Báo cáo Y học: Refolding of the Escherichia coli expressed extracellular domain of a7 nicotinic acetylcholine receptor Cys116 mutation diminishes aggregation and stabilizes the b structure pdf

Refolding of the
Escherichia coli
expressed extracellular domain
of a7 nicotinic acetylcholine receptor
Cys116 mutation diminishes aggregation and stabilizes the b structure
Victor I. Tsetlin
1
, Natalia I. Dergousova
1
, Ekaterina A. Azeeva
1
, Elena V. Kryukova
1
, Irina A. Kudelina
1
,
Elena D. Shibanova
1
, Igor E. Kasheverov
1
and Christoph Methfessel
2
1
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia;
2
Central Research Biophysics, Bayer, Leverkusen, Germany
Heterologous expression of the extracellular domains
(ECDs) of the nicotinic acetylcholine receptor (AChR)
subunits may give large amounts of proteins for studying the
functional and spatial characteristics of their ligand-binding
sites. The ECD of the a7 subunit of the homo-oligomeric a7

neuronal AChR appears to be a more suitable object than
the ECDs of other heteromeric neuronal or muscle-type
AChRs. The rat a7 ECDs (amino-acid residues  1–210)
were recently expressed in Escherichia coli as fusion proteins
with maltose-binding protein [Fischer, M., Corringer, P.,
Schott, K., Bacher, A. & Changeux, J. (2001) Proc. Natl
Acad.Sci.USA98, 3567–3570] and glutathione S-trans-
ferase (GST) [Utkin, Y., Kukhtina, V., Kryukova, E.,
Chiodini, F., Bertrand, D., Methfessel, C. & Tsetlin, V.
(2001) J. Biol. Chem. 276, 15810–15815]. However, these
proteins exist in solution mostly as high-molecular mass
aggregates rather than monomers or oligomers. In the pre-
sent work it is found that refolding of GST–a7-(1–208)
protein in the presence of 0.1% SDS considerably decreases
the formation of high-molecular mass aggregates. The
C116S mutation in the a7moietywasfoundtofurther
decrease the aggregation and to increase the stability of
protein solutions. This mutation slightly increased the
affinity of the protein for a-bungarotoxin (from K
d
 300 to
150 n
M
). Gel-permeation HPLC was used to isolate the
monomeric form of the GST–a7-(1–208) protein and its
mutant almost devoid of SDS. CD spectra revealed that the
C116S mutation considerably increased the content of
b structure and made it more stable under different condi-
tions. The monomeric C116S mutant appears promising
both for further structural studies and as a starting material

for preparing the a7 ECD in an oligomeric form.
Keywords: a7 nicotinic acetylcholine receptor; extracellular
domains; expression in Escherichia coli; Cys116 mutation;
CD spectroscopy.
Nicotinic acetylcholine receptors (AChRs), belonging to the
family of ligand-gated ion channels, are divided into two
major groups: muscle-type and neuronal receptors. The
muscle-type AChR from the electric organ of the Torpedo
ray has been studied most comprehensively. It is composed
of five subunits, namely two a subunits, and one b, c,andd
subunits. Mammalian muscle AChRs are similar, the only
difference being the presence of e subunits in the mature
receptors instead of c in the fetal versions. Neuronal AChRs
are composed of a subunits (a2–a10) and b subunits (b2–
b4), either as hetero-oligomers of a/b combinations or as
homo-oligomers, like pentaoligomeric a7 AChRs (reviewed
in [1–3]).
The current ideas on the spatial organization of the whole
family of nicotinic AChRs are based mainly on the electron
microscopy data for the Torpedo AChR. The subunits are
arranged pseudosymmetrically along the central axis form-
ing a channel. A general shape of the molecule and its
disposition in the membrane were established by electron
microscopy two decades ago [4]. Recent cryo-electron
microscopy data provided a better view of the channel
structure and of the extracellular portions. In particular, a
resolution of 4.6 A
˚
has been achieved for the extracellular
domain (ECD) moiety of the membrane-bound Torpedo

AChR [5]. This domain accommodates the binding sites for
agonists and competitive antagonists. Biochemistry and
molecular biology data also suggest that all other nicotinic
AChRs should have a structural/functional organization
similar to that of the Torpedo AChR (reviewed in [1–3]). In
principle, heterologous expression of ECDs may provide
sufficient amounts of proteins necessary for establishing
their high-resolution spatial structure with the aid of X-ray
analysis or NMR.
The ECD (amino-acid residues  1–210) of the mouse
muscle and Torpedo a subunits were obtained by heterolo-
gous expression in mammalian cells [6] and in E. coli [7–9],
respectively. The secondary structure of these proteins was
determined by CD spectroscopy. However, these proteins
apriorilack the contacts with other non-a subunits that also
participate in forming the ligand-binding sites in the intact
receptors. This drawback has been overcome by expressing
the ECDs of all Torpedo subunits that assemble into a
Correspondence to V. I. Tsetlin, Shemyakin-Ovchinnikov
Institute of Bioorganic Chemistry, Russian Academy of Sciences,
16/10 Miklukho-Maklaya Str., V-437 Moscow GSP-7, 117997 Russia.
Fax/Tel.: + 7095 335 57 33, E-mail:
Abbreviations: AChR, acetylcholine receptor; AChBP, acetylcholine-
binding protein; ECD, extracellular domain; MBP, maltose-binding
protein; GST, glutathione S-transferase; aBgt, a-bungarotoxin; IPTG,
isopropyl thio-b-
D
-galactoside; GdnHCl, guanidine hydrochloride.
(Received 6 December 2001, revised 8 April 2002,
accepted 29 April 2002)

