Mesitylene-Cored Glucoside Amphiphiles (MGAs) for Membrane Protein Studies: Importance of Alkyl Chain Density in Detergent Efficacy

Article abstract of DOI:10.1002/chem.201603338

Detergents serve as useful tools for membrane protein structural and functional studies. Their amphipathic nature allows detergents to associate with the hydrophobic regions of membrane proteins whilst maintaining the proteins in aqueous solution. However

Full text of DOI:10.1002/chem.201603338

A Journal of
Accepted Article
Title: Mesitylene-cored glucoside amphiphiles (MGAs) for membrane
protein studies: importance of alkyl chain density in detergent
efficacy
Authors: Pil Seok Chae, Kyung Ho Cho, Orquidea Ribeiro, Yang
Du, Elena Tikhonova, Jonas Mortensen, Kelsey Markham,
Parameswaran Hariharan, Claus Løland, Lan Guan, Brian
Kobilka, and Bernadette Byrne
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201603338
Link to VoR: http://dx.doi.org/10.1002/chem.201603338
Supported by

Chemistry - A European Journal
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FULL PAPER
Mesitylene-cored glucoside amphiphiles (MGAs) for membrane
protein studies: importance of alkyl chain density in detergent
efficacy
[
a]
[b]
[c]
[d]
[e]
Kyung Ho Cho, Orquidea Ribeiro, Yang Du, Elena Tikhonova, Jonas S. Mortensen, Kelsey
[
d]
[d]
[e]
[d]
[c]
Markham, Parameswaran Hariharan, Claus J. Loland, Lan Guan, Brian K. Kobilka, Bernadette
[
b]
[a]
Byrne, and Pil Seok Chae*
Dedication ((optional))
[
2]
Abstract: Detergents serve as useful tools for membrane protein
structural and functional studies. Their amphipathic nature allows
detergents to associate with the hydrophobic regions of membrane
proteins whilst maintaining the proteins in aqueous solution.
However, widely used conventional detergents have major
limitations in maintaining the structural integrity of membrane
proteins and thus there are major efforts to develop novel agents
with improved properties. We prepared mesitylene-cored glucoside
amphiphiles (MGAs) with three alkyl chains and compared these
agents with previously developed xylene-linked maltoside agents
are of major interest in drug discovery , but only a few hundred
novel membrane protein structures are currently available while
[
3]
there are more than 120,000 structures of soluble proteins.
The slow progress in membrane protein structural study is
strongly associated with their high propensity to aggregate and
denature in an aqueous medium. Conventional detergents such
as
n-octyl--D-glucopyranoside
(OG),
n-dodecyl--D-
maltopyranoside (DDM) and lauryldimethylamine-N-oxide
(LDAO) are widely used to avoid such protein aggregation and
[
4]
denaturation. However, many membrane proteins surrounded
even by these popular detergents tend to undergo structural
degradation over time. This is particularly true for eukaryotic
membrane proteins and membrane protein complexes. Thus,
new detergents with enhanced properties are necessary to
advance membrane protein science.
number of non-conventional amphiphiles have been
developed over the last two decades. Representatives include
polymer-based nano-assemblies (amphiphilic polymers
, nanodics (NDs) and nanolipodisq particles )
(
XMAs) with two alkyl chains and a conventional detergent (DDM).
[
5]
When these agents were evaluated for four membrane proteins
including a G protein-coupled receptor (GPCR), some agents such
as MGA-C13 and MGA-C14 displayed markedly enhanced
behaviours toward membrane protein stability relative to both DDM
and the XMAs. This favourable behaviour is due likely to the
increased hydrophobic density provided by the extra alkyl chain.
Thus, this study not only describes new glucoside agents with
potential for membrane protein research, but also introduces a new
detergent design principle for future development.
