) shows alternating layers of
polar moieties (the N-oxide groups) and nonpolar moieties (the
alkyl groups, which adopt a fully extended conformation). This
spatialarrangementmaximizespolar-polarandnonpolar-nonpolar
contactsandminimizespolar-nonpolarcontacts.Thepolar-nonpolar
layers arise because the molecules themselves are arranged in
sheets in which all molecules are parallel to one another, and
the molecules in adjacent sheets have antiparallel orientation
relative to one another. The nonpolar layer is formed by
interleaved alkyl groups from two layers of TDAO molecules.
The nonpolar layer thickness can be defined as the distance from
a nitrogen in one molecular sheet to the nearest nitrogen in the
other sheet; this distance is 18.1 Å in the crystal of TDAO.
Figure 3 (lower part) shows the packing pattern in crystals
of T-N-oxide grown from CH2Cl2/Et2O. The pattern of alternat-
ing polar and nonpolar layers is analogous to that observed for
TDAO. The polar group of T-N-oxide is identical to the polar
group featured in all of the tripod amphiphiles discussed below:
the N-oxide nitrogen is connected via a trimethylene unit to
the nitrogen of a secondary amide group. Within the polar layers
of crystalline T-N-oxide, each N-oxide oxygen forms an
intramolecular H-bond to the amide NH. Each nonpolar layer
in this crystal is formed by the tail-to-tail packing of two sheets
Figure 4. The lattices observed for tripod amphiphile 1 crystallized from
water (top) or from organic solvents (bottom). The parallelepipeds represent
the unit cells. In the upper image, the red lines represent the borders between
polar and nonpolar layers. In the lower image, alternating red and green
lines are used to represent the borders between polar and nonpolar layers,
because in this structure there are two independent molecules of 1 in the
lattice. Layer thicknesses given in Table 1 are based on separations between
red lines, or separations between green lines.
of T-N-oxide molecules. Defining a nonpolar layer thickness
requires one to identify a point of reference within the polar
portion of the amphiphile. For TDAO, the nitrogen atom was
an obvious point of reference, but for T-N-oxide (and the
amphiphiles discussed below), the choice is less clear. We use
the carbonyl carbon atom as the reference point for determining
nonpolar layer thicknesses for T-N-oxide and amphiphiles 1-8
(Table 1). The nonpolar layer thickness in crystalline T-N-oxide
is 17.7 Å, which is quite similar to that found for TDAO.
The tripod and dipod amphiphile crystal structures (Figures
4-6) display packing patterns that feature alternating polar and
nonpolar layers, as seen for detergents TDAO and T-N-oxide
(Figure 3). The nonpolar layers in the tripod and dipod
amphiphile crystals are formed by two sheets of molecules that
approach one another in tail-to-tail fashion. Within the tripod
or dipod segments, that is, the segments up to the carbonyl
(23) Allen, F. H. Acta Crystallogr. 2002, B58, 380–388.
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N-Oxide Tripod Amphiphiles
A R T I C L E S
Figure 5. Crystal structures of tripod amphiphiles 2 (top left), 3 (top right), 4 (middle left), 5 (middle right), and 6 (bottom left). The parallelepipeds
represent the unit cells. The red lines represent the borders between polar and nonpolar layers.
The polar segments common to 1-8 and T-N-oxide,
C(dO)CH2CH2CH2N(fO)(CH3)2, display considerable con-
formational variation. Presumably this variation arises at least
in part from the drive to satisfy H-bonding potential. In a few
cases, potential H-bonding groups include water molecules. One
of the crystalline forms of 1 was grown from aqueous solution
and contains 2.5 water molecules per amphiphile molecule. All
other tripod crystals were grown from CH2Cl2/Et2O, and most
do not contain any water molecules; however, the crystal of 1
grown from CH2Cl2/Et2O contains 0.5 water molecule per
amphiphile, and the crystal of 6 contains 3 water molecules
per amphiphile. In many cases, the trimethylene unit within the
polar segment contains a gauche torsion angle. Several different
H-bonding patterns are seen, including intramolecular N-O--
H-N (as in T-N-oxide), and intermolecular N-O--H-N, H2O--
H-N, and CdO--H-N. The variations in the polar group
conformation and polar group arrangement motivated us to use
the carbonyl carbon rather than the N-oxide nitrogen as the point
Figure 6. Crystal structures of dipod amphiphiles 7 (top) and 8 (bottom).