Eur. J. Biochem. 269, 2801–2809 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02961.x
pentaoligomer in a baculovirus system [10].
1
The usefulness
of the ÔdomainÕ approach became even more clear after
solving the X-ray structure of the acetylcholine-binding
protein (AChBP) from Lymneae stagnalis
2
[11,12], a major
breakthrough in the field. This water-soluble protein has the
size of the AChR subunit ECD and forms homopentamers.
In view of this finding, it seems especially interesting to
prepare for structural analysis the ECD of the a7 AChR, i.e.
of the receptor known to function as a homopentamer [13].
In fact, there are already several publications on the a7ECD
heterologous expression. The chicken a7 ECD (including
also the transmembrane fragment M1) was isolated after
expression in Xenopus oocytes [14]. It formed an oligomer,
but the protein was obtained only in minute amounts. Large
amounts of the rat a7 ECD (residues 1–196) were obtained
recently in E. coli as a water-soluble fusion protein with the
N-terminally attached maltose-binding protein (MBP) [15].
However, the major obstacle for structural studies appears
to be the presence of aggregates with molecular masses
considerably exceeding that of pentaoligomers. A similar
problem was encountered in our laboratory with rat a7
ECD (residues 1–208) fused to glutathione S-transferase
(GST) [16] or to MBP.
Here we report optimization of refolding conditions,
which for the GST–a7-(1–208) protein resulted in the

diminished tendency for aggregation. The aggregation
could be further decreased and, under certain conditions
completely prevented, by blocking free SH group(s) in the
refolded protein or by mutating the Cys116 residue. It
allowed us to obtain the monomeric forms at relatively high
concentrations. This more stable C116S monomer appears
to be useful for structural studies and may serve as an
intermediate on the way to an oligopentamer. As a first step,
we determined its secondary structure by CD spectroscopy
and found a predominance of b structure, similar to what
was found in the extracellular moiety of the intact Torpedo
AChR [5], muscle a subunits [6–9] and what is characteristic
of AChBP [12].
EXPERIMENTAL PROCEDURES
Construction of expression vectors
The gene encoding the rat a7 AChR ECD was amplified by
PCR using the a7 cDNA cloned in the pBS SK(–) (provided
by H J. Kreienkamp) as template and primers 1/2 for
GSTa7-(1–208) and 5/6 for MBP-a7-(1–208) (see Fig. 1).
The PCR products were digested with appropriate restric-
tion enzymes (BamHI and HindIII for GST fusion proteins;
XmnIandBamHI for MBP fusion protein), gel-purified
using the QIAquick gel extraction kit (Qiagen) and ligated
with linearized pGEX-KG vector (Amersham Pharmacia
Biotech) for GST fusion proteins and pMAL-c2 vector
(New England Biolabs) for MBP-a7-(1–208). The ligation
reaction mixtures were used to transform E. coli cells
JM109 for GST fusion proteins and TB1 cells for MBP-a7-
(1–208). Potential clones were screened with colony PCR,
and the presence of the insert was confirmed by restriction

analysis.
To obtain the C116S mutant of the fusion protein GST–
a7-(1–208), designated GST–a7m-(1–208), site-directed
mutagenesis was performed in a two step PCR. The
forward mutagenic primer 3 (see Fig. 1) contained an SspI
restriction site. The codon CAG for Gln117 was exchanged
for CAA and the codon CTC for Leu119 for TTG to
generate an SspI restriction site. The reverse primer 4 also
contained an SspI restriction site. Each construct was
verified by DNA sequencing.
Proteins expression, purification and refolding
GST fusion proteins. JM109 cells carrying appropriate
constructs were grown in Luria–Bertani medium with
ampicillin (100 lgÆmL
)1
)at37°C (about 3 h). When the
D
600
reached a value of 0.4–0.6, isopropyl thio-
b-
D
-galactoside (IPTG) was added to final concentration
of 0.3 m
M
, and the bacteria were further cultured for 3 h at
37 °C. Cells were pelleted by centrifugation (15 min,
10 000 g) and stored at )20 °C. Both fusion proteins
GST–a7-(1–208) and its C116S mutant GST–a7m-(1–208)
were found in the inclusion bodies. The cells were suspended
in 10 m

M
Tris/HCl, 150 m
M
NaCl, 1 m
M
EDTA, pH 7.5,
and then disrupted by sonication (10 pulses by 30 s, 10 °C).
After centrifugation (10 min, 14 000 g), the inclusion bodies
were washed intensively three times with the above buffer
containing in a series 0.1% Triton X-100, 2
M
NaCl, and
then 2
M
urea. At each washing step the pellet was
resuspended by sonication. Finally, the pellet was washed
with 10 m
M
Tris/HCl, pH 7.5, harvested by centrifugation
(15 min, 20 000 g) and stored at )20 °C. Partially purified
inclusion bodies were solubilized in 50 m
M
Tris/HCl, 8
M
urea or 6
M
GdnHCl, 10 m
M
dithiothreitol at room
temperature, with gentle stirring overnight. The concentra-

