Circular dichroism (CD) and attenuated total
reflection Fourier transform infrared (ATR-FTIR) spectroscopy are used
to establish the secondary structure of peptides containing one or more
transmembrane segments (M1-M4) of the Torpedo californica
nicotinic acetylcholine receptor (