Tandem Facial Amphiphiles for Membrane Protein Stabilization
Pil Seok Chae,†Kamil Gotfryd,‡Jennifer Pacyna,§Larry J. W. Miercke,|Søren G. F. Rasmussen,⊥
Rebecca A. Robbins,|Rohini R. Rana,§Claus J. Loland,‡Brian Kobilka,*,⊥Robert Stroud,*,|
Bernadette Byrne,*,§Ulrik Gether,*,‡and Samuel H. Gellman*,†
Department of Chemistry, UniVersity of Wisconsin, Madison, Wisconsin 53706, United States, Department of
Neuroscience and Pharmacology, The Faculty of Health Sciences, UniVersity of Copenhagen,
DK-2200 Copenhagen, Denmark, Department of Life Sciences, Imperial College London, London, SW7 2AZ, U.K.,
Department of Biochemistry and Biophysics, UniVersity of California, San Francisco, California 94158, United States,
and Molecular and Cellular Physiology, Stanford UniVersity, Stanford, California 94305, United States
Received August 13, 2010; E-mail: email@example.com; firstname.lastname@example.org; email@example.com;
Abstract: We describe a new type of synthetic amphiphile that
is intended to support biochemical characterization of intrinsic
membrane proteins. Members of this new family displayed
favorable behavior with four of five membrane proteins tested,
and these amphiphiles formed relatively small micelles.
Membrane proteins (MPs) play crucial roles in biology, but these
proteins are difficult to handle and analyze because of their physical
properties.1The native conformations of MPs display extensive
nonpolar surfaces, which is necessary for residence in a lipid bilayer
but leads to denaturation and/or aggregation in an aqueous medium.
Detergents, such as dodecyl-?-D-maltoside (DDM), are typically
employed to render MPs soluble by coating nonpolar protein
surfaces.2However, not all MPs can be maintained in native-like
conformations when solubilized with conventional detergents.3
Moreover, even when a native conformation can be achieved, the
MP-detergent complex may manifest unfavorable properties with
regard to structural analysis (inability to crystallize and/or too large
for NMR). Since our understanding of membrane protein structure
and function remains poorly developed relative to understanding
of soluble proteins, there is a persistent need for new amphiphilic
“assistants” that can promote solubilization and manipulation of
Several groups have reported creative implementations of the
“facial amphiphile” concept for the design of novel agents that
display favorable behavior with selected membrane proteins.5
McGregor et al., for example, have reported lipopeptides that are
intended to match the width of a lipid bilayer and to form a sheath
around nonpolar surfaces of MPs.5cZhang et al. have developed
cholate-based amphiphiles in which hydrophilic maltose units
project from one side of the rigid and hydrophobic steroidal
skeleton.5dHere we disclose the design of “tandem facial am-
phiphiles” (TFAs), which contain a pair of maltose-functionalized
deoxycholate units. Unlike previous cholate-based designs, the
TFAs are long enough to match bilayer width,6and unlike
lipopeptides, the TFAs are readily synthesized in large quantities.
We show that one TFA forms micelles containing only six
molecules and that simple TFAs can be used to maintain a variety
of MPs in native-like states in aqueous solution.
A set of four TFAs was generated from a deoxycholate-bis-
maltoside building block via linkage with a diaminopropane unit
(Figure 1). Molecular mechanics calculations suggest that an
extended conformation of the TFA backbone has a length that is
comparable to the width of a typical lipid bilayer (∼30 Å).6These
TFAs vary in the appendage on the amide nitrogen atoms. Each
amphiphile could be obtained in excellent purity (>98%) and good
overall yield (∼65%) in five straightforward synthetic steps with
two chromatographic purifications.6Multigram quantities are readily
The TFAs displayed interesting behavior in water. TFA-0 forms
a hydrogel at concentrations >0.4 wt %, and this compound was
not studied further. The other three TFAs are soluble to 5-10 wt
% in aqueous media. Critical micelle concentrations (CMCs) were
determined by monitoring solubilization of a hydrophobic fluores-
cent dye, dicyclohexatriene,7and the hydrodynamic radii (Rh) of
the micelles were determined via dynamic light scattering (DLS).6
Table 1 compares the data for TFAs with those for DDM, a
conventional detergent that is very widely used for MP applications;
DDM and our TFAs share maltose as their hydrophilic moieties.
CMC values of the three TFAs are smaller than that of DDM,
whether the CMC is measured in units of mM or wt %. The micelles
†University of Wisconsin.
‡University of Copenhagen.
§Imperial College London.
|University of California.
Figure 1. Chemical structures of DDM (top), tandem facial amphiphiles
(TFAs, middle), and schematic representation of membrane proteins
interacting with DDM (bottom left) and TFAs (bottom right).