tion of fusion protein at this step was no more than
1mgÆmL
)1
. Denaturing and reducing agents were removed
by dialysis against 20 m
M
Tris/HCl, pH 8.0, at 10 °C, 24 h.
Three different conditions were tested for refolding of the
GST fusion proteins: in the presence of 0.1% Chaps, 0.1%
SDS, or without any detergent in the protein solution and in
dialysis buffer. The concentration of each detergent was
Fig. 1. Schematic representation of recombinant fusion proteins con-
taining the rat a7ECD.The positions of the disulfide bridges in GST
and a7 ECD, as well as of the unpaired Cys116 in a7, are indicated
under the rectangles. 1–6, primers used for preparing the respective
cDNAs (reconstituted restriction enzyme recognition sites are in bold,
italics indicate the mutation sites).
2802 V. I. Tsetlin et al. (Eur. J. Biochem. 269) Ó FEBS 2002
below critical micellar concentration. Oxidation of the fully
reduced protein was performed on air
3
,at7°Candwas
monitored by titration of free SH groups [17]. If a precipitate
was formed, it was removed by centrifugation (15 min,
20 000 g), and the protein solution was stored at 4 °C.
GST was expressed in E. coli strain BL21 as a soluble
protein, and was purified by chromatography on glutathi-
one–agarose as recommended by manufacturer (Amersham
Pharmacia Biotech). The purified protein was denatured in
50 m

M
Tris/HCl, 8
M
urea, 10 m
M
dithiothreitol and
refolded under the conditions described above.
MBP fusion protein. Different E. coli strains were used to
optimize the MBP-a7-(1–208) expression. The best results
were obtained with a protease-deficient strain CAG597. The
cells with recombinant plasmid were grown in Luria–
Bertani medium with 0.2% glucose and ampicillin
(100 lgÆmL
)1
)at37°C (about 6 h). IPTG was added to
final concentration of 0.3 m
M
(D
600
¼ 0.4–0.6), and the
bacteria were further cultured overnight at 30 °C. Cells were
pelleted by centrifugation (10 min, 10 000 g) and stored at
)20 °C. MBP-a7-(1–208) was expressed mostly as a soluble
protein, smaller amounts being detected by SDS/PAGE in
the inclusion bodies.
MBP-a7-(1–208) was purified by chromatography on
amylose resin as recommended by manufacturer (New
England Biolabs), using 20 m
M
maltose for eluting the

fusion protein. After purification, the protein obtained was
unstable. However, it could be stored for over a month at
4 °C in the following buffer: 20 m
M
Tris/HCl, pH 7.5,
200 m
M
NaCl, 1 m
M
EDTA, 0.05% octyl-b-
D
-glucoside,
1m
M
sodium azide, 1 l
M
pepstatin A, 10 l
M
leupeptin,
10 l
M
chymostatin, 10 l
M
antipain, 10 l
M
bestatin, 1 m
M
phenylmethanesulfonyl fluoride.
MBP was expressed and purified under the same
conditions.

With the same protocol, the expression of the C116S
mutant of MBP-a7-(1–208) was carried out to give mostly a
soluble protein. However, in contrast to all other expressed
products, this mutated protein was found considerably
more toxic for E. coli and the level of expression was very
low; insufficient amounts of the purified MBP-a7m-(1–208)
protein were obtained to perform further studies.
Determination of protein concentration
Protein concentrations were determined using the Bradford
Protein assay (Bio-Rad) with bovine serum albumin as
reference, and by UV spectra. Extinction coefficients
(k 278 nm) for each fusion protein were determined as a
sum of all the extinction coefficients of the protein aromatic
amino acids.
SDS/PAGE and Western blotting
All protein samples were analyzed by SDS/PAGE according
to Laemmli [18] in a 12% gel. Samples were prepared under
denaturating reducing conditions (by boiling in a sample
buffer containing 1% 2-mercaptoethanol and 2% SDS) or
under nonreducing conditions (without boiling and with no
2-mercaptoethanol in a sample buffer containing 0.1%
SDS). For Western blotting, the proteins were transferred
from unstained SDS gels to a nitrocellulose membrane using
a TransBlot SemiDry Electrophoretic Transfer (Amersham
Pharmacia Biotech) at 30 V in 2 h. After blocking with 1%
bovine serum albumin in phosphate buffered saline, pH 8.2,
membranes were incubated with
125
I-labelled a-cobratoxin
from the Naja kaouthia cobra venom at 10 °Covernight.

Unbound toxin was removed by washing, and labeled
proteins were detected by autoradiography.
Gel-permeation HPLC
Gel-permeation HPLC was carried out on a Super-
dex 200 HR column (10 · 300 m
M
; Amersham Pharmacia
Biotech) by isocratic elution at a flow rate 0.5 mLÆmin
)1
in
20 m
M
Tris/HCl buffer, pH 8.0, containing 150 m
M
NaCl,
either in the presence of 0.1% detergent (SDS or Chaps) or
in the absence of any detergent. The column was calibrated
with standard proteins dissolved in the same buffers as the
expressed proteins under examination.
Binding experiments
125
I-Labelling of a-bungarotoxin (aBgt) and a-cobratoxin
(for Western blotting experiments) was carried out with the
chloramine T method followed by desalting on a G15
Sephadex column in 50 m
M
Tris/HCl buffer, pH 8.0, as
described previously [19]. Equilibrium binding was analyzed
on anion-exchange filters DE81 [20] in a fast filtration
modification [21]. Various amounts of

