Tripod amphiphiles for membrane protein manipulation
Pil Seok Chae,aPhilip D. Laible*band Samuel H. Gellman*a
Received 24th July 2009, Accepted 9th September 2009
First published as an Advance Article on the web 14th October 2009
Integral membrane proteins (IMPs) are crucial biological components, mediating the transfer of
material and information between cells and their environment. Many IMPs have proven to be
difficult to isolate and study. High-resolution structural information on this class of proteins is
limited, largely because of difficulties in generating soluble forms of such proteins that retain
native folding and activity, and difficulties in generating high-quality crystals from such
preparations. Isolated IMPs typically do not dissolve in aqueous solution, a property that arises
from the large patches of hydrophobic surface necessary for favorable interactions with the core
of a lipid bilayer. Detergents are generally required for IMP solubilization: hydrophobic segments
of detergent molecules cluster around and shield from water the hydrophobic protein surfaces.
The critical role played by detergents in membrane protein manipulation, and the fact that many
IMPs are recalcitrant to solubilization and/or crystallization with currently available detergents,
suggest that it should be valuable to explore new types of amphiphiles for these purposes. This
review constitutes a progress report on our long-term effort to develop a new class of organic
molecules, collectively designated ‘‘tripod amphiphiles,’’ that are intended as alternatives to
conventional detergents for membrane protein manipulation. One long-range goal of this research
is to identify new types of amphiphiles that facilitate IMP crystallization. This review should help
introduce an important biochemical need to organic chemists, and perhaps inspire new
approaches to the problem.
Integral membrane proteins (IMPs) account for about 25% of
all open reading frames in the genomes of living organisms and
play vital roles in diverse cellular processes, such as signal
transduction, transport, and cell recognition and communication.
More than 50% of drugs target IMPs.1Therefore, structural
and functional knowledge of IMPs is essential for fundamental
biological understanding as well as for rational drug design.
High-resolution structural data are available for o0.5% of
IMPs despite extensive effort; the few hundred IMP crystal
structures currently available stand in contrast to the tens of
thousands of structures that have been determined for soluble
proteins.2–4This scarcity of IMP structural information is
mainly attributable to the difficulty of manipulating IMPs. It
is often impossible to generate aqueous solutions of functional
IMPs without the assistance of low-molecular-weight amphiphilic
agents. Conventional detergents are widely used for this
purpose. IMPs must display large patches of hydrophobic
surface in order to reside in a membrane, and detergent
molecules apparently coat these hydrophobic surfaces, rendering
the protein compatible with an aqueous environment.5,6
However, conventional detergents are not compatible with
all IMPs.7,8Therefore, newtypes of amphiphile merit exploration
for the ability to maintain IMPs in a native-like state after
removal from the membrane.9–12Ultimately, such amphiphiles
might facilitate IMP crystallization.
Over the past two decades, a few research groups have
examined unusual synthetic amphiphiles for the ability
to stabilize IMPs in aqueous solution. Examples include
peptitergents,13amphiphilic polymers (amphipols),14–16hemi-
fluorinated amphiphiles,17,18lipopeptide detergents (LPDs),19
and deoxycholate-based facial amphiphiles.20,21Peptitergents
are peptides designed to be lipophilic on one side and hydro-
philic on the other upon folding to an a-helical conformation;
this design has its roots in earlier fundamental exploration of
amphiphilic secondary structures.22Amphipols are co-polymers
that have been shown to stabilize a number of IMPs after they
have been extracted from the membrane with conventional
detergents.23Some hemifluorinated amphiphiles were observed
of a few of IMPs, particularly delicate assemblies composed of
multiple subunits, such as cytochrome b6f complexes.17,18
LPDs, featuring an amphiphilic a-helix, have been reported
to keep several IMPs (e.g., bacteriorhodopsin, PagP, Lac
permease-cytochromeb562 fusion protein) soluble in water
while maintaining native structure.19However, few of these
novel amphiphiles are commercially available, and there is no
report of their use for IMP crystallization so far. Thus, there is
a continuing need for synthetically accessible amphiphiles that
can serve as tools for manipulating IMPs.