[
6]
A
[7a,b]
[7c]
[7d]
(
amphipols)
and peptide-based amphiphiles (e.g., lipopeptide detergents
[
8a]
[8b]
(
LPDs)
and -peptides (BPs) ). Small amphipathic
molecules have also been invented as exemplified by tripod
[
9a-c]
[9d]
amphiphiles (TPAs)
,
facial amphiphiles (FAs)
, glyco-
Introduction
[
9e]
diosgenin (GDN) , neopentyl glycol (NG) class detergents
glucose neopentyl glycols (GNGs)
[
9f,g]
(
,
maltose neopentyl
Membrane proteins play a variety of roles for proper cellular
functions, including molecular transport, signal transduction and
cell-to-cell communication. Malfunctions of membrane proteins
are implicated in many diseases, reflected by the fact that more
than half of all pharmaceuticals target these important
[9h-j]
glycols (MNGs)
,
neopentyl glycol-derived triglucosides
[9k]
[9l]
(
NDTs) ), and penta-saccharide detergents (PSEs) . Of these
small agents, NG class agents (GNGs and MNGs) have proved
particularly interesting, as class members (e.g., MNG-3 and
GNG-3) have contributed to the determination of more than 25
crystal structure membrane proteins. This clearly illustrates the
important role of new amphiphiles in membrane protein
[
1]
molecules. Three dimensional structures of membrane proteins
[
10]
research.
maltoside amphiphiles (XMAs)
In a recent study we described xylene-linked
[
[
[
[
a] K. H. Cho, Prof. P. S. Chae
[11]
Department of Bionanotechnology, Hanyang University, Ansan,
with a p-xylene linker that
1
5588 (Korea) E-mail: pchae@hanyang.ac.kr
showed a favorable stabilizing effect on some membrane
proteins, but were suboptimal for other membrane proteins such
b] O. Ribeiro, Dr. B. Byrne
Department of Life Sciences, Imperial College London
London, SW7 2AZ (UK)
c] Dr. Yang Du, prof. B. K. Kobilka
as the leucine transporter (LeuT) and the 
2
adrenergic receptor
(
2
AR) (Figure S1a). We hypothesize that the presence of two
Molecular and Cellular Physiology, Stanford
CA 94305 (USA)
alkyl chains as the hydrophobic group in the XMAs is not ideal
for tight packing of detergent alkyl chains around a target
membrane protein, thereby limiting the protein stability conferred
by these detergents in an aqueous environment. One strategy to
address this issue is to enhance the hydrophobic density of the
detergent molecules by increasing the number of alkyl chains.
Thus, we developed three alkyl chains-bearing amphipathic
agents, designated mesitylene-cored glucoside amphiphiles
d] Dr. E. Tikhonova, K. Markham, Dr. P. Hariharan, Prof. L. Guan
Department of Cell Physiology and Molecular Biophysics, Center for
Membrane Protein Research, School of Medicine, Texas Tech
University Health Sciences Center Lubbock, TX 79430 (USA)
e] J. S. Mortensen, Prof. C. J. Loland
[
Department of Neuroscience and Pharmacology, University of
Copenhagen, DK- 2200 Copenhagen (Denmark)
Supporting information for this article is given via a link at the end of
the document. See DOI: 10.1039/x0xx00000x
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201603338
FULL PAPER
(
MGAs) which contain the mesitylene group as a linker. These
p-xylene to mesitylene, three alkyl chains and three branched
diglucosides could be successfully implemented into the
periphery of the central benzene ring. Each agent could be
prepared without synthetic difficulty via a protocol comprising
five synthetic steps, giving overall yields of ~25%. Interestingly,
water-solubility was dramatically increased for the MGAs; the di-
alkylated glucosides (XGAs) were poorly water-soluble with a C7
alkyl chain (<1%) while the tri-alkylated glucosides (MGAs) were
more than 20% water-soluble even with a C15 alkyl chain. A
similar trend was observed in a comparison with the di-alkylated
maltoside versions (XMAs); in this scaffold, a C12 alkyl chain
was the maximum chain length for good water-solubility. Note
that the XMAs contain a higher number of glucose units than the
MGA counterparts (8 vs 6). Thus, these results indicate that the
MGAs possess a more water-soluble architecture than the
XMAs/XGAs, a feature likely contributing to their favorable
properties for membrane protein manipulation.
agents bear one additional alkyl chain as well as one more
branched diglucoside headgroup around the central benzene
ring compared to the previously developed XMAs.
evaluation with four membrane protein systems, these new
agents displayed markedly enhanced behaviors for all target
proteins relative to the previously reported XMAs
[
11]
In the
[
11]
and DDM,
indicating a critical role of alkyl chain density in detergent
behaviors toward membrane protein stability.