125
I-labelled aBgt
(from 1.5 to 70 pmol, specific radioactivity  25 CiÆmmol
)1
)
were incubated with 20 pmol of different proteins for 2 h at
room temperature in 50 lLof50m
M
Tris/HCl buffer,
pH 8.0. The final concentration of detergents in the reaction
mixtures was 0.004 or 0.06% for the proteins refolded in
0.1% SDS or 0.1% Chaps, respectively. The SDS concen-
tration was determined in reaction with p-rosaniline chloride
[22]. Nonspecific binding was determined by preincubation
for 1 h of the expressed products with a 100-fold molar
excess of a-cobratoxin isolated from the Naja kaouthia
cobra venom. As the additional controls, binding experi-
ments with the expressed GST or MBP were carried out
under the same conditions. Incubation mixtures were
applied on DE81 filters presoaked in 50 m
M
Tris/HCl
buffer, pH 8.0, containing 0.1% Triton X-100, and quickly
washed under vacuum with 5 mL of the same buffer. The
analysis of binding experiments was carried out using
ORIGIN
v5.0 (MicroCal Software, Inc.).
GST–a7-(1–208) fusion protein modification
with
N

-ethylmaleimide
A 0.5-mL volume of refolded fusion protein GST–a7-
(1–208) solution (0.2 mgÆmL
)1
)in20 m
M
Tris/HCl, pH 8.0,
was incubated with 10 m
M
dithiothreitol for 3 h at room
temperature. The reduced protein was dialyzed against the
same buffer without dithiothreitol for 20 h at 4 °Cand
oxidized by air. A decrease in the free SH content was
monitored by the Ellman’s method. The protein solution
containing 1.0 ± 0.1 SH per mol protein was obtained.
N-ethylmaleimide was added to a final concentration of
1m
M
, incubated for 15 min at room temperature, and then
the excess of N-ethylmaleimide was removed by dialysis.
The residual content of SH groups was found to be less than
0.15 mol SH per mol protein.
Ó FEBS 2002 Mutation and refolding of a7 extracellular domain (Eur. J. Biochem. 269) 2803
CD spectroscopy
CD spectra were recorded on a JASCO J-500A spectro-
polarimeter (Japan). The results were expressed as molar
ellipticity, [Q](degÆcm
2
Ædmol
)1

), with the average mean
amino-acid residue weight (MRW) of 115. The molar
ellipticity was determined as [Q] ¼ Q·100MRWÆc
)1
Æl
)1
,
where c is the protein concentration in mgÆmL
)1
, l is the
light path length in centimeters, and Q is the measured
ellipticity in degrees at a wavelength k. The instrument was
calibrated with (+)-10-camphorsulfonic acid, assuming
[Q]
291
¼ 7820 degÆcm
2
Ædmol
)1
[23]. Secondary structure
was calculated according to the program
CONTIN
for
globular proteins [24].
RESULTS
Expression in
E. coli
and refolding
of the GST– a7-(1–208) protein, its C116S mutant,
and MBP-a7-(1–208) protein

These fusion proteins are schematically depicted in Fig. 1.
Figure 2 shows the SDS/PAGE analyses in reducing
conditions of the (GST)-a7-(1–208) (lane 1) and of the
respective C116S mutant (lane 2) after refolding from
inclusion bodies in the presence of 0.1% SDS. A similar
picture was observed when these proteins were refolded
either in the presence of 0.1% Chaps or in aqueous buffer
without any detergents (data not shown). The 1–208
fragment was also successfully expressed as a soluble
MBP-a7-(1–208) protein and isolated with the aid of affinity
chromatography on an amylose resin (lane 3). A compar-
ison with the standards (lane 4) shows that all the proteins
obtained have apparent molecular masses in the expected
range.
On storage of the GST–a7-(1–208) protein refolded in the
absence of any detergents, precipitation usually occurred.
This protein could be kept in solution in concentrations up
to  1.2 mgÆmL
)1
if supplemented with 0.1% Chaps [16].
Somewhat higher concentrations ( 1.5–2.0 mgÆmL
)1
)
were achieved with refolding in the presence of 0.1%
SDS. The solutions in 0.1% SDS-containing buffers could
be stored at 4 °C for over a month, the C116S mutant being
especially stable. Although the MBP-a7-(1–208) was pro-
duced as a water-soluble protein, after purification it could
be kept for prolonged period in solution at concentrations
below 0.2 mgÆmL

)1
only if supplemented with 0.05% octyl-
b-
D
-glucoside and a cocktail of protease inhibitors.
Analysis of a-bungarotoxin binding
As seen from Fig. 3A, the expressed proteins specifically
bind
125
I-labelled a-cobratoxin on blots. Data on the
125
I-labelled aBgt equilibrium binding in solution are
compiled in Table 1 and illustrated in Fig. 3B for GST–
a7-(1–208) and its C116S mutant. The K
d
values are in the
range of 100–820 n
M
anddependontherefoldingcondi-
tions. When refolded in the presence of 0.1% SDS, the
GST–a7-(1–208) and its C116S mutant have very similar K
d
values (310 and 160 n
M
, respectively). The difference was
larger (180 and 820 n
M
) when refolding was carried out in
0.1% Chaps. For MBP-a7-(1–208) protein, K
d