Organic chemists can readily envision new types of low-
molecular-weight amphiphiles and plan synthetic routes to
generate such molecules, but there have been relatively few
aDepartment of Chemistry, University of Wisconsin-Madison,
1101 University Avenue, Madison, WI 53706, USA.
E-mail: email@example.com; Fax: +1 608-265-4534;
Tel: +1 608-262-3303
bBiosciences Division Argonne National Laborotory, 9700 South Cass
Avenue, Argonne, IL 60439, USA. E-mail: firstname.lastname@example.org;
Fax: +1 630-252-3387
This journal is ? c The Royal Society of Chemistry 2010Mol. BioSyst., 2010, 6, 89–94 | 89
REVIEW www.rsc.org/molecularbiosystems | Molecular BioSystems
efforts of this type to date. In our view, this situation arises for
several reasons. (1) Very few organic chemists are aware of the
problem. (2) Specialized biochemical skills are required to
work with IMPs, and few, if any, organic chemistry groups
have the necessary skills. Therefore, pursuing the development
of new amphiphiles for use with IMPs requires long-term
collaborations. (3) Assessing structural and functional integrity
of an IMP in an amphiphile-solubilized state is often challenging.
(4) Each integral membrane protein has its own peculiarities as
a subject of experimental analysis, and expertise developed
with one particular IMP does not necessarily transfer directly
to the study of other IMPs. Most laboratories that explore
IMP structure and function are focused on just one or a few
examples. This situation makes it challenging to assess the
extent to which new agents for membrane protein manipulation
might have broad utility.24,25
2. Tripod amphiphile design
Classical detergents, as exemplified by n-dodecyl-b-D-maltoside
(DDM) and lauryl dimethylamine-N-oxide (LDAO) (Fig. 1),
have a relatively simple architecture in which a hydrophilic
‘‘head group’’ is attached to a long lipophilic ‘‘tail’’ (the latter
is usually a straight-chain alkyl group).12The classical detergent
motif is generalized in structure I of Fig. 2. A few widely used
detergents have different architectures (e.g., 3-[(3-cholamindo-
shown in Fig. 1). CHAPS is an instructive example. This
non-classical and commercially available detergent is widely used
in protein science. CHAPS has been used to promote or assist
crystallization of several soluble proteins, and occasionally
specific CHAPS molecules are observed in the resulting
structures.28–31To our knowledge, however, there is no example
of an integral membrane protein that has been crystallized from a
CHAPS-solubilized state.32One IMP, lactose permease LacY
from Escherichia coli, has been crystallized from a DDM-
solubilized state with the use of CHAPS as an additive; the
protein structure was determined from these crystals (3.5 A˚
resolution). In general, it remains an open question whether
amphiphiles with non-classical architectures can promote IMP
crystallization. In our view, this uncertainty arises at least in part
because relatively few non-classical amphiphiles have received
careful attention from membrane protein scientists.
Tripod amphiphile architecture,33–35illustrated in structures
II and III of Fig. 2, differs fundamentally from that of
classical detergents in that the tripods contain a branch point
within the hydrophobic portion (II) or within both the
hydrophobic and hydrophilic portions (III). A branch point,
particularly a quarternary center, provides a subtle restriction
on conformational mobility relative to linear molecular
fragments,36–38such as the n-alkyl tails common among
conventional detergents. (Of course, a steroidal structure, as
in CHAPS, has several branch points, but in this case
conformational restriction is more severe, resulting from the
cyclic constraints.) Our motivation for including branch points
was to try to decrease the flexibility that characterizes most
conventional detergents, which we believed might contribute
to the difficulty of maintaining membrane proteins in an
active state in solution and, perhaps, to the crystallization of
membrane protein–detergent complexes.
detergents, deoxycholate-based amphiphile, amphipol (A8-35; n E 80), and LPD).
