Trehalose-cored amphiphiles for membrane protein stabilization: Importance of the detergent micelle size in GPCR stability

Article abstract of DOI:10.1039/c8ob03153c

Despite their importance in biology and medicinal chemistry, structural and functional studies of membrane proteins present major challenges. To study diverse membrane proteins, it is crucial to have the correct detergent to efficiently extract and stabilize the proteins from the native membranes for biochemical/biophysical downstream analyses. But many membrane proteins, particularly eukaryotic ones, are recalcitrant to stabilization and/or crystallization with currently available detergents and thus there are major efforts to develop novel detergents with enhanced properties. Here, a novel class of trehalose-cored amphiphiles are introduced, with multiple alkyl chains and carbohydrates projecting from the trehalose core unit are introduced. A few members displayed enhanced protein stabilization behavior compared to the benchmark conventional detergent, n-dodecyl-β-d-maltoside (DDM), for multiple tested membrane proteins: (i) a bacterial leucine transporter (LeuT), (ii) the R. capsulatus photosynthetic superassembly, and (iii) the human β₂ adrenergic receptor (β₂AR). Due to synthetic convenience and their favourable behaviors for a range of membrane proteins, these agents have potential for membrane protein research. In addition, the detergent property-efficacy relationship discussed here will guide future design of novel detergents.

Full text of DOI:10.1039/c8ob03153c

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Biomolecular Chemistry
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Trehalose-cored amphiphiles for membrane
protein stabilization: importance of the detergent
Cite this: DOI: 10.1039/c8ob03153c
micelle size in GPCR stability†
a
b
c
d
Manabendra Das, ‡ Yang Du,
Jonas S. Mortensen,
Manuel Ramos,
a
a
a
e
Lubna Ghani,d
Ho Jin Lee,
Claus J. Loland,
Hyoung Eun Bae,
Brian K. Kobilka
Bernadette Byrne,
and Pil Seok Chae
c
b
a
Lan Guan,
*
Despite their importance in biology and medicinal chemistry, structural and functional studies of mem-
brane proteins present major challenges. To study diverse membrane proteins, it is crucial to have the
correct detergent to efficiently extract and stabilize the proteins from the native membranes for bio-
chemical/biophysical downstream analyses. But many membrane proteins, particularly eukaryotic ones,
are recalcitrant to stabilization and/or crystallization with currently available detergents and thus there are
major efforts to develop novel detergents with enhanced properties. Here, a novel class of trehalose-cored
amphiphiles are introduced, with multiple alkyl chains and carbohydrates projecting from the trehalose core
unit are introduced. A few members displayed enhanced protein stabilization behavior compared to the
benchmark conventional detergent, n-dodecyl-β-D-maltoside (DDM), for multiple tested membrane pro-
teins: (i) a bacterial leucine transporter (LeuT), (ii) the R. capsulatus photosynthetic superassembly, and (iii)
Received 19th December 2018,
Accepted 25th February 2019
the human β
2
2
adrenergic receptor (β AR). Due to synthetic convenience and their favourable behaviors for a
DOI: 10.1039/c8ob03153c
range of membrane proteins, these agents have potential for membrane protein research. In addition, the
rsc.li/obc
detergent property–efficacy relationship discussed here will guide future design of novel detergents.
the structures and functions of these biomacromolecules is of
key importance for biology and rational drug design.
Introduction
3
Integral membrane proteins are involved in all aspects of vital Unfortunately, membrane proteins are inherently challenging
cellular activities including inter- or intra-cellular material to study in vitro because of their low natural abundance,
transfer, signal transduction, photosynthesis, cell adhesion, problematic overexpression, and tendency to misfold/denature
cytoskeletal organization, protein trafficking, etc. Additionally, once extracted from the native membranes into aqueous
membrane proteins account for up to one third of the human buffer. These bio-macromolecules are insoluble in aqueous
1
proteome and make up approximately half of all presently solutions due to the presence of their hydrophobic transmem-
2
marketed therapeutic targets. Thus, in depth information on brane domains (TMs). To circumvent these issues, convention-
al detergents are used for membrane protein extraction and
stabilization. Detergents shield the protein hydrophobic
a
exterior from the polar aqueous environment. However,
detergent micellar assemblies do not often adequately mimic
some of the hallmarks of the native membranes. For example,
n-octyl-β-D-glucoside (OG), n-nonyl-β-D-glucoside (NG) and
lauryldimethylamine-N-oxide (LDAO) are widely used for mem-
brane protein crystallization mainly due to the fact that
Department of Bionanotechnology, Hanyang University, Ansan, 155-88, Korea.
E-mail: pchae@hanyang.ac.kr
b
Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305, USA.