values were
in the low micromolar range (data not shown).
It is known that aBgt binds to the full-size a7AChRin
the low nanomolar range [13,25]. A K
d
of 1.6–2.0 n
M
was
reported for the extended chicken a7 ECD expressed in the
Xenopus oocytes [14], whereas a water-soluble protein
MBP-a7-(1–196) produced in E. coli had a K
d
of 2.5 l
M
[15]. The highest affinity for GST–a7-(1–208) proteins was
 150 n
M
, which is still considerably weaker than for the
full-size a7AChR
4
. However, the GST–a7-(1–208) refolded
in 0.1% Chaps was able to distinguish the long-chain
a-neurotoxins from the short ones, as well as the a7AChR
targeting a-conotoxin ImI from the muscle-type targeting
a-conotoxin GI [16]. These properties of the intact a7
AChRs are preserved in the GST–a7-(1–208) refolded in
0.1% SDS (data not shown).
Analysis of aggregation state
The aggregation state of the fusion proteins containing the
rat a7 ECD was examined by SDS/PAGE (Fig. 4) and gel-

permeation HPLC (Fig. 5). In the presence of 2-mercapto-
ethanol, the GST–a7-(1–208) protein refolded in 0.1% SDS
is predominantly a monomer, whereas in the absence of the
reducing agent it contains also a considerable portion of
oligomers and high-molecular mass aggregates (cf. lanes 1
and 2 in Fig. 4).
For the chick a7 AChR heterologously expressed in a
mammalian cell line, it was recently shown that cysteines of
the ECD are involved in aggregation, which can be
considerably decreased by reducing agents or by treatment
with N-ethylmaleimide [26]. We decided to check whether
Fig. 2. SDS/PAGE of recombinant fusion proteins containing the rat a7
ECD. (1 and 2) GST–a7-(1–208) and GST–a7m-(1–208) fusion pro-
teins, respectively, after refolding from the inclusion bodies in the
presence of 0.1% SDS; (3) MBP-a7-(1–208) fusion protein after
affinity purification on amylose resin; (4) molecular mass markers (in
kDa).
2804 V. I. Tsetlin et al. (Eur. J. Biochem. 269) Ó FEBS 2002
modification with N-ethylmaleimide gave a similar effect for
the rat a7ECDexpressedinE. coli and the consequences of
deleting the SH group of the specified residue, Cys116, by
mutating the latter into a Ser.
After reduction–reoxidation of the GST–a7-(1–208) and
dialysis, only 1 SH group per mol was detected by Ellman’s
reagent. Because of the absence of free SH groups in GST
(see Fig. 1), there were reasons to believe that it was that of
Cys116. The N-ethylmaleimide treatment of the fusion
protein GST–a7-(1–208) refolded in 0.1% SDS leads to an
clear decrease in aggregates and oligomers on nonreducing
gels (cf. lanes 2 and 6 in Fig. 4). The effect of mutating

Cys116 is even more dramatic: there is almost no difference
between the SDS/PAGE under reducing and nonreducing
conditions (lanes 3 and 4 in Fig. 4), the monomer prevailing
in both cases. This result explains why the solutions of the
C116S mutant in 0.1% SDS are much less prone to
aggregation and precipitation on prolonged storage as
compared to the GST–a7-(1–208) protein itself.
The dependence of the aggregation state on the refolding
conditions and on the introduced mutation was examined
by gel-permeation HPLC on a Superdex 200 column
(Fig. 5). The GST–a7-(1–208) proteins, refolded in the
presence of 0.1% SDS, were subjected to chromatography
in the presence of 0.1% SDS, which is similar to SDS/
PAGE under nonreducing conditions. It was found that the
fraction of aggregates (marked with an asterisk) is consid-
erably smaller for the C116S mutant (Fig. 5A). The major
broad peak centred at  70 kDa originates mainly from a
monomeric fraction as indicated from its position on SDS/
PAGE under nonreducing conditions (data not shown; the
shoulders at < 43 kDa are the concomitant E. coli pro-
teins). The consequences of mutation are very pronounced
for the GST–a7-(1–208) proteins refolded in 0.1% Chaps.
Chromatography in a buffer containing the same detergent
showed in the GST–a7-(1–208) protein a large peak of
aggregates overlapping the oligomeric and monomeric
fractions, while for the C116S mutant a broad peak of
oligomers dominated (Fig. 5B).
The proteins obtained were also analyzed on a Super-
dex 200 column equilibrated in purely aqueous buffers. For
the GST–a7-(1–208) protein refolded in Tris/HCl buffer

containing 1 m
M
dithiothreitol and dialysed against the
same buffer without dithiothreitol, the intense peak of high-
molecular mass aggregates (m > 600 kDa) and a broad
peak with a centre at  450 kDa corresponding to oligo-
mers and monomers are present (Fig. 5C). It should be
noted that the resolution of the column is better here than in
the presence of 0.1% SDS, the standards on average elute
later (cf. Fig. 5A,C). For the GST–a7m-(1–208) protein,
also refolded in the absence of 0.1% SDS, the peak of
aggregates is decreased by  25%, the centre of broad peak
of oligomers shifts from 450 to 300 kDa (corresponding to
oligomers of five or six units). When the MBP-a7-(1–208)
protein, produced in a water-soluble form and not subjected
to action of such denaturants as 8
M
urea or GdnHCl, was
chromatographed under the conditions of Fig. 5C, only one
sharp peak of high-molecular mass aggregates was obtained
(Fig. 5D) showing that this protein is not promising for
further physicochemical studies. On the other hand, the
chromatography on Superdex 200 in the absence of 0.1%
SDS revealed a dramatic difference between the GST–a7-
(1–208) protein and its C116S mutant when they were
refolded in the presence of 0.1% SDS (Fig. 5E). Whereas
for GST–a7-(1–208) the peak of aggregates is much larger
than the broad peak centred at  70 kDa, the latter is
predominant for GST–a7m
5