The parallelepipeds represent the unit cells. The red lines represent the
borders between polar and nonpolar layers.
of reference for determining nonpolar layer thicknesses in these
crystals (Table 1).
The packing of tripod segments within the nonpolar layers
in the crystals of tripod amphiphiles 1-6 appears to display a
carbon, the conformations observed appear to be those expected
common trend (Figures 4 and 5). In each case, two of the three
to have the lowest conformational energy. All CH2-CH2 bonds
that can adopt an anti torsion angle do so, and the cyclohexyl
units in 4-7 display chair conformations with substituents in
equatorial positions (Figures 5 and 6).
nonpolar groups attached to the quaternary carbon lie ap-
proximately parallel to the plane of the layer. These two groups
fill the space between neighbors within each sheet of molecules.
The third nonpolar group projects into the nonpolar layer, toward
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A R T I C L E S
Chae et al.
Table 2. bR Solubilization Efficiency of the Amphiphiles 1, 5, 7,
the other sheet of molecules that participates in this layer. The
orientation of this “projecting” nonpolar group relative to the
plane of the nonpolar layer varies from structure to structure.
In 2 and 4, the projecting nonpolar group (butyl in both cases)
is approximately perpendicular to the plane of the nonpolar
layer; this orientation is particularly noticeable for 2 (Figure
5). In most other cases, the projecting nonpolar group has an
oblique orientation relative to the plane of the nonpolar layer.
This projecting group is butyl for both crystal forms of 1, p-t-
butylphenyl for 3, cyclohexyl for 5, and methyl for 6.
In contrast to the seven structures for tripod amphiphiles, we
have only two for dipod amphiphiles (7 and 8; Figure 6). The
packing trend noted above seems to hold for 8 in that one phenyl
group lies approximately parallel to the plane of the nonpolar
layer, while the other phenyl group projects obliquely into the
nonpolar layer. In contrast, for 7 both of the cyclohexyl rings
seem to project obliquely, but at a shallow angle, into the
nonpolar layer.
It is impossible to draw firm conclusions regarding the ways
in which tripod amphiphiles self-associate in aqueous solution
based on the packing patterns observed in the crystals of 1-6,
or regarding the ways that these amphiphiles might arrange
themselves around nonpolar surfaces displayed by a membrane
protein; nevertheless, we believe that the trends observed in these
crystal structures offer a basis for speculation on these important
issues. The seven tripod amphiphile packing patterns display a
range of nonpolar larger thicknesses, from 9.3 to 13.5 Å. These
nonpolar layers are consistently thinner than those formed by
the detergent with a conventional amphiphilic architecture,
TDAO, or T-N-oxide. Consideration of the seven packing
patterns collectively suggests that the tripod architecture offers
flexibility in the way that a collection of these molecules might
adapt themselves to a nonpolar surface. The data suggest that
it might be possible to optimize such interactions by changing
the identities of the nonpolar units that are directed toward
neighboring amphiphiles and toward the surface and/or by
changing the “tilt” of individual tripod units. The tilt of a tripod
can be defined on the basis of the plane that contains the carbon
atoms from the three nonpolar units that are directly bonded to
the quaternary carbon, and the mean plane of the nonpolar
surface with which the tripod amphiphile interacts; the tilt is
the angle between these two planes. The ways in which a tripod
amphiphile layer accommodates itself to the hydrophobic surface
displayed by another amphiphile layer in these crystals may
bear some resemblance to the ways in which clusters of tripod
amphiphiles in solution accommodate themselves to hydropho-
bic surfaces displayed by membrane proteins.
and T-N-Oxide
1
5
7
T-N-oxide
CMC (mM)
4.4
8.8
0.32
87
4.5
9
0.31
4.9
9.8
0.33
70
1.1
8.9
0.33
50
concentration (mM)
concentration (%)
solubilization yield (%)
91
reported in Table 1. Thus, a nonpolar layer in this crystal of 1
could be defined by the leftmost red line and the second green
line from the left, and the corresponding polar layer could be
defined by the second green line from the left and the third red
line from the left. In this case, the nonpolar layer thickness is
6.96 Å and the polar layer thickness is 8.23 Å; this nonpolar
layer thickness would be by far the smallest among the structures
considered. The origin of the difference between the packing
of 1 in the two crystal forms is impossible to identify by
inspection, but perhaps one important factor is the angle at which
butyl groups project into the nonpolar layers. Overall, the
differences between the two packing patterns displayed by 1
suggest that variations in relative positioning of neighboring
tripod amphiphiles allow these molecules to accommodate
themselves in diverse ways to nonpolar surfaces.