Results and Discussion
Detergent structures and physical characterizations
Table 1. Molecular weights (MWs) and critical micelle concentrations
(
CMCs) of MGAs (MGA-C10, MGA-C11, MGA-C12, MGA-C13,
MGA-C14 and MGA-C15) and a conventional detergent (DDM), and
hydrodynamic radii (R ; n = 5) of their micelles.
h
[
a]
[b]
Detergent
MGA-C10
MGA-C11
MGA-C12
MGA-C13
MGA-C14
MGA-C15
DDM
M
W
CMC [M]
~8
CMC [wt%]
h
R [nm]
1736.1
1778.2
1820.3
1862.3
1904.4
1946.5
510.1
~0.0014
~0.009
2.9±0.01
3.0±0.04
3.1±0.03
3.2±0.03
3.4±0.04
3.6±0.02
3.4±0.02
~5
Scheme 1. Chemical structures of MGAs (MGA-C10, MGA-C11, MGA-C12,
MGA-C13, MGA-C14 and MGA-C15) with alkyl chain length variation from
C10 to C15. The number of carbons in the alkyl chain was utilized for
detergent designation.
~3.5
~3
~0.0006
~0.0006
~0.0004
~0.0003
~0.0087
~2
~1.5
~170
Initially, we prepared glucoside versions of the previously
reported XMAs, designated xylene-linked glucoside amphiphiles
(
XGAs) (Figure S1b), however the initial characterization
[a] Molecular weight of detergents. [b] Hydrodynamic radius of detergents
identified a number of issues with these agents. First, XGA
synthesis was much more difficult than that for the XMAs, giving
overall yields of less than 10%. The synthetic efficiency was
significantly lower than that of the XMAs (~60%), due to poor
levels of glycosylation (~15%). Second, the XGAs showed
unexpectedly low water-solubility, with the C7 alkyl chain XGA
measured at 1.0 wt% by dynamic light scattering.
Detergent micelles were characterized in terms of critical micelle
concentrations (CMCs) and micelle size, both of which were
estimated
via
fluorophore
encapsulation
using
[
12]
(
XGA-C7) only marginally soluble in water (<1%). Thus, we
diphenylhexatriene (DPH) and dynamic light scattering (DLS),
respectively. The results are summarized in Table 1. The CMC
values of all MGAs (1.5~8.0 M) were significantly smaller than
that of DDM (~170 M), indicating a strong tendency to form
self-assemblies. Within the MGA series, the detergent CMC
values decreased with alkyl chain length, giving MGA-C10 with
the shortest alkyl chain the largest values (~8.0 M) and MGA-
C15 with the longest alkyl chain the smallest value (~1.5 M).
This is likely due to the increased hydrophobicity of the lipophilic
groups with increasing alkyl chain length. Micelles formed by all
the MGAs were smaller than or comparable to those formed by
DDM (2.9~3.6 vs 3.4 nm). Detergent micelle size tends to
increase with alkyl chain length in the architecture because of a
gradual change in the geometry from conical to cylindrical shape.
Yet, all of the MGAs prepared in this study self-assembled into
could use only shorter alkyl chain agents (XGA-C4, XGA-C5 and
XGA-C6) for evaluation with membrane proteins. In addition, the
preliminary results of these evaluations indicated that the
glucoside versions of XMAs (i.e., XGAs) are unfavorable for
membrane protein solubilisation and stabilization (see below).
These agents tend to form large self-aggregates with diameters
of 150 ~ 200 nm, significantly deviating from detergent micelles
(
Table S1 & Figure S2a), which is likely to be associated with
their limited applicability for membrane protein manipulation.
These unfavorable results prompted us to develop an additional
class of glucoside amphiphiles with a mesitylene linker rather
than p-xylene linker (MGAs; Scheme 1). Detergent alkyl chain
length varied from C10 to C15 and this is incorporated into the
detergent designation. Owing to the core structure change from
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Chemistry - A European Journal
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micellar structures with a diameter smaller than or comparable
to DDM, mainly due to the presence of the large headgroup (i.e.,
three branched diglucosides). As can be seen in the DLS
profiles, the micelles formed by these MGAs showed a single set
of size populations with a narrow distribution, indicative of high
homogeneity (Figure S2b).
differences between the MGAs became more obvious. Of the
MGAs, MGA-C13 and MGA-C14 were most effective followed
by MGA-C15. All the other MGAs (MGA-C10, MGA-C11 and
MGA-C12) having a rather short alkyl chain were less favorable
in this regard although being still better than DDM. This result
indicates the favorable architecture of the MGAs for preserving
the folded state of the transporter.