-(1–208).
Thus, the presented SDS/PAGE and gel-permeation
HPLC data show that the problem of aggregation can be
partly solved by chemical modification of accessible sul-
fhydryl groups or by mutating the Cys116 residue. How-
ever, formation of intermolecular disulfides with the
participation of the Cys116 sulfhydryl is one of the factors
leading to aggregation, but not the sole one. A further
decrease in the aggregation extent depends only weakly on
the state of SH groups and requires the addition of 0.1%
SDS.
The monomeric fractions 2 (Fig. 5E) were collected for
further analysis. The K
d
values characterizing their inter-
Fig. 3. Interaction of fusion proteins containing a7 ECD with iodinated
a-neurotoxins. (A) Autoradiography of
125
I-labelled a-cobratoxin
binding by MBP-a7-(1–208) (lane1) and GST–a7-(1–208) (lane 2) on
blots. Lane 3, protein standards as detected by Coomassie staining of
the respective portion of the gel. (B)
125
I-Labelled aBgt binding curves
for GST–a7-(1–208) protein (filled circles) and GST–a7m-(1–208)
protein (open circles) refolded in 0.1% SDS. Before the binding assay,
the SDS concentration was decreased to 0.004% by dilution with
aqueous buffer. The indicated K
d
values are averaged from two inde-

pendent experiments and calculated using
ORIGIN
5.0.
Ó FEBS 2002 Mutation and refolding of a7 extracellular domain (Eur. J. Biochem. 269) 2805
action with
125
I-labelled aBgt (see Table 1) are very close to
those of the starting GST–a7-(1–208) and GST–a7m-
(1–208) proteins, confirming a somewhat higher affinity of
the C116S mutant. The SDS content in the pooled fractions
in the reaction with p-rosaniline chloride [22] was estimated
to be 0.0001%. When these fractions were reapplied to the
same column equilibrated without 0.1% SDS, only the
peaks of monomers were observed, virtually without traces
of aggregates (Fig. 5F, fraction 2). For comparison,
rechromatography of the pooled aggregates peaks is shown
(Fig. 5F, fraction 1).
CD spectra analysis
CD spectra of the GST–a7-(1–208) protein and its C116S
mutant look similar (Fig. 6), having the contributions from
both a helices and b structure
6
. Analysis of the CD curves
(Table 2) revealed that the content of the secondary
structure varies depending on the protein and the refolding
conditions. In general, these variations are smaller for the
C116S mutant: the secondary structure is very similar for
the protein refolded either in purely aqueous solution, or
refolded and analyzed in 0.1% SDS, or isolated from the
latter by HPLC in the absence of detergent. The average

content of the a helices, b structure and unordered confor-
mation is estimated to be 22, 45, and 33%, respectively. It is
also clear from the Table 2 that under all similar conditions,
the C116S mutant is characterized by almost a twofold
higher content of b structure than the starting GST–a7-
(1–208) protein. In contrast, the latter has a 1.5-fold higher
content of a helices and a somewhat higher percentage of
unordered structure. In view of the more stable secondary
structure of the mutant and its decreased tendency to
aggregation, we assume that the C116S mutant is a better
model of the monomeric a7 ECD than the starting GST–
a7-(1–208) protein. If we further assume that GST, which
does not change its secondary structure under different
conditions (Table 2), also preserves it in the a7ECDfusion
proteins, a rough estimate of the secondary structure can be
made for the a7 ECD moiety. Because GST and a7ECD
have very close molecular masses, their contributions to CD
curves should be almost equal. Using the averaged values
for the C116S mutant, we obtained the following values for
the a7 ECD: 17% a helix, 56% b structure and 27% of
unordered structure.
DISCUSSION
The results obtained, along with other recently reported
data [15,16], show that ECD of the rat a7AChRcanbe
heterologously expressed in E. coli as different fusion
proteins soluble, either in purely aqueous solutions or in
the presence of detergents. The respective proteins bind
aBgt and other long-chain a-neurotoxins with an affinity
which is not strongly dependent on the chosen variant of the
a7 ECD, production of proteins in water-soluble form or

their recovery from the inclusion bodies, or on refolding
with or without detergents. The earlier reported affinities for
MBP-a7-(1–196) in aqueous solution [15] and for GST–a7-
Table 1. Radioligand assay data of
125
I-labelled iodinated a-bungarotoxin binding by the proteins refolded under different conditions.
Protein
Detergent content in
refolding buffer (%)
125
I-Labelled aBgt binding parameters
K
d
(n
M
) B
max
(%)
b
GST–a7-(1–208) – 260 ± 170 6.6 ± 3.8
SDS, 0.1 310 ± 70 12.5 ± 3
Chaps, 0.1 180 ± 75 3.0 ± 0.5
Monomeric form
a
SDS, 0.0005 240 ± 160 1.7 ± 0.4
GST–a7m-(1–208) – 130 ± 75 3.3 ± 0.6
(C116S mutant) SDS, 0.1 160 ± 40 10 ± 1
Chaps, 0.1 820 ± 175 3.0 ± 0.4
Monomeric form
a