Chemical structures of representative classical detergents (LDAO, DDM) and selected non-classical amphiphiles (CHAPS, tween
90 | Mol. BioSyst., 2010, 6, 89–94 This journal is ? c The Royal Society of Chemistry 2010
3. Synthetic approaches to tripod amphiphiles
Evaluation of new amphiphiles for solubilization and stabili-
zation of membrane proteins requires access to multi-gram
quantities of these compounds. Widespread use by biochemists
would require kilogram quantities. Conventional detergents
such as DDM, along with cholate derivatives such as CHAPS,
can be readily prepared in these amounts, but more exotic
amphiphiles such as LPDs and peptitergents cannot. Our
reduction of the tripod amphiphile design to practice has
been guided by the importance of synthetic accessibility.
The synthetic scheme summarized in Fig. 3 shows how a
short sequence of reactions gives rise to an intermediate
tripod-carboxylic acid33,34that can then be converted into a
variety of different tripod amphiphiles. We have focused on
uncharged polar moieties (N-oxide or carbohydrate) because
these polar groups are dominant among conventional
detergents employed for membrane protein manipulation.32
(Detergents with polar groups that bear a net charge, such as
SDS, tend to denature proteins.) The modularity of the
synthetic approach illustrated in Fig. 3 supports a broad
survey of structure–property relationships among tripod
amphiphiles. Fig. 3 shows variation of only the polar group,
but straightforward modifications of this route (e.g., use of a
different ketone starting material or a different Grignard
reagent in step b) allow alteration of the hydrophobic portion.
The tuning of hydrophile–lipophile balance (HLB)39,40and
conformational flexibility41enabled by this modularity should
be important in the search for optimal performance with
specific proteins. (HLB has been defined as 20 times the
molecular weight of the hydrophilic portion divided by the
molecular weight of the compound.39,40)
The ease with which tripod amphiphile structure may be
varied is important because it is unlikely that any single
version will be a ‘‘magic bullet’’ that succeeds with all or even
a large fraction of membrane proteins. The relationship
between conventional detergent HLB and efficacy in extraction
of IMPs from their native membranes illustrates this
point.42–46Detergents within a narrow range of HLB values
(12 to 15) display high efficiency and selectivity for the
extraction of human adenosine A3 receptor in a functional
state.47Comparable optimal HLB ranges have been found for
other IMPs from prokaryotic and eukaryotic sources. For
tripod amphiphiles (TPAs; II and III).
Schematic representation of conventional detergents (I) and
ethylene glycol, reflux; (d) EDC?HCl, HOBt, 3-(dimethylamino)-1-propylamine, DMF, then m-CPBA, CHCl3; (e) EDC?HCl, HOBt, serinol,
DMF; (f) AgOTf, 2,3,4,6-tetra-O-benzoyl-a-D-glucopyranosyl bromide, DCM, then NaOMe, MeOH; (e) EDC?HCl, HOBt, ethanolamine, DMF;
(h) 1,2-trans-peracetylated maltose, BF3?Et2O, DCM, then NaOMe, MeOH.