Published on Web 11/04/2010
10.1021/ja1072959 2010 American Chemical Society
16750 9 J. AM. CHEM. SOC. 2010, 132, 16750–16752
formed by TFA-1 and TFA-2 (Rh≈ 2.0 nm) are somewhat smaller
than those formed by DDM, while micelles formed by TFA-3 are
comparable to those of DDM (Rh≈ 3.4 nm).
Micelles formed by DDM or by TFA-1 in pH 7.0 buffer (20
mM HEPES, 150 mM NaCl) were further characterized by gel
filtration using a triple-detector system8(light scattering, refractive
index, and differential pressure) (Table 2). In both cases the micelles
are globular and monodisperse. TFA-1 micelles contain only 6
molecules, which contrasts with the ∼175 molecules in a DDM
micelle. TFA-2 (R ) ethyl) seems to behave similarly to TFA-1
(R ) methyl), given the similarity in Rh, but TFA-3 (R ) butyl)
forms larger micelles. A related trend was observed among
lipopeptides, with increasing length of the alkyl appendages leading
to increasing micelle size.5c
Bacteriorhodopsin (bR) has been widely employed for assessment
of new amphiphiles because this membrane protein is readily
available, and stability can be assessed via spectrophotometry
(absorbance at 554 nm). Following standard protocols,9we used
2.0 wt % octyl-?-D-thioglucoside (OTG) to extract bR from the
native purple membrane. After removal of insoluble debris via
ultracentrifugation, the bR solution was diluted with amphiphile-
containing solutions to generate samples containing 0.2 wt % OTG
+ 0.8 wt % TFA. A control sample had OTG added to give a total
of 1.0 wt %. Figure 2a shows that all three TFA-containing samples
were much more effective at maintaining native bR absorbance over
20 days relative to the sample containing only OTG. The bR was
almost completely denatured by day 10 in the OTG-only sample
but ∼80% intact at day 20 when solubilized with TFA-1.10,11
The promising results with bR stabilization led us to investigate
a more challenging system, the photosynthetic superassembly
formed by the light harvesting I (LHI) and reaction center (RC)
complexes from Rhodobacter capsulatus.12This superassembly
contains 30-40 protein molecules (five different components), and
maintenance of native quaternary structure can be assessed via
spectrophotometry. The LHI-RC superassembly was extracted from
native membranes with 1.0 wt % DDM and purified with DDM at
its CMC (0.009 wt %). This preparation was diluted 20-fold with
solutions containing TFA-1, TFA-2, or TFA-3, so that residual
DDM was far below its CMC (0.0004 wt %). The final TFA
concentrations were 0.043 wt % (well over the CMC in each case).
A control sample had DDM added to a total concentration of 0.049
wt % (all samples were CMC + 0.04 wt %). Figure 2b shows that
the LHI-RC superassembly solubilized with any of the TFAs was
more stable over 20 days than was the superassembly solubilized
by DDM. Controls involving other common biochemical detergents
(lauryldimethylamine oxide or octyl-?-D-glucoside) showed rapid
degradation of the superassembly.10
Each membrane protein (such as bR) or membrane protein
assembly (such as LHI-RC) has unique requirements for mainte-
nance in a native-like state in aqueous solution; therefore, it is
important to assess the capabilities of new amphiphiles in multiple
systems, in order to establish the breadth of their utility. We turned
next to cytochrome bo3ubiquinol oxidase (Cyt bo3), the structural
stability of which was assessed at elevated temperature (40 °C)
with a reactive probe, N-[4-(7-diethylamino-4-methyl-3-coumari-
nyl)phenyl]maleimide (CPM).13This maleimide derivative reacts
with the thiol groups of sterically accessible Cys side chains. The
coumarin moiety of CPM is internally quenched by the maleimide
unit, but thiol reaction causes the unit to become fluorescent. CPM
can therefore be used to detect thermally induced protein unfolding,
via an increase in fluorescence, if the protein contains Cys residues
that are buried in the native state but accessible upon unfolding.
Cyt bo3was initially extracted from the native membrane with DDM
and then diluted to generate solutions containing 0.043 wt % TFA-
1, TFA-2, or TFA-3 (residual DDM ) 0.0008 wt %). A control
sample had DDM added to a total concentration of 0.049 wt %
(CMC + 0.04 wt % for each amphiphile). Figure 3a shows that
TFA-solubilized Cyt bo3samples were more resistant to thermal
denaturation than was the DDM-solubilized control.