SDS, 0.0005 100 ± 60 1.6 ± 0.3
a
The monomeric forms were prepared by ultrafiltration of the HPLC purified monomeric fractions (see Fig. 5E,F) using an Amicon 8010
ultrafiltration membrane YM10. SDS content in the concentrated samples was determined in reaction with p-rosaniline chloride [22].
b
The
B
max
values are presented as a percentage ratio of the calculated B
max
in n
M
to the concentration of the respective protein (in n
M
) in the
incubation mixture. The data presented were calculated with the use of
ORIGIN
5.0.
Fig. 4. Analysis of the a7 ECDs aggregation state by SDS/PAGE in the
presence (+) or absence (–) of 1% 2-mercaptoethanol (ME). Lanes 1
and 2, GST–a7-(1–208); lanes 3 and 4, GST–a7m-(1–208) mutant;
lanes 5 and 6, GST–a7-(1–208) modified with N-ethylmaleimide, lane
7, molecular mass markers, kDa.
2806 V. I. Tsetlin et al. (Eur. J. Biochem. 269) Ó FEBS 2002
(1–208) in 0.1% Chaps [16] are in the low micromolar range.
The K
d
values obtained in this work are in the range of 100–
850 n
M

, the highest affinity being shown by the C116S
mutant of GST–a7-(1–208) protein refolded from the
inclusion bodies in the presence of 0.1% SDS (Table 1).
Although even this affinity is much weaker than that of the
intact a7 AchR, which binds aBgt with K
d
 1–5 n
M
[13,25], such important features of the a7 AChR selectivity
as the capacity to discriminate long- and short-chain
a-neurotoxins and various a-conotoxins, are retained in
the expressed a7 ECD (see Results).
High molecular mass aggregates, whose molecular mas-
ses are considerably larger that of a pentamer, are the major
obstacle to obtainaing soluble and correctly folded hetero-
logously expressed ECDs of AChRs. Aggregation usually
leads to insoluble precipitates. However, a7ECDsmay
contain high molecular mass aggregates even in solution
(Fig. 5). The major cause of poor solubility might have been
the necessity of isolating the proteins from inclusion bodies
under denaturing conditions and then to refold them.
Therefore, much effort has been made to produce the
AChR ECDs or their large portions as soluble fusion
proteins [15,27
7
]. In particular, this goal was achieved for the
rat a7 ECD in [15] and in the present work using similar
fusion proteins with MBP. However, these soluble proteins
contained various amounts of aggregates.
Aggregation and precipitation of the proteins obtained

from the inclusion bodies could be partly overcome by
optimizing the refolding conditions. If 0.1% SDS was
present from the stage of dissolving the inclusion bodies, the
GST–a7-(1–208) protein could be kept in solution (Tris/
HCl buffer, pH 8.0, 0.1% SDS) for a long time at
concentrations about 1 mgÆmL
)1
. Under these conditions
the major fraction of the protein is a monomer, but the
fraction of high-molecular mass aggregates is also quite
large (Fig. 5A). It is known that SDS is capable of inhibiting
the aggregation of bacterially expressed or denatured
proteins and therefore is widely used in refolding studies
[28–30]. It is assumed that the masking of hydrophobic
protein interfaces by detergent molecules results in
8
this
reduction in aggregation. There are also indications that
SDS diminishes the aggregation by inhibiting the formation
Fig. 5. Gel-permeation HPLC of the a7 ECD-containing fusion proteins
on a Superdex 200 HR column. (A) GST–a7-(1–208) (solid line) and
GST–a7m-(1–208) (dashed line) refolded in the presence of 0.1% SDS
are analyzed on the column equilibrated in 20 m
M
Tris/HCl, pH 8.0,
containing 150 m
M
NaCl (elution buffer) supplemented with 0.1%
SDS. Proteins (30–50 lg) were eluted at a flow rate of 0.5 mLÆmin
)1

.
The same conditions, with the exception of presence or absence of
detergents in the elution buffers, and designations apply to (B), (C) and
(E). (B) GST–a7-(1–208) and GST–a7m-(1–208) refolded in the pres-
ence of 0.1% Chaps, the column is equilibrated and run in the elution
buffer containing 0.1% Chaps. (C) GST–a7-(1–208) and GST–a7m-
(1–208) proteins refolded in the absence of detergents, the elution
buffer is without detergents. (D) MBP-a7-(1–208) protein analyzed in
the conditions of (C) (E) GST–a7-(1–208) and GST–a7m-(1–208)
proteins refolded in the presence of 0.1% SDS are analyzed as in (C)
and (D). (F) Rechromatography of the fractions marked with bars 1
(aggregates) and 2 (monomer) collected on chromatography of GST–
a7-(1–208) (E). In this figure solid and dashed lines correspond to
chromatography of the fractions 1 and 2 from (E), respectively. In all
figures, vertical short lines correspond to the elution times of protein
standards (with masses in kDa) under the chosen chromatographic
conditions, the asterisk marking the exclusion volume.
Fig. 6. CD spectra of fusion protein GST–a7-(1–208) (solid line) and
GST–a7m-(1–208) mutant (dashed line) refolded in 20 m
M
Tris/HCl,
pH 8.0, containing 0.1% SDS.
Ó FEBS 2002 Mutation and refolding of a7 extracellular domain (Eur. J. Biochem. 269) 2807
of intermolecular disulfide bridges [31]. Note that aggrega-
tion, oligomerization and the acquisition of aBgt binding
capacity by the a7andTorpedo AChRs were shown by
Green and coworkers to depend on the state of cysteine
residues in the ECDs of the a7anda subunits, respectively
[26,32]. The major role of the redox state of the disulfide
Cys128–Cys142 was assumed for the a7AChR[26],