As a complement to the crystallographic results presented
above, we examined the new amphiphiles for the ability to
extract bR from its native membrane environment. Many of the
new amphiphiles (2, 3, 4, and 6) were not sufficiently soluble
in aqueous buffer to support extraction studies; amphiphile 8
proved to be too soluble for this purpose (CMC ) 78 mM).
We used 1 as a positive control, because this tripod has
previously been shown to extract bR efficiently.18a,b Results are
summarized in Table 2. Critical micelle concentration (CMC)
values were determined for 5, 7, and T-N-oxide via fluorescent
dye solubilization, as previously described.24 Solubilization of
bR by 1, 5, or 7 was evaluated at an amphiphile concentration
of 2 × CMC; because the CMC values are quite similar for
these three compounds, the amphiphile concentrations in the
extraction solutions were comparable, in terms of molarity or
weight %. T-N-oxide was examined at an absolute concentration
similar to that of the other compounds, even though this
detergent has a somewhat lower CMC than does 1, 5, or 7.
The data in Table 2 show that tripod amphiphile 5 is
comparable to 1 in its ability to extract intact bR from the native
membrane. Dipod amphiphile 7 is less effective, and detergent
T-N-oxide displays a further decrease in efficacy. Previous work
has shown that the conventional detergent LDAO causes bR
denaturation upon attempted solubilization.18a
Comparison of the two forms of 1 seems to offer a hint of
the way in which adjustments proposed in the previous
paragraph might be realized. The form crystallized from water
has a nonpolar layer thickness of 12.1 Å, while the crystal from
organic solvents has a 9.3 Å nonpolar layer thickness. This range
of nonpolar thicknesses spans almost the entire range observed
among crystalline 1-6 (only 3, with a 13.5 Å nonpolar layer
thickness, lies outside this range). The packing pattern of 1 as
crystallized from organic solvents is more complex than the
other packing patterns presented here, because there are two
independent molecules of 1 in this crystal. This complexity
makes it more challenging to define the border between polar
and nonpolar layers in this form of 1 relative to the other
crystals. The lattice view in the lower part of Figure 4 suggests
that there is an alternative way to define the layer thicknesses
in this crystal, relative to the method used to generate the values
Conclusions
We have presented a set of crystal structures that show how
tripod amphiphiles assemble into a regular lattice. Trends
observed among these packing patterns are suggestive with
regard to the way such amphiphiles may associate with exposed
hydrophobic surfaces of membrane proteins. In particular, the
data raise the possibility that tripod amphiphiles can accom-
modate themselves to a nonpolar surface by altering their overall
tilt relative to that surface, and by varying which of the nonpolar
appendages is oriented toward the surface. These observations
help to identify new design strategies. For example, the ability
of tripod amphiphiles to solubilize membrane proteins in their
native states may be optimized when the three hydrophobic
(24) Chattopadhyay, A.; London, E. Anal. Biochem. 1984, 139, 408–412.
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1958 J. AM. CHEM. SOC. VOL. 132, NO. 6, 2010

N-Oxide Tripod Amphiphiles
A R T I C L E S
crystal of 1 from water, and Dr. D. Powell for solving this crystal
structure. We thank Dr. P. Laible of the Argonne National
Laboratory for providing purple membrane preparations.
appendages differ from one another, which could enhance the
ability of a tripod layer to adapt to local topological variations
in the protein surface. Examples explored to date have had at
least two identical hydrophobic appendages, and it will be
interesting to evaluate new tripod amphiphiles with more diverse
sets of hydrophobic groups.
Supporting Information Available: Synthetic protocols and
characterization data for new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. This research was supported by the NIH
(P01 GM75913). We thank Dr. D. T. McQuade for growing the
JA9085148
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J. AM. CHEM. SOC. VOL. 132, NO. 6, 2010 1959