The six MGAs were also evaluated with Salmonella typhimurium
Detergent evaluation with multiple membrane proteins
[15]
melibiose permease (MelBSt).
E. coli membranes containing
MelBSt were treated with 1.5 wt% individual MGAs or DDM and
incubated for 90 min at 0°C for direct protein extraction. The
amounts of soluble MelBSt after ultracentrifugation were
determined by Western blot. The amount of soluble MelBSt in
individual detergents was expressed as a percentage of total
MelBSt in the untreated membrane sample (Figure 2). At 0°C,
DDM showed high efficiency in the extraction of the permease
while all MGAs were inferior to DDM, indicating the suboptimal
behavior of MGAs. When the incubation temperature was
increased to 45°C, however, the amount of soluble MelBSt
significantly increased for all the MGAs; particularly, these with a
long alkyl chain (MGA-C13, MGA-C14, or MGA-C15) maintain
the soluble fractions to a level comparable to that with DDM.
Detergent efficacy between DDM and the MGAs could be clearly
discerned by a further temperature increase to 55°C. At this
higher temperature, DDM yielded only a small amount of soluble
MelBSt (<10%), implying that most DDM-solubilized permeases
aggregated or denatured during the 90 min-incubation. In
contrast, all the MGAs substantially retained soluble MelBSt, with
the remarkable performance for MGA-C13 and MGA-C14 (90-
Figure 1. Thermal denaturation profile of UapA solubilized in a MGA (MGA-
C10, MGA-C11, MGA-C12, MGA-C13, MGA-C14, or MGA-C15) and DDM
used at two different concentrations: CMC+0.04 wt% (a) and CMC+0.2 wt%
(b). The relative amount of foled protein was monitored as a function of time
using the CPM assay carried out at 40°C for 120 min. The data is
representative of two independent experiments.
In order to characterize the MGAs in terms of detergent efficacy
100%) because no appreciable decrease in the amount of
for membrane protein stabilization, the new agents were first
soluble MelBSt was detected. In the case of MGA-C15, the
amount of soluble MelBSt was slightly decreased at 55°C. This
result suggests that MGA-C13 and MGA-C14 have optimal
structures for retaining MelBSt solubility. When the di-alkylated
versions of MGAs (i.e., XGAs; XGA-C4, XGA-C5 and XGA-C6)
were tested for this protein (Figure S3), all the XGAs were
inferior to DDM with regard to extracting MelBSt from the
membranes at 0°C and retaining the permease solubility at the
elevated temperatures. These results clearly demonstrate that
the tri-alkylated MGA architecture is superior to that of the di-
alkylated XGAs at preserving MelBSt solubility/stability.
+
evaluated with
symporter (UapA)
a protein transporter, uric acid-xanthine/H
[
13]
from Aspergillus nidulans. The protein
expressed in Saccharomyces cerevisiae was extracted by DDM
from the membranes and then purified in 0.03% (w/v) of the
same detergent. The DDM-purified UapA protein was diluted
into buffer solutions containing individual MGAs or DDM to reach
the final detergent concentration of the individual CMCs+0.04
wt%. After the dilution, the residual DDM concentration was
0.0002 wt%, far smaller than the CMC of DDM (0.0087 wt%)
Protein thermostability was assessed over time using the dye,
N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide
[
14]
The favorable behaviors for UapA and MelBSt encouraged us
(
CPM).