The wild type of bacterial leucine transporter (LeuT WT) was
examined because the functional state of this membrane protein is
readily assessed by using a scintillation proximity assay (SPA)14
to monitor binding of radiolabeled leucine. LeuT was initially
extracted with DDM and then diluted with amphiphile-containing
solutions to generate final TFA concentrations of 0.04 or 0.2 wt %
(residual DDM ) 0.005 wt %). Control samples had 0.05 or 0.2
wt % DDM (overall, the final concentrations were CMC + 0.04
wt % or CMC + 0.2 wt %). At the lower amphiphile concentrations,
DDM was slightly better than the TFAs at maintaining LeuT WT
Table 1. Critical Micelle Concentration (CMC) of TFAs and
Hydrodynamic Radii (Rh) of Their Micelles (Mean ( SD, n ) 3)
13 ( 1.4
13 ( 1.8
7 ( 2.3
CMC (wt %)
0.0028 ( 0.00030
0.0028 ( 0.00039
0.0016 ( 0.00051
1.9 ( 0.08
2.0 ( 0.03
3.3 ( 0.12
3.4 ( 0.03
aMolecular weight of detergents.bHydrodynamic radius of micelles
measured by dynamic light scattering.
Table 2. Detailed Characterization of TFA-1 (Mean ( SD, n ) 6)
and DDM Micelles (Mean ( SD, n ) 8)
3.42 ( 0.04 0.028 ( 0.009 1.01 (
DDM 89 982 ( 663 176.4 ( 1.3
TFA-1 13 279 ( 157 6.2 ( 0.07 1.96 ( 0.01 0.036 ( 0.005 1.00 (
aMolecular weight of micelles.bAggregation number of micelles.
number-averaged molecular weight.
dWeight-averaged molecular weight divided by
eSpecific refractive index in-
Figure 2. Time course of the stability of bR (a) and R. capsulatus
superassembly (b) at room tempearture. Detergents were tested at 0.2 wt
% OTG + 0.8 wt % TFA and CMC + 0.04 wt % for bR and R. capsulatus
J. AM. CHEM. SOC. 9 VOL. 132, NO. 47, 2010
function over 12 days (Figure 3b), but the TFAs were clearly Download full-text
superior at the higher concentrations (Figure 3c). TFA-1 and TFA-3
at the higher concentration matched DDM at the lower concentration
in maintaining LeuT WT activity over the time period.
As a final test, we examined the TFAs for the ability to stabilize
a GPCR, the human ?2adrenergic receptor (?2AR).15This assay
employs a ?2AR-T4-lysozyme fusion protein (?2AR-T4L) com-
plexed to the inverse agonist carazolol; stability is assessed by
following the fluorescence emission maximum of carazolol, which
shifts from 341 nm in the bound state to 356 nm in aqueous solution
(i.e., after release upon ?2AR denaturation). Monitoring the 341:
356 nm peak intensity ratio upon heating yields cooperative
denaturation data. In this assay, the TFAs proved to be inferior to
We have introduced a new class of molecules, “tandem facial
amphiphiles”, that contain two deoxycholate-derived subunits and
that are sufficiently long to span a lipid bilayer.6These molecules
can be easily prepared on a scale that would support biochemical
research. One of the new amphiphiles, TFA-1, was shown to form
small, discrete micelles in water (MW ≈ 13 kD). In contrast, DDM,
a popular biochemical detergent, forms much larger micelles (MW
≈ 90 kD). Three TFAs have been evaluated for the ability to
maintain intrinsic membrane proteins or protein assemblies in
native-like forms in aqueous solution. In four of the five cases we
examined, the TFAs proved to be comparable or superior to a
conventional detergent for stabilizing the membrane proteins. Given
the great variation in structure and physical properties among
membrane proteins, no single amphiphile or amphiphile family will
be maximally effective for every case. Because the TFAs manifest
favorable solubilization/stabilization behavior with several diverse
membrane protein systems, relative to widely used conventional
detergents (DDM or OTG), and because this new amphiphile class
can form small assemblies, it seems likely that TFAs will be
valuable tools for characterization of membrane proteins, perhaps
including high-resolution structural analysis.3b
Acknowledgment. This work was supported by NIH Grant P01
GM75913 (S.H.G.), membrane protein expression center Grant
2P50 GM073210 (R.S.), the European Community’s Seventh
Framework Programme FP7/2007-2013 under grant agreement No.
HEALTH-F4-2007-201924, EDICT Consortium (K.G., B.B., U.G.),
the Lundbeck Foundation (S.G.F.R., C.J.L., U.G.), and the Danish
National Research Council (C.J.L., U.G.). R.R.R. was funded by
the Defence Science and Technology Laboratory (DSTL), Porton
Down, U.K. We thank Dr. P. Laible and N. Abbott for providing
materials and allowing us use of a DLS instrument, respectively.
Supporting Information Available: Experimental procedures for
characterizations of new compounds, micelle characterization, and
membrane protein stability assays. This material is available free of
charge via the Internet at http://pubs.acs.org.
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Figure 3. Time course of changes in stability of solubilized Cyt bo3and
activity of LeuT WT. (a) CPM assay for Cyt bo3was performed at 40 °C
for 130 min using CMC + 0.04 wt % amphiphile. LeuT WT was kept at
room temperature up to 12 days in the presence of CMC + 0.04 wt % (b)
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16752J. AM. CHEM. SOC. 9 VOL. 132, NO. 47, 2010