Communication
between DDM and XMA-4- or XMA-5-solubilised protein were
observed (Figure 3a and Figure S4 in the Supporting Informa-
tion). The ability of b AR solubilised in XMA-4 or XMA-5 to
2
properly activate G-protein was characterized by G-protein-
[14a]
coupling assay.
As can be seen in Figure 3a, the bimane
spectra of XMA-4-solubilized receptor–G-protein complexes are
similar to that of the DDM-solubilized complex. A similar trend
was observed using XMA-5-solubilized receptor (Figure S4).
The results indicate that these two agents behave well for re-
ceptor activation by agonist binding and G-protein coupling.
The reduction in fluorescence intensity and the shift in maxi-
mal emission wavelength observed is ascribed to conforma-
tional changes of b AR associated with the transition from the
2
inactive to active state caused by the binding of both ISO and
[14a,n]
G-protein.
Detergent efficacy (XMAs vs. DDM) was further
compared by diluting these agents far below their respective
CMC values. As shown in Figure 3b, DDM-solubilized b AR un-
2
derwent an obvious conformational change by this dilution
while XMA-4- and XMA-5-solubilized receptors underwent only
minor changes, suggesting a slow off-rate for these new
agents from the receptor compared to DDM (Figure S5 in the
Supporting Information). These intriguing results prompted us
Figure 2. SDS-PAGE and Western blot analysis of MelBSt. Identical amounts
of membrane containing MelBSt were treated with the individual detergents
(XMA-1–5, and DDM) at 1.5 wt% for 90 min at the specified temperatures,
and the samples were analysed by SDS (16%)-PAGE following ultracentrifu-
gation. The amount of soluble protein was detected using Western blotting
with anti-His tag antibody. An untreated membrane sample (“Memb”) was
included as a control.
to carry out the ligand-binding assay for b AR after detergent
2
exchange. The receptor activity purified in DDM and XMAs
3
3
were assessed by binding of [ H]dihydroalprenolol ([ H]DHA).
The receptor purified in both XMA-4 and XMA-5 showed
a level of radioligand binding affinity similar to DDM-purified
receptor, indicating that these two XMAs could be useful alter-
protein solubilisation could be increased by
a longer
incubation time. For example, we found a substantial increase
in solubilisation efficiency of the novel agents when the
experiment was carried out at 48C, overnight (Figure S2).
These promising results of the novel agents for membrane
protein stabilisation prompted us to test them with human b2
natives to DDM, the best conventional detergent for b AR
2
study. Next, we moved to the leucine transporter (LeuT) from
[
24]
Aquifex aeolicus, for the evaluation of the novel agents to
stabilise a secondary active transporter. Protein activity in
aqueous solutions supplemented with individual XMAs or
DDM was measured by a scintillation proximity assay (SPA) at
regular intervals over 12 day incubation period at room tem-
adrenergic receptor (b AR), a G-protein coupled receptor
2
[
21]
(
GPCR). In order to explore the effect of the novel agents on
[25]
À1
the conformational change of b AR, we measured fluorescence
perature. DDM-purified transporter at 1.5 mgmL was used
as a stock solution. At a detergent concentration of CMC+
0.04 wt%, all XMAs were inferior to DDM (Figure S6a in the
Supporting Information). When we increased detergent con-
centration to CMC+0.2 wt%, only XMA-4 and XMA-5 showed
comparable efficacy to DDM only in the latter part of the
incubation period (i.e., from day 5 to 12), indicating that most
XMAs have limited stabilising effect on this particular
membrane protein as compared to DDM (Figure S6b).
2
changes in a bimane fluorophore associated with alterations in
[22]
receptor conformation upon ligand and G-protein binding.
The bimane moiety is covalently attached to cysteine 265 lo-
cated at the cytoplasmic end of transmembrane helix 6 (TM6).
Thus, receptor conformation as well as conformational changes
associated with inactive and active states of the receptor could
be precisely detected by the changes of fluorescence emission
spectrum of the monobromobimane-labelled b AR (mBBr-
2
[
23]
b2AR).
For this experiment, DDM-purified mBBr-b AR at
Detergent efficacy is known to be membrane protein
specific. When we evaluated XMAs for four membrane
proteins systems and compared with DDM, the most common
2
À1
1
mgmL was diluted into individual detergent solutions at
a concentration of CMC+0.04 wt%, and the bimane fluores-
cence spectra were recorded in the absence or presence of
a high-affinity agonist, BI-167107 (Figure S3 in the Supporting
Information). Of the five new agents, XMA-4 and XMA-5 result-
ed in bimane spectra similar to that of DDM, indicating effec-
tive preservation of the receptor activity by these two XMAs.
Binding of a full agonist (e.g., BI) to the receptor is known to
be insufficient to fully activate the receptor, which further re-
[26]
conventional detergent for membrane protein study,
in
terms of membrane protein stabilisation efficacy, some new
agents appeared to be superior to DDM for Bor1 and MelBSt
while those agents were comparable to DDM for b AR and in-
2
ferior to DDM for LeuT. Of the XMAs, XMA-4 and XMA-5
showed generally favourable behaviour for protein stability;
even for LeuT, these agents are the most comparable to DDM.
We believe that these agents with C11 and C12 alkyl chains, re-
spectively, are the most hydrophobic of XMAs and thus have
optimal hydrophile–lipophile balance (HLB). In contrast, XMA-
[
14a]
quires G-protein binding.
A similar result was found in this
study for mBBr-b AR in the presence of the full agonist, isopro-
2
terenol (ISO), although slight differences in the bimane spectra
Chem. Eur. J. 2015, 21, 10008 – 10013
10011
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Cho, Kyung Ho