E-mail: kobilka@stanford.edu
c
Department of Neuroscience, University of Copenhagen, DK-2200, Copenhagen,
Denmark. E-mail: cllo@sund.ku.dk
d
Department of Cell Physiology and Molecular Biophysics, Center for Membrane
4
they form small protein-detergent complexes (PDCs).
Protein Research, School of Medicine, Texas Tech University Health Sciences Center,
Lubbock, TX 79430, USA. E-mail: lan.guan@ttuhsc.edu
However, many membrane proteins are not stable in these
detergents, resulting in irreversible nonspecific aggregation
due to exposure of the hydrophobic TMs to a polar medium or
e
Department of Life Sciences, Imperial College London, London, SW7 2AZ UK.
E-mail: b.byrne@imperial.ac.uk
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8ob03153c
Current address: Molecular Biophysics, Technische Universität Kaiserslautern,
5
penetration of detergent molecules between TM helices. On
the other hand, n-dodecyl-β-D-maltoside (DDM) is generally a
‡
Erwin-Schrödinger -Str. 13, 67663, Kaiserslautern, Germany.
mild detergent preferred for maintaining the native states
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of proteins in solution, but this maltoside tends to form rela-
tively large PDCs unfavorable for protein crystallization and
6
NMR studies. In addition, many membrane proteins solubil-
ized in a commercially available detergent are not stable
enough for the downstream protein structure determination.
Thus, the development of novel membrane-mimetic systems
with improved efficacy is of importance for structural and
functional characterization of membrane proteins.
Notable examples of several membrane-mimetics such as
7
8
amphipols (Apols), nanodiscs (NDs), lipopeptide detergents
(
9
10
LPDs) and β-peptides (BPs) have been developed. These
agents have been demonstrated to be effective at stabilizing
multiple membrane proteins but form large PDCs or are
ineffective at protein extraction. More importantly, these
systems have had limited success in producing protein crys-
tals. With carbohydrate-based head groups and structurally
modifiable hydrophobic segments, several small amphipathic
Fig. 1 Background for this study. Schematic chemical structures of pre-
20
viously reported dodecyl trehaloside detergents (DDTre) (a) and new
trehalose-cored amphiphiles (TCGs/TCMs) (b). DDTre has a trehalose
unit used as the head group while TCGs/TCMs used this disaccharide
unit as the central core of the amphiphiles. (b, right) Side view of the
agents have also been invented, as exemplified by neopentyl structure of a trehalose-cored amphiphile optimized by DFT calculations
1
1
(
B3LYP/6-31G*) in which the alkyl chains are segregated from the hydro-
glycol (NG) class amphiphiles (MNGs/GNGs), facial amphi-
1
2
13
philic backbone in contact with water. These non-ionic trehalose-cored
detergents do not interfere with optical spectroscopy in the far-UV
range, and contain a dense hydrophobic group for effective protein
stabilization.
philes (FAs), mannitol-based amphiphiles (MNAs), neopen-
tyl glycol triglucosides (NDTs), resorcinarene-based gluco-
sides (RGAs), penta-saccharide amphiphiles (PSEs) and
vitamin E-based glucosides (VEGs). Some small detergents
1
4
1
5
16
1
7
1
8a
such as butane-1,2,3,4-tetraol-based maltosides (BTMs)
and
1
8b
norbornane-based maltosides (NBMs)
exploit molecular
chirality to modulate detergent efficacy for protein stabilization. (TCG-Ls). Introduction of the glycerol linker allowed us to
Of these small amphipathic agents, GNG-3 and MNG-3 have prepare water-soluble TCG-Ls with the alkyl chain length
contributed to the determination of more than 35 new mem- extended to C12. This chain extension is important as the alkyl
brane protein crystal structures including the β2 adrenergic, chain lengths of the TCGs with no linker are likely too short to
acetylcholine and opioid G-protein coupled receptors (GPCRs) stabilise membrane proteins. Of note, an optimal alkyl chain
19
in the last several years. Using FA-5 or FA-7 alone or mixed length for protein stability is variable within the range of C10
with lipids (DMPC), Lee and co-workers obtained crystals of the to C13 depending on the target protein. A good match in hydro-
ATP-binding cassette transporter (MsbA) and the GPCR-like bac- phobic dimensions between detergent molecules and mem-
1
2b
teriorhodopsin, respectively.
coside and maltoside amphiphiles with trehalose as a confor- glucoside and maltoside head groups, along with the glycerol
mationally restricted core unit, designated TCGs and TCMs, linker, we varied the detergent alkyl chain length from C to
respectively (Fig. 1b). Three sets of trehalose-cored amphiphiles
12. Furthermore, the head group architecture of these amphi-
Herein, we introduced new glu- brane proteins is necessary to maintain protein stability. Using
5
C
with structural variation were prepared to evaluate their stabiliz- philes varied from tetra-glucoside to tetra-maltoside to octa-
ation efficacy for different membrane proteins including a glucosides. We hypothesized that these TCGs/TCMs/TCG-Ls
G-protein coupled receptor (GPCR). We found that some of with their large range of structural variations may have
these agents conferred markedly enhanced stability to target different efficacies for membrane protein stabilization
proteins compared to DDM.