whereas the involvement of the disulfide Cys192–Cys193
was demonstrated for the Torpedo a subunit [32]. The a7
ECD has the Cys128–Cys142 disulfide bond
9
characteristic
for the whole family of ligand-gated ion channels, as well as
the vicinal disulfide bond
10
Cys192–Cys193 (see Fig. 1)
present also in all other a subunits of neuronal and
muscle-type AChRs. On the other hand, Cys116 is present
only in a7anda8 subunits. We thought that the presence of
a free SH group of Cys116 might be one of the factors
leading to formation of intermolecular disulfides and
aggregates in the bacterially expressed a7ECDs.
Indeed, the GST–a7m-(1–208) protein on SDS/PAGE
under nonreducing conditions was present mainly as a
monomer, whereas the starting protein contained a large
proportion of aggregates (Fig. 4). A similar effect could be
achieved by blocking free sulfhydryl groups in GST–a7-
(1–208) protein by N-ethylmaleimide (Fig. 4). The consid-
erable decrease in aggregation caused by the C116S
mutation with the proteins refolded and analyzed in the
presence or absence of various detergents is illustrated by
HPLC data (Fig. 5). For the proteins refolded in the
presence of 0.1% SDS, chromatography on a column
equilibrated in a purely aqueous buffer allowed us to obtain
the monomeric fractions (Fig. 5E,F). These fractions bind
125
I-labelled aBgt with the affinities similar to those of the

starting proteins (Table 1). Because the mutant revealed
even higher affinity, the presence of the Cys116 free SH
group is not essential for aBgt binding.
CD spectra (Fig. 6 and Table 2) indicate that the GST–
a7-(1–208) and GST–a7m-(1–208) proteins refolded under
different conditions contain varying amounts of regular
secondary structure. Interestingly, under all similar condi-
tions the C116S mutant has about a twofold higher content
of b structure than the GST–a7-(1–208) protein. The C116S
mutant is less prone to aggregation (see Figs 4 and 5), binds
aBgt even better than the starting protein (Table 1), and its
CD curves are not very sensitive to the conditions of
refolding or measuring the spectra. Therefore, the confor-
mation of the a7 moiety in the mutant may be more similar
to the ECD conformation of the intact a7 AChR. Although
calculation of the secondary structure of the 1–208 fragment
from the CD data for the fusion protein GST–a7-(1–208)
and GST might seem arbitrary, we believe that it gives a
qualitatively correct conclusion: the measured relatively
high content of b structure is not largely due to the
contribution of GST.
A high content of b structure found experimentally for
the C116S mutant and the even higher content calculated
for the a7 ECD moiety is important. It is presumed that the
ECDs in different, hetero-oligomeric or homo-oligomeric
AChRs, have a similar spatial organization. CD analyses of
the mouse muscle a subunit ECD heterologously expressed
in mammalian cells [6] and of the Torpedo a subunit ECD
expressed in E. coli [7–9] gave an estimate of  50% for
b structure. These results are in good agreement with the

subsequently published data of cryo-electron microscopy,
which revealed a high content of b structure in the ECD of
the intact Torpedo californica AChR [5]. Our present results
suggest that b structure is an important element of spatial
organization of the a7ECD.
When our work was in progress, isolation of acetylcho-
line-binding protein (AChBP) from a mollusc Lymneae
stagnalis and X-ray analysis of the respective protein
heterologously expressed in yeast have been published
[11,12]. This protein, with 24% homology to a7ECD,
contains practically all amino-acid residues of the AChR
ligand-binding sites, has two invariant disulfides and lacks
extra Cys116 of a7. It has an immunoglobulin-like topology
(rich in b structure) and forms homopentamers [12]. There-
fore, at least in terms of secondary structure, the monomeric
form of the GST–a7m-(1–208) protein resembles the
protomer of the AChBP. This would justify further efforts
to prepare the oligomeric a7 ECD using the obtained
monomers as the starting material.
ACKNOWLEDGEMENTS
The authors are grateful to Dr H J. Kreienkamp for a7 cDNA clone, to
O. Ustinova for help with CD measurements and to Dr J. Freigang for
fruitful discussion. The work was supported by grants (to V. T.) from
Bayer AG (Leverkusen) and Russian Foundation for Basic Research.
Table 2. CD data of the different expressed proteins refolded under various conditions.
Protein
Detergent content in
refolding buffer
Concentration
(mgÆmL

)1
)
Calculated secondary structure (%)
abR
GST–a7-(1–208) – 0.20 43 12 45
– 0.10 44 27 29
0.1% SDS 0.16 29 36 35
Monomeric form
a
0.0005% SDS 0.13 39 23 38
GST–a7m-(1–208) (C116S mutant) – 0.21 21 44 35
– 0.84 20 47 33
0.1% SDS 0.20 19 47 34
Monomeric form
a
0.0005% SDS 0.12 29 40 31
GST – 0.20 28 35 37
0.1% SDS 0.90 27 31 42
a
See the respective note in the Table 1.
2808 V. I. Tsetlin et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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