This non-fluorescent maleimide-containing molecule
[
16]
to evaluate the MGAs with the leucine transporter (LeuT) from
Aquifex aeolicus. MGA-C10 was inferior to the other MGAs in
the evaluation with UapA and MelBSt and thus was not further
evaluated. The transporter expressed in E. coli membranes was
first extracted from the membranes using 1.0 wt% DDM and
purified by the use of 0.05 wt% same detergent. The DDM-
purified transporter was mixed with buffer solutions including
individual MGAs or DDM to give a final detergent concentration
of the individual CMCs+0.04 wt%. The residual DDM
concentration was 0.005 wt%. The protein samples solubilized in
the individual detergents were incubated for 12 days at room
temperature and substrate binding activity of the transporter was
measured at regular intervals via scintillation proximity assay
becomes highly fluorescent following specific conjugation with
the sulfhydryl group of cysteine residues. Upon protein unfolding,
a sulfhydryl group embedded in the protein interior tends to
become exposed on the protein surface, leading to an increase
in fluorescence intensity. Thus, the CPM assay provides a
convenient means to estimate the change in the amounts of
accessible sulfhydryl groups in a reaction medium, directly
associated with the change in the relative amounts of unfolded
target protein. As shown in Figure 1, all six MGAs were superior
to DDM in terms of maintaining the folded structure of the
protein at the individual CMCs+0.04 wt%. No noticeable
differentiation was observed between the MGAs in this rather
low detergent concentration. When detergent concentration was
increased to the individual CMCs+0.2 wt%, however, efficacy
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Chemistry - A European Journal
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Figure 2. Themostability of MelBSt solubilized in a MGA (MGA-C10, MGA-C11, MGA-C12, MGA-C13, MGA-C14, or MGA-C15) or DDM. The protein was
extracted from E. coli membranes by treatment with 1.5 wt % individual detergents at four different temperatures (0, 45, 55, and 65°C) for 90 min. The solubilized
MelBSt was separated by ultracentrifugation and visualized by Western blot (a). The amounts of the soluble MelBSt were expressed as percentages of total MelBSt
in the untreated membrane (Memb) and presented in histograms (b). Error bars, SEM, n = 3.
3
[17]
(
SPA) using a radio-labelled substrate ([ H]-Leu).
As can be
DDM-purified receptor was diluted with individual MGAs- or
DDM-containing buffers to give a final detergent concentration of
the individual CMCs+0.2 wt%. After dilution, the residual DDM
concentration in the MGA samples was 0.0007 wt%. The
seen in Figure 3a, the DDM-solubilized transporter gave a
gradual loss in substrate binding activity over time, resulting in ~
0% retention of the initial activity after the 12-day incubation. All
4
the MGAs were better than DDM in this context, with the best
performance achieved for MGA-C11 followed by MGA-C12 and
MGA-C13. The MGA-C11-solubilized transporter gave almost
full retention in the substrate binding activity (~90%) after the 12-
day incubation. Of the tested MGAs, MGA-C15 with the longest
alkyl chain was the poorest at retaining transporter activity, but
still slightly better than DDM. As detergent concentration was
increased to the individual CMCs+0.2 wt%, the DDM-solubilized
transporter more rapidly lost the substrate binding activity
receptor stability was addressed by measuring the ligand
3
binding activity using
dihydroalprenolol, DHA). As a preliminary study, the activity of
the receptor solubilized in the individual MGAs or DDM was
a
radio-labelled antagonist ([ H]-
[
19]
measured after
a
30 min dilution to allow detergent
reconstitution. The MGAs (MGA-C12, MGA-C13 and MGA-C14)
showed an initial receptor activity comparable to both each other
and DDM (Fig. S5). However, a clear difference in detergent
efficacy was observed between the MGAs and DDM when the
receptor stability was monitored at regular intervals over a three-
day incubation at room temperature (Fig. 3c). The DDM-
solubilized receptor rapidly lost activity over time, resulting in
only ~15 % retention of receptor activity at the end of the three-
day incubation. A similar activity loss was observed for MGA-
C12-solubilized receptor. In contrast, the MGA-C13 or MGA-
C14-solubilized receptor lost ligand binding capacity much
slower and additionally there was greater activity at the end of
the incubation period (50-60%), with better performance for
MGA-C14 relative to MGA-C13. In order to provide greater
insights into the properties of the MGAs, DDM- or MGA-C13-
(
Figure 3b). A similar trend was observed for the MGAs with a
long alkyl chain (MGA-C14 and MGA-C15); these agents
resulted in a significant decrease in the transporter activity
(
40~50% after 12 day incubation). In contrast, the MGAs with a
short alkyl chain (MGA-C11, MGA-C12 and MGA-C13) were still
very effective at preserving the activity of the transporter (~90%
after the 12-day incubation). Thus, the outcome of the MGAs
was dependent on detergent alkyl chain length. When the di-
alkylated versions (XGAs; XGA-C4, XGA-C5 and XGA-C6) were
evaluated with the transporter, none of these agents were better
than DDM (Figure S4), consistent with the XGA results for
MelBSt
.
solubilized
2
 AR was analyzed by size exclusion
chromatography (SEC) following detergent exchange. MGA-C13
was chosen for this assessment as it showed effective
stabilization of all four targeted membrane proteins. As can be
seen in Fig. S6a, MGA-C13 formed smaller PDCs with the
receptor than DDM, which may be favorable for membrane
protein crystallization. Application of the DDM- or MGA-C13-
We next moved to the human 
G protein-coupled receptor (GPCR), for detergent evaluation.
2
2
adrenergic receptor ( AR), a
[
18]
For this evaluation, the three MGAs (MGA-C12, MGA-C13 and
MGA-C14) were selected as MGA-C11 and MGA-C15 showed
the worst behavior for UapA and LeuT, respectively. The
receptor was first expressed in Sf9 insect cells and then
solubilized from the membranes using a conventional detergent
solubilized 
2
AR to the SEC column in a detergent-free buffer
revealed a significant efficacy difference between DDM and
(
DDM). The receptor was further purified in 0.1 wt% DDM. The
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Chemistry - A European Journal
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MGA-C13; a large decrease in intensity of the monodisperse
peak (~13 mL) and a concomitant appearance of the new broad
aggregation peak (~10 mL) were observed for the DDM-
solubilized receptor in detergent-free buffer (Fig. S6b). In
density) relative to the XGAs/XMAs with only two alkyl chains.
Accordingly, the MGAs would generate a detergent micelle
interior with increased hydrophobic density compared to the
XGAs/XMAs. This may result in a stronger and thus more
favorable detergent interaction with the protein hydrophobic
surface, thus providing an optimal platform for effective protein
encapsulation. The increased alkyl chain density also seemed to
endow the MGAs with additional advantages. First, the MGAs
were more water-soluble with increasing alkyl chain length than
the equivalent XGAs and XMAs. Second, the MGAs formed
relatively small micelles with a very low CMC value (1.5~8.0 M),
in stark contrast with the XGAs which formed large liposomes
with a high critical aggregation value (~800 M). Collectively, the
current study revealed that the diverse detergent properties such
as water-solubility, self-assembly morphology and, more
importantly, membrane protein stabilization efficacy could be
significantly improved by modulating the alky chain density (i.e.,
hydrophobic density) of detergent micelles. This detergent
structure-property-efficacy relationship has not been studied in
detail so far.
contrast, only
a slight change in the chromatogram was
observed for the MGA-C13-solubilized receptor under the same
condition. Thus, this MGA agent maintained receptor integrity
even in a detergent-free buffer, presumably due to this detergent
forming stronger interactions with the receptor than DDM.
It is notable that the MGAs bear glucosides instead of
maltosides as headgroups. In general, maltoside detergents
(
e.g., DDM) are more effective at stabilizing membrane proteins
than glucoside agents (e.g., OG). However, glucoside agents
are also widely used for membrane protein study, presumably as
a result of the small headgroup (i.e., glucoside), which results in
the formation of small PDCs. Small PDCs are often favorable for
generation of high quality protein crystals and for nuclear
magnetic resonance (NMR)-based protein structural study. Thus,
glucoside detergents have distinctive advantages over maltoside
agents even if those agents have relatively limited ability to
stabilize membrane proteins. A similar conclusion could be
reached from the comparison of the GNGs with MNGs. For
example, GNG-3 was generally inferior to the maltoside analog
Figure 3. Long-term substrate or ligand binding activity for LeuT (a,b) and

2
AR (c) solubilized in individual MGAs and DDM. Five MGAs (MGA-C11,
MGA-C12, MGA-C13, MGA-C14 and MGA-C15) and three MGAs (MGA-C12,
MGA-C13 and MGA-C14) were used for the evaluation with LeuT and  AR,
respectively. Two different detergent concentrations ((a) CMC+0.04 wt% and
b) CMC+0.2 wt%) were used for LeuT stability measurement while a single
detergent concentration (CMC+0.2 wt%) was used for AR stability
2
(
[
9f,h,j]

2
(MNG-3) in terms of maintaining membrane protein stability
,
experiment. LeuT activity was measured at regular intervals over the course of
but this agent has successfully facilitated the high resolution
structure determinations of several membrane proteins.
a 12-day incubation at room temperature via scintillation proximity assay (SPA)
[10o,p,21]
3
using a radio-labelled substrate ([ H]-Leucine). 
2
AR ligand binding activity
3
This analysis (glucosides vs maltosides) implies that it is
challenging to develop novel glucoside detergents with
enhanced efficacy toward membrane protein stabilization
relative to DDM, reflected by the fact that most novel detergents
effective in this regard contain maltoside headgroups (e.g.,
MNGs, FAs, and GDN). Very recently, a class of glucosides
called NDTs have attracted substantial attention as these
was assessed at regular intervals using the antagonist [ H]-dihydroalprenolol
(DHA) over a 3-day incubation at room temperature. Error bars, SEM, n = 3.
In this study, we introduced mesitylene-cored glucosides (MGAs)
with three alkyl chains and found that these agents were
consistently more effective than DDM and di-alkylated versions
(
XGAs) for membrane protein stability. These agents were also
glucosides were shown to be superior to DDM for multiple
clearly superior to the previously-reported di-alkylated
maltosides (XMAs) at stabilizing membrane proteins, particularly
[9k]
membrane proteins.
As a hydrophilic neopentyl glycol (NG)
linker was used to connect the glucose group with the
hydrophobic alkyl chain, however, the headgroup of this agent is
an NG-glucose conjugate rather than glucoside alone. The
2
for LeuT and  AR. The previous study had shown that the
[
11]
XMAs were inferior to DDM for these membrane proteins. The
well-behaved MGAs (MGA-C13 and MGA-C14) proved to be
even superior to MNG-3, one of the most successful new
current MGAs utilized
a hydrophobic linker derived from
dimethylmalonate to bridge the head and tail groups. Thus,
these agents are the first examples of glucoside detergents
conferring enhanced protein stability for the multiple membrane
proteins relative to DDM.
[
5c,20]
amphiphiles
, in terms of stabilizing UapA (Fig. S7). This
[9h,j]
finding appeared to be true for LeuT as well.
It is hard to
narrow down which structural features are responsible for the
enhanced efficacy of the MGAs. However, due to the presence
of three alkyl chains around a central ring structure, the MGA
molecules have increased alkyl chain density (i.e., hydrophobic
Conclusions
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Chemistry - A European Journal
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Detergent efficacy tends to vary depending on the
characteristics of target membrane proteins. Consequently, it is
often the case that a detergent effective for one membrane
protein is not favorable for another membrane protein. Thus, it is
extremely important to evaluate a newly-developed agent for
multiple membrane proteins to show general utility in membrane
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This work was supported by the National Research Foundation
of Korea (NRF) funded by the Korean government (MSIP) (grant
number 2008-0061891 and 2016R1A2B2011257 to P.S.C. and
K.H.C.). This work was also supported by the National Science
Foundation (grant number MCB-1158085 to L.G.) and by the
National Institutes of Health (grant number R01 GM095538 to
L.G.).
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Keywords: amphiphile design • membrane proteins • detergents
•
protein stabilization • protein solubilisation
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FULL PAPER
Mesitylene-cored glucoside
Kyung Ho Cho, Orquidea Ribeiro, Yang
Du, Elena Tikhonova, Jonas S.
Mortensen, Kelsey Markham,
Parameswaran Hariharan, Claus J.
Loland, Lan Guan, Brian K. Kobilka,
Bernadette Byrne, and Pil Seok Chae*
amphiphiles (MGAs) with three alkyl
chains and six glucose units proved
markedly superior to both a
conventional detergent (DDM) and di-
alkylated xylene-linked agents (XMAs)
in terms of membrane protein
stabilization. This study indicates that
detergent alkyl chain density plays a
crucial role in determining detergent
efficacy.
Page No. – Page No.
Mesitylene-cored glucoside
amphiphiles (MGAs) for membrane
protein studies: importance of alkyl
chain density in detergent efficacy
length
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