(Fig. 2b), providing insights into detergent structure property–
efficacy relationships. Trehalose is widely used in life sciences
as an additive for stabilizing proteins with a mechanism attrib-
uted to vitrification, water replacement and water entrap-
Results and discussion
Detergent structures and physical characterization
2
1
ment. Trehalose and its derivatives have found use in bio-
22,23
24
medical applications,
DNA amplification
and the
2
5
First, we prepared two sets of trehalose-cored detergents with pharmaceutical industries. This trehalose unit has also been
either a glucoside (TCG) or maltoside (TCM) hydrophilic group used as an amphiphile scaffold for membrane protein studies
2
0
directly attached to the trehalose core (Fig. 2a). Along with four (Fig. 1a), but a previous study utilized the disaccharide unit
glucose/maltose units, both TCGs and TCMs share four alkyl as the head group rather than the central core unit connecting
chains as the hydrophobic group, but the alkyl chain length the head and tail groups. Thus, this study is the first example
varies from C to C for the glucosides and from C to C for of a disaccharide unit used as the detergent core for mem-
5
7
8
10
the maltosides, as indicated in the detergent designation. For brane protein studies. Due to the presence of two central pyra-
an additional set of amphiphiles, eight glucose units were nose rings, the new detergents have reduced conformational
attached to the central trehalose unit using a glycerol linker flexibility of the head and tail groups in aqueous solution. At
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Fig. 2 (a) Synthetic scheme and chemical structures of newly prepared trehalose-cored amphiphiles (TCGs/TCMs/TCG-Ls). Novel amphiphiles
were derived from α,α-trehalose via dibenzylidenated trehalose (A) and tetra-alkylated tetra-ol intermediates (B). The tetra-alkylated tetra-ol inter-
mediates and TCGs/TCMs/TCG-Ls contain a C axis passing through the central part of the molecules, indicated by the blue dotted line on the
2
chemical structures of the tetra-ol intermediate. The inset within circle (black) illustrates a known mechanism of β-selective glycosylation involving
neighboring group participation (NGP). (b) Schematic representation of a membrane protein interacting with one of the new detergents following
extraction from the membrane.
the same time, due to the presence of multiple flexible alkyl C7 to C10 to C12. In addition, a high hydrophilic density
chains, the detergent molecules should readily adopt an appeared to be attained by an intramolecular hydrogen bond
optimal conformation for protein stability when associated network within the head groups (Fig. 1b, right).
with membrane proteins. Thus, the new agents differ from
Both TCG and TCM sets were prepared from D-(+)-trehalose
both conventional detergents which contain either a very flex- according to a protocol comprising straightforward synthetic
ible alkyl chain (e.g. DDM) or facial amphiphiles (FAs) with steps (Fig. 2a): acid-catalyzed formation of 4,6,4′,6′-dibenzylide-
1
2
conformationally locked hydrophobic groups. In addition, as nated trehalose (A), tetra-alkylation and benzylidene acetal
the current trehalose-cored amphiphiles contain no aromatic removal (B), stereo-selective glycosylation and global de-
group, membrane proteins can be studied via UV spectro- protection (see Scheme S1† for details). In the case of TCG-Ls
scopic methods, in comparison with aromatic group-contain- containing the glycerol linker, two additional steps of allyla-
ing detergents such as calix[4]arene based detergents tion and syn-dihydroxylation using OsO were necessary to syn-
4
2
6
27
(
C4Cn), ganglio-tripod amphiphiles (TPAs), Chae’s glyco- thesize tetra-alkylated octa-ol intermediates (see Scheme S2†
xylene-linked maltoside amphiphiles for details). High anomeric purity of all the new detergents
XMAs) and vitamin E-based glycosides (VEGs). Energy- was attained by stereo-selective β-glycosylation via neighboring
2
8
tritons (CGTs),
2
9
17
(
minimized conformations of the new agents obtained by group participation (i.e. anchimeric assistance) of the benzoyl
3
0
density functional theory (DFT) calculation show that the four group (inset within the circle in Fig. 2a). The high purity of
1
alkyl chains are well segregated from the carbohydrate-based each new detergent was confirmed by their individual H NMR
head groups (Fig. 1b & Fig. S1). The hydrophobic length in spectra (Fig. S2–S5†). For example, when measured in CD
3
OD,
these energy-minimized conformations increases from 7.7 to the axial anomeric protons of TCG-C5 attached to the anome-
1
1.0 to 13.2 Å when the detergent alkyl chain is extended from ric carbons, designated H and H (Fig. 3a & S2†), produced
a a′
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the glycerol linker (see supplementary Scheme S2†) and there
are multiple β-anomeric protons with dissimilar chemical
environments in these linker-bearing detergents, as observed
by the individual spectra (Fig. S4 & S5†). Peak appearance in
the range of 4.3 to 4.7 ppm with an integration value of eight
in the spectra, however, indicates that all newly formed glycosi-
dic bonds have β-stereochemistry.
The identity of the new detergents was further confirmed
1
3
by their C NMR spectra. Note that the TCGs and TCMs are
optically active because of a lack of a symmetry plane yet they
contain a molecular symmetry axis (i.e. C2 axis) vertically
passing through the central part of the molecules. Consistent
1
3
Fig. 3 (a) The chemical structure of TCG-C5 is given to illustrate the with this molecular symmetry, TCG-C5 showed three C NMR
axial anomeric protons (H
a
and Ha’) and equatorial anomeric protons peaks at 94.9, 104.5, and 103.5 ppm corresponding to the
(
(
e
H ) and their couplings with the neighboring protons (H in blue color).
anomeric carbons (C1, C17 and C23), respectively (Fig. 3a
and c). The upfield chemical shift of the anomeric carbon C1
1
b) Partial H NMR spectrum in the anomeric region for TCG-C5
showing its high anomeric purity. The NMR spectrum of TCG-C5 gave
(
(
δ = 94.9 ppm) with respect to the other anomeric carbons
C17 and C23) is probably due to the shielding effect (also
two doublets at 4.64 and 4.34 ppm, along with a coupling constant
3
(
J
aa) of 8.0 Hz, typical peak characteristics of β-anomeric protons.
TCG-C5 also contains α-anomeric proton (H
e
), giving doublets at termed an anomeric effect) of a lone pair on the nearby oxygen
3
5
.18 ppm with a reduced coupling constant ( Jae = 4.0 Hz). (c) A partial
C NMR spectrum of TCG-C5. Only anomeric carbon signals for
atom donating electron density into the σ* molecular orbital
of the C–O bond as indicated by a red arrow in Fig. 3a.
Most of the new agents were water-soluble up to more than
1
3
TCG-C5 are assigned.
2.5 wt%, with no observed precipitation over a month at
room temperature (Table 1). With moderate water-solubility
two separated peaks at 4.64 and 4.34 ppm as doublets, respect- (∼2.0 wt%), TCG-C7, TCM-C10 and TCG-L12 required a brief
3
ively. In addition, the vicinal coupling constants ( J ) for these sonication for complete dissolution. Critical micelle concen-
aa
anomeric protons (Ha and H ) were 8.0 Hz, typical of a trations (CMCs) were determined by monitoring solubilization
a′
β-anomer, representing an exclusive formation of β-glycosidic of a hydrophobic fluorescent dye (i.e., diphenylhexatriene
3
1
bonds in the glycosylation step. Note that the α-anomeric (DPH)) and the hydrodynamic radii (R ) of the self-assem-
h
proton (H ) present in the central part of the trehalose moiety blies were estimated through dynamic light scattering (DLS)
e
in TCG-C5 produced a peak that is downfield shifted to measurements. The results obtained for the novel agents are
3
5
.18 ppm with a relatively small coupling constant ( J = 4.0 summarized in Table 1, along with DDM. Of the three sets, the
ae
1
Hz) (Fig. 3a and b). As for the TCMs, the H NMR spectra were TCGs with relatively short alkyl chains gave the highest CMCs.
6 3
measured in DMSO-d due to their solubility issues in CD OD TCG-C5 and TCG-C6 gave CMCs (0.20 and 0.15 mM, respect-
and gave no clear indication of the stereochemistry of the ively) comparable to that of DDM (0.17 mM), while that of
newly formed glycosidic bonds because of peak overlap TCG-C7 was about six times lower than that of DDM (0.03 vs.
(Fig. S3†). In the cases of TCG-Ls with the good solubility in 0.17 mM). The CMCs of the TCMs and TCG-Ls (from 0.004 to
CD OD, the anomeric peaks were broader and more complex 0.020 mM) were much lower than that of DDM, which indi-
3
than the TCGs. This is because the OsO -catalyzed dihydroxyla- cates a stronger tendency of these agents to self-assemble com-
4
tion produced epimeric isomers at the asymmetric carbons in pared to DDM. As expected from the increased hydrophobicity,
Table 1 Molecular weights (MWs), critical micelle concentrations (CMCs), water-solubility of novel agents (TCGs, TCMs and TCG-Ls) along with a
conventional detergent (DDM) and hydrodynamic radii (R
h
; n = 4) of their micelles in water at room temperature
a
b
Detergent
MW
CMC (mM)
CMC (wt%)
R
h
(nm)
Solubility (wt%)
TCG-C5
TCG-C6
TCG-C7
TCM-C8
TCM-C9
TCM-C10
TCG-L9
TCG-L10
TCG-L11
TCG-L12
DDM
1271.4
1327.5
1383.6
2088.3
2144.4
2200.5
2440.7
2496.8
2552.9
2609.0
510.1
∼0.20
∼0.15
∼0.03
∼0.02
∼0.009
∼0.005
∼0.01
∼0.008
∼0.006
∼0.004
0.170
∼0.025
28 ± 1.0
35 ± 1.0
39 ± 1.1
20 ± 1.0
22 ± 1.0
25 ± 1.0
2.9 ± 0.0
3.0 ± 1.0
3.1 ± 0.1
3.2 ± 0.0
3.4 ± 0.0
∼5.0
∼2.5
∼2.0
∼2.5
∼2.5
∼2.0
∼10
∼0.020
∼0.0042
∼0.0042
∼0.0019
∼0.0011
∼0.0024
∼0.0020
∼0.0015
∼0.0010
0.0087
∼10
∼5.0
∼2.5
>10
a
b
Molecular weight of detergents. Hydrodynamic radius of detergent micelles measured at 1.0 wt% by dynamic light scattering.
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within a set of detergents, CMC values were decreased
with increasing alkyl chain length. For instance, the CMCs of
the TCMs reduced from 0.02 to 0.01 to 0.005 with the alkyl
chain length increasing from C8 to C9 to C10. A comparison
of detergents with the same chain length revealed only minor
differences in the CMCs. For example, the CMC of TCM-C9
was marginally lower than that of TCG-L9 (∼0.009 vs.
∼
0.01 mM). This result indicates that the hydrophilic group
has little effect on the CMC. Overall the new detergents, par-
ticularly TCMs and TCG-Ls, gave lower CMCs than detergents Fig. 4 (a) DLS profile and (b) TEM image of TCG-L10. Size distribution
0.01 to 20 mM) popularly used for membrane protein crystalli- of TCG-L10 micelles was determined by DLS. The detergent was used at
(
3
2
1.0 wt% for DLS and TEM experiments. A minor discrepancy in the
micelle size of TCG-L10 observed between DLS and TEM data could be
an artifact originating from different sampling conditions (wet vs. dry).
DLS: dynamic light scattering; TEM: transmission electron microscope.
zation. Thus, the new agents can be used in relatively low
concentrations for membrane protein studies, but are likely to
form strong interactions with the protein and thus maybe
difficult to exchange.
The TCGs with a tetra-glucoside head group formed rather
large self-assemblies ranging from 28 to 39 nm. Head group
conversion from a tetra-glucoside (TCGs) to a tetra-maltoside light-harvesting complex I and the reaction centre complex
3
4,35
(
TCMs) resulted in a substantial reduction in the sizes of deter- (LHI-RC).
This LHI–RC super-assembly contains a large
gent self-assemblies to the range of 20 to 25 nm although number of protein subunits, and is thus highly susceptible to
these were still significantly larger than the size of DDM self- protein denaturation. In addition, the complex contains mul-
assemblies (3.4 nm). When a bulky octa-glucoside was intro- tiple cofactors such as chlorophylls and carotenoids in the
duced as a head group (TCG-Ls), a marked decrease in the self- interior. In an intact structure, the LHI-RC complex gives
assembly size was observed. The micelle sizes of the TCG-Ls rise to a strong absorbance at 875 nm (A875), which decreases
2
7b,c
were even smaller than that of DDM (2.9 to 3.2 nm vs. 3.4 nm). dramatically with protein denaturation.
Thus, we utilized
This change in the self-assembly size dependent on the deter- optical spectroscopy to assess the complex stability over time.
gent head group is likely to be due to a change in the geometry Here, the super-assembly was extracted from the native mem-
(
i.e., change in the relative volume ratio of the head and tail brane with 1.0 wt% DDM and purified in 0.0087 wt% of the
3
3
2+
groups). The TCGs with the smallest head group gave the same detergent (i.e. 1xCMC) using Ni -NTA affinity chromato-
largest self-assemblies while the TCG-Ls with the largest head graphy. The DDM-purified LHI-RC complexes were diluted
group formed the smallest self-assemblies. Thus, the current into buffer solutions containing the individual detergents
study illustrated a way to systematically vary the self-assembly (TCGs, TCMs, and TCG-Ls) to give final detergent concen-
size by introducing stepwise changes in the head group trations of CMCs + 0.05 wt%. OG and DDM were used as
volume. The sizes of self-assemblies formed by the individual control agents as these are representative glucoside and malto-
sets of detergents also tended to vary with the alkyl chain side detergents, respectively. The protein sample was incu-
length although the size variations were relatively small. For bated at 25 °C for the first 10 days and then incubated at 35 °C
instance, the self-assembly size was increased from 20 to 22 to for the next 10 days. This temperature variation allowed us to
2
5 nm when alkyl chain lengths of the TCMs were increased investigate how sensitive the integrity of the LHI-RC complex
from C8 to C9 to C10. Similar trends were observed for the is to an elevated temperature (Fig. 5a, S7†).
TCGs and TCG-Ls. When we investigated the size distribution
Utilizing the strong absorbance of the native complex at
of detergent assemblies, all new agents showed only one set 875 nm (A875), we monitored the complex integrity at regular
of micellar populations, indicative of high homogeneity intervals during the 20-day incubation at room temperature.
(Fig. S6†).
As can be seen in Fig. 5a, the LHI-RC complex in OG under-
TCG-L10 micelles were further characterized by trans- went rapid degradation, while DDM-solubilized complexes
mission electron microscopy (TEM) analysis. The TEM image showed a gradual decrease in A875 over time. The rate of
reveals that TCG-L10 forms spherical micelles with an esti- protein degradation was faster when the incubation tempera-
mated size of ∼7 nm (Fig. 4b) in diameter, roughly consistent ture was increased to 35 °C. In DDM, less than 5% complex
with DLS data (D
observed from the TEM images of TCG-C7 and TCM-C10 The TCGs were better at maintaining the native structure of
Fig. S7†). Wet and dry samples were used for DLS and TEM the complexes than DDM (Fig. S8a†) but the most stable
measurements, respectively, which would be a reason for such complex was obtained using the TCMs and TCG-Ls. These
h
= 6.0 nm) (Fig. 4a). Similar trends were integrity was retained at the end of the incubation (day 20).
(
a minor deviation in the micelle size between these two data.
agents retained 75–80% complex integrity at the end of the
incubation period. An increase in detergent concentration
from CMCs + 0.05 wt% to CMCs + 0.2 wt% resulted in a less
Detergent evaluation with membrane proteins
The novel agents were evaluated using the photosynthetic stable complex in both DDM and OG (Fig. 5b) but little effect
Rhodobacter (R.) capsulatus super-assembly, comprising the was observed in the case of the new agents (Fig. 5b & S8b†). It
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Fig. 5 Long-term stability of R. capsulatus superassembly comprising
light harvesting complex I and reaction centre (LHI-RC) solubilized in
individual detergents (DDM, OG, TCMs and TCG-Ls). Detergents were
used at two different concentrations (CMC + 0.05 wt% (a) and CMC +
0
.2 wt% (b)). Protein integrity was assessed by measuring the absor-
bance value at 875 nm (A875) over the course of a 20-day incubation.
The samples were incubated at 25 °C for the first 10-days, followed by
another 10-day incubation at an elevated temperature of 35 °C, as indi-
cated by the vertical dotted lines. The results are expressed as percen-
tages of an absorbance value measured at the 0-day time point. Error
bars, SEM, n = 2.
2
Fig. 6 Time course stability of LeuT (a and b) and β AR (c) solubilized in
the novel detergents. A conventional detergent (DDM) was used as a
control. For LeuT stability, TCMs and TCG-Ls were tested at CMCs +
0
.04 wt% (a) and CMCs + 0.2 wt% (b) while two selected TCMs (TCM-C9
is noteworthy that all the TCMs and TCG-Ls were far more and TCM-C10) at CMCs + 0.2 wt% were used for β AR stability analysis
2
effective than DDM and the TCGs at stabilizing the LHI–RC (c). LeuT stability was assessed by monitoring the ability of the transpor-
3
ter to bind a radiolabeled substrate ([ H]-leucine (Leu)) at regular inter-
long-term.
vals over a 13-day incubation at room temperature. Error bars, SEM, n =
These novel agents were further evaluated with the leucine
transporter (LeuT), a prokaryotic homologue of the mamma-
2
–3. β
to bind a radio-labeled ligand ([ H]-dihydroalprenolol (DHA)) at regular
lian neurotransmitter/sodium symporter (NSS) family from intervals over a 3-day incubation at room temperature. Error bars, SEM,
2
AR stability was assessed by measuring the ability of the receptor
3
3
6
Aquifex aeolicus. The transporter was initially extracted from n = 3.
membranes with 1.0 wt% DDM and subsequently purified in
0
.05 wt% of the same detergent. The DDM-purified LeuT was
diluted into buffer solutions containing individual agents stability over the course of the incubation. At this higher deter-
TCGs/TCMs/TCG-Ls) or DDM to give final detergent concen- gent concentration, however, the TCG-Ls appeared to be better
(
trations of CMCs + 0.04 or CMCs + 0.2 wt%. We assessed than the TCMs, with the best performance observed for
3
protein stability by monitoring radiolabeled leucine ([ H]-Leu) TCG-L12 (Fig. 6b). Although unclear in the TCM cases, deter-
3
7
binding via scintillation proximity assay (SPA) at regular gent efficacy of the TCG-Ls for transporter stability tended to
intervals over a 13-day incubation at room temperature. At increase with increasing alkyl chain length at this high deter-
both detergent concentrations, the DDM-solubilized LeuT gent concentration. Of the new agents, TCG-L12 with the
underwent a gradual loss in substrate binding ability over longest alkyl chain was most effective at retaining the transpor-
the incubation period (Fig. 6a and b). The TCG-solubilized ter activity at both concentrations (Fig. 6a and b).
transporters were substantially worse than DDM at maintain-
ing transporter activity (Fig. S9†). The lipophilic length of this coupled receptor (GPCR), the human β
set (C5 to C7) seems to be too short to stabilize the transpor- (β
We further evaluated the new agents using a G-protein
adrenergic receptor
2
AR). The receptor was first extracted from the membranes
2
3
8
ter. In contrast, all the TCMs and TCG-Ls were markedly better using DDM and purified in the same detergent. The DDM-pur-
than DDM at maintaining transporter stability long-term. ified receptor was diluted in buffer solutions containing either
There was no noticeable difference in detergent efficacy for the individual new agents or DDM. The final detergent concen-
protein stabilization between the TCMs and TCG-Ls under the trations were CMCs + 0.2 wt%. As a direct assessment of recep-
conditions tested, but detergent efficacy tended to improve tor stability in the individual agents, the ability of the receptor
3
with increasing alkyl chain length: the best detergent was to bind the radioactive antagonist ([ H]-dihydroalprenolol
3
9
TCG-L12 and the worst detergent was TCM-C8. A similar deter- (DHA)) was measured. Preliminary analysis obtained by
gent efficacy order was obtained when detergent concen- measuring the initial ligand binding of the receptor following
trations were increased to CMCs + 0.2 wt%. Again, the TCMs 30 min sample dilution indicated that only two compounds
and TCG-Ls were superior to DDM and the TCGs for LeuT (TCM-C9 and TCM-C10) had detergent efficacy comparable to
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that of DDM (Fig. S10†) with the rest of the amphiphiles efficacy for receptor stabilization was enhanced with increas-
showing reduced efficacy. Detergent efficacy for receptor stabi- ing alkyl chain length. Due to a strong binding to the protein
lity tended to increase with increasing alkyl chain length, as surface, a long alkyl chain detergent would be generally most
observed for LeuT. For the two selected amphiphiles (TCM-C9 suitable for membrane protein stability, especially for aggrega-
and TCM-C10), detergent efficacy was further investigated by tion-susceptible proteins. This is consistent with the popular
monitoring ligand binding activity of the receptor at regular use of DDM, a C12 alkyl chain conventional detergent, com-
intervals over a 3-day incubation at room temperature (Fig. 6c). pared to n-decyl-β-D-maltoside (DM) and OG for membrane
The DDM-solubilized receptor showed high initial activity, but protein research. However, this long alkyl chain preference for
a rapid loss in receptor activity was observed, giving only protein stability could not explain the inferior behavior of
2
∼10% residual activity at the end of the incubation (day 3). TCG-L11/L12 to TCM-C9/C10 for β AR stability observed here.
TCM-C9 was a little worse than DDM at preserving receptor This is because the detergent head group also plays an impor-
activity long term. However, TCM-C10 showed a more gradual tant role in detergent efficacy for protein stabilization. For
decrease in receptor activity than DDM, retaining nearly 30%
2
β AR stability, the tetra-maltoside TCMs appeared to be better
activity at the end of the incubation (Fig. 6c). Collectively, the than the octa-glucoside TCG-Ls, as exemplified by the com-
detergent efficacy order for receptor stability was TCMs > parison of the TCM-C9/C10 with TCG-L9/L10. Despite having
TCG-Ls ≈ TCGs, with the best performance observed for the same alkyl chain length, TCM-C9/C10 induced greater
TCM-C10.
β2AR stability than the TCG-L analogs. In the case of LeuT, the
octa-glucoside detergents (TCG-Ls) appeared to be best for
transporter stability, followed by tetra-maltoside TCMs and
tetra-glucoside TCGs. Therefore, both the detergent alkyl chain
length and head group identity are key players in stabilizing
Discussion
In this study, we introduced trehalose-cored glucosides (TCGs/ membrane proteins.
TCG-Ls) or maltosides (TCMs) with four alkyl chains and mul-
tiple carbohydrate units. When evaluated with multiple mem- stability in long alkyl chain detergents.
Most previous studies have demonstrated enhanced β AR
2
1
5,18,29
Thus, we
brane proteins, we found a large variation in detergent efficacy expected that the TCG-Ls, particularly TCG-L11 and TCG-L12,
for protein stabilization depending on the target protein tested would be better than the TCM-C9/C10 for β AR stability. This
2
here. All three sets of the new agents were significantly better was our reasoning for the preparation of this third set of
than DDM at stabilizing the LHI-RC complex. A similar result amphiphiles. However, the TCG-Ls were inferior to the TCMs
was observed with the TCMs and TCG-Ls for LeuT stability, for receptor stability, indicating the presence of an additional
but the TCGs were worse than DDM for this transporter. When factor critical for detergent efficacy with respect to GPCR stabi-
the new agents were evaluated with β
pound (TCM-C10) was better than DDM at stabilizing the β2AR stability was a small CMC.
2
AR, only one new com- lity. An additional feature previously reported as favourable for
1
8,29
As the TCG-Ls and TCMs
receptor. The protein-specific nature of detergent efficacy have very similar CMCs, this is unlikely to be the reason for
observed here is consistent with a general notion that there is the different receptor stabilities obtained. Notably, due to the
no single solution to effective maintenance of membrane pro- large volume of the head group (i.e., octa-glucoside), the
teins in aqueous solution. This is due to large variations in TCG-Ls formed small micelles even in the case of the C12 alkyl
dimensions and properties of membrane proteins. For chain detergent (TCG-L12; 3.2 nm), significantly deviating
example, LHI-RC is known to be denaturation-sensitive while from the tetra-maltoside TCMs which formed substantially
4
0
2
LeuT and β AR are prone to aggregation. Despite the protein- larger self-assemblies (20–25 nm). Thus, the small micelle size
specificity, it is notable that TCM-C10 displayed favorable is likely to be responsible for the poor receptor stability seen
behaviors for all three membrane proteins (LHI-RC, LeuT and in the TCG-Ls. When micelle sizes of the TCG-Ls were
β AR) tested here compared to DDM, a gold standard conven- increased from 2.9 (TCG-L9) to 3.0 (TCG-L10) to 3.1 (TCG-L11)
2
tional detergent. TCG-L12 was very effective at stabilizing two to 3.2 nm (TCG-L12), these linker-bearing agents showed
of three membrane proteins (LHI-RC and LeuT). Detergent rapidly enhanced receptor stability, further supporting the
efficacy for protein stabilization tended to be strongly depen- importance of the micelle size in receptor stability. As small
dent on the alkyl chain length (Fig. S1†). In the case of LeuT, a micelles are less able to shield the hydrophobic surfaces of
detergent with a long alkyl chain was generally better than one membrane proteins, a small micelle-forming detergent might
with a short chain. The set of TCG-Ls with the relatively long be suboptimal at stabilising membrane proteins, particularly
alkyl chains (C11 to C12) was the best for LeuT stability, fol- aggregation-prone ones. Small LDAO and OG micelles are typi-
lowed by the set of TCMs with an intermediate chain length cally far less effective than relatively large DDM micelles at sta-
(C8 to C10). The TCGs with the short alkyl chain (C5 to C7) bilising membrane proteins. Thus, one way to improve TCG-L
were the least effective. Within each set of TCGs/TCMs/TCG-Ls, efficacy for β AR stabilization is to use a branched alkyl chain
2
in addition, detergent efficacy for protein stabilization tended instead of a straight chain, which will increase the volume of
to improve with increasing alkyl chain length. A similar trend the detergent hydrophobic group, thereby resulting in enlarge-
was found in detergent evaluation with β
2
AR. Within individ- ment of the TCG-L micelles. Based on our analysis of the
ual detergent sets, particularly for the TCG-Ls, detergent current results, at least four key factors (i.e., alkyl chain length,
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Organic & Biomolecular Chemistry
head group identity, CMC and micelle size) must be con-
sidered when designing a novel detergent for membrane
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novel detergent with optimal behaviour for multiple mem-
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6
7
8
We have prepared UV-transparent trehalose-cored glucosides
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Conflicts of interest
The authors declare the following competing financial interest
(c) K. H. Cho, H. E. Bae, M. Das, S. H. Gellman and
(
(
s): P. S. C. and M. D. are inventors on a patent application
number: 10-2018-0155778) that covers the TCGs, TCMs and
P. S. Chae, Chem. – Asian J., 2014, 9, 632–638.
1
2 (a) Q. Zhang, X. Ma, A. Ward, W.-X. Hong, V.-P. Jaakola,
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Acknowledgements
This work was supported by the National Research Foundation
of Korea (NRF) funded by the Korean government
2013, 110, E1203–E1211.
1
3 H. Hussain, Y. Du, N. J. Scull, J. S. Mortensen, J. Tarrasch,
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