Modular synthesis of tripod amphiphiles: (a) CH2(CN)2, AcOH, NH4OAc, benzene, reflux; (b) PhMgBr, CuCN, THF, 0 1C; (c) KOH,
This journal is ? c The Royal Society of Chemistry 2010 Mol. BioSyst., 2010, 6, 89–94 | 91
instance, detergents with HLB E 13 successfully extracted
D-alanine carboxypeptidase from Bacillus subtilis cells.45A
similar value was found for protein extraction from mitochondrial
membrane from bovine heart.48It is notable that optimum
HLB values vary somewhat from one IMP to the next. For
instance, mitochondrial porin solubilization from bovine heart
was more effective with detergents that have a smaller HLB,
while total membrane protein solubilization from the same
source was better achieved with the detergents of higher
IMP solubilization with N-oxide tripod
Our initial tripod amphiphile studies focused on solubilization
of bacteriorhodopsin (bR) and bovine rhodopsin (Rho) from
their native membranes. These IMPs are attractive testbeds
because the native membranes are relatively easy to obtain,
and the quality of the extracted protein can be assessed via
simple optical spectroscopic measurements. bR occurs naturally
in a two-dimensional crystalline protein–lipid array (‘‘purple
membrane’’) in Halobacterium salinarum. The high degree of
order in the purple membrane assembly renders bR resistant
to solubilization by many detergents, and only a few classical
detergents (e.g., octyl glucoside, Triton X-100) have been
successful.49,50Triton X-100 is the most effective among
classical detergents, but 20 hours are required for complete
solubilization.51In contrast, more than 95% of the bR
was solubilized from purple membrane by N-oxide tripod
amphiphiles 1 and 4 within 30 min (Fig. 4).33,34In addition,
these two tripod amphiphiles proved to be effective for
solubilization of Rho, a G-protein coupled receptor, from
bovine rod outer segments.33,34
We were initially surprised by the success of N-oxides 1
and 4 for bR solubilization, because treatment of purple
membrane with LDAO (Fig. 1), a widely used conventional
N-oxide detergent, causes rapid bR denaturation, as indicated
by a loss of the characteristic purple color.52This contrast
enabled us to probe the functional importance of amphiphile
architecture. We prepared 5 and 6, isomers of tripod
amphiphile 4, in which the lipophilic portion is incrementally
transformed into a conventional detergent ‘‘tail’’. Both 5 and 6
behaved similarly to LDAO, causing rapid bR denaturation.33
This early result provided strong support for our hypothesis
that the tripod amphiphile architecture represents an advanta-
geous complement to conventional detergent architecture.
Comparisons among a small set of N-oxide tripod amphiphiles
revealed that 1 was somewhat superior to 4 in terms of
behavior with bR and Rho. Solubilized forms of each protein
could be readily purified (including removal of endogenous
lipids), and the resulting preparations were stable for several
weeks, as assessed by optical absorbance.34Stability of
solubilized membrane proteins on this time scale is essential
for crystallization efforts. Two proteins have been crystallized
from the 1-solubilized state, bR53and a form of the potassium
channel from Streptomyces lividans;33however, structure-
determination has not been carried out in either case.
IMP solubilization with glyco-tripod
The promising behavior of N-oxide TPAs with bR and Rho
encouraged us to expand the application of tripod amphiphiles
to more delicate systems. We focused on the transmembrane
protein superassembly formed by the light harvesting-I (LHI)
complex and the reaction center (RC) complex of the photo-
synthetic bacterium Rhodobacter capsulatus. This LHI–RC
superassembly has not been crystallized, but its architecture
is believed to be similar to that of the LHI–RC superassembly
from Rhodopseudomonas palustris, previously solved to 4.8 A˚
resolution,54and the LHI–RC superassemblies from other
purple non-sulfur bacteria.55–57The LHI–RC superassembly
represents a challenging system. To solubilize the superassembly
in a native form, an amphiphile should be mild enough to
preserve the tertiary structures of at least five different
component proteins as well as the quaternary association
of B30–40 protein molecules. The fragile nature of the
R. capsulatus LHI–RC superassembly may underlie the lack
of crystallographic analysis for this system. Unique spectral
signatures arising from cofactors enable one to detect the
native state of the LHI–RC superassembly and partially
denatured states; this capability is very useful for evaluation
of superassembly solubilization and stability over time.
Attempted solubilization of the LHI–RC superassembly
from R. capsulatus membranes with N-oxide tripod amphiphile
1 led to extensive disruption.35However, tripod amphiphile 2,
which has a branched diglucoside head group, could extract
the intact LHI–RC superassembly from the native membrane.
Tripod amphiphile 3 is very similar to 2, but there is no branch
point in the hydrophilic portion of 3. This subtle structural
difference leads to a substantial functionaldifference: considerable
degradation of LHI was observed when solubilization was
undertaken with 3. The contrast between 2 and 3 suggests that
a branch point in the hydrophilic portion can complement a
branch point in the hydrophobic portion in terms of membrane
protein stabilization in aqueous solution. Interestingly, this
functional distinction does not arise from HLB, which is quite
similar for the two tripod amphiphiles, nor is the distinction
reflected in critical micelle concentrations (CMC), which are
almost identical for 2 and 3 (3.6 and 4.0 mM). It should be
pointed out that 3, lacking a branch point in the hydrophilic
portion, is significantly less soluble than analogue 2.
amphiphiles 5 and 6, which have more classical architectures.
Chemical structures of tripod amphiphile 4 and isomeric
92 | Mol. BioSyst., 2010, 6, 89–94This journal is ? c The Royal Society of Chemistry 2010
We prepared a series of detergents (7a–e) bearing the branched
diglucoside head group found in 2 connected to a linear alkyl tail.
Among this series, the version with a 12-carbon tail (7c) was
most effective at extracting the LHI–RC superassembly from
R. capsulatus membranes. However, the extraction efficiency of
this detergent was much lower than that observed with tripod
amphiphile 2. This comparison seems to complement the
comparison of N-oxides 4–6 discussed above in suggesting that
the tripod architecture displays distinctive advantages in the
context of membrane protein solubilization and stabilization.
Interestingly, some conventional detergents feature branching in
the hydrophilic portion, as exemplified by the tween series
(Fig. 1). Among non-classical amphiphiles, amphipols and
LPDs (Fig. 1) can be viewed as containing branch points.
We could not compare 7c with the analogue bearing a maltose
(i.e., non-branched) head group because the latter was insoluble.
Thus, even in the context of a conventional hydrophobic tail,
behavior. Collectively, these observations raise the possibility
placements of internal branch points and manifesting useful
properties for IMP manipulation, remain to be discovered by
seems toconfer favorable
A recent comparison of 4120 commercially available
detergents in terms of LHI–RC superassembly solubilization
from R. capsulatus membranes indicates that DDM is one of
the most effective among conventional detergents.58This
demonstration of the value of DDM as a tool for membrane
protein manipulation complements results obtained from
studies with other IMPs, including Rho,59diacyl glycerol
kinase,60lactose permease,61and human apelin receptor.62
In this context, it is noteworthy that we found DDM to be
less effective than 2 for long-term stabilization of the
R. capsulatus LHI–RC superassembly in aqueous solution;35
this comparison highlights the promise of tripod amphiphiles
for membrane protein research.
The results summarized above suggest that a simple molecular
design strategy, summarized by cartoons II and III in Fig. 2,
represents a useful addition to the toolkit available for the
study of integral membrane proteins. Three tripod amphiphiles
have recently become commercially available, which should
enable a full-fledged test of their utility. In our view, the most
important conclusion to be drawn so far from this effort
involves branch points: their placement in either the hydro-
phobic portion or the hydrophilic portion can be productive,
and incorporation in both portions can be synergistic, with
regard to membrane protein solubilization and stabilization.
A molecular design that is conducive to facile variation
enables exploration of structure–property relationships. Com-
parisons among tripod amphiphiles having closely related
hydrophilic portions, as reported in our publications,34,35have
shown that performance is quite sensitive to these variations.
More recently we have explored the impact of variations in the
hydrophobic portions and observed profound effects on
function. The two TPAs shown in Fig. 5, related structurally
to 2, proved to be inferior to 2 for solubilization of the
LHI–RCsuperassembly from R.capsulatus(unpublishedresults).
These findings show that variations in the TPA hydrophobic
group can have a significant influence on performance.
The many integral membrane proteins that remain difficult
to isolate and purify, or that are refractory to crystallization,
constitute a strong impetus for the invention of new synthetic
amphiphiles. This work requires the creativity and skill set of
the organic chemist, which must be deployed in concert with
the techniques and insights of membrane protein biochemists.
In addition to solubilization, stabilization and ultimately
crystallization of membrane proteins, novel amphiphiles can
be applied to solution NMR spectroscopy63–65and might
support exciting new methods for study of membrane proteins,
such as mass spectrometry.66It is hoped that this review will
inspire more chemists to apply their imaginations to the
development of new types of amphiphilic agents for applications
in membrane protein science.
This work was supported by NIH grant P01 GM75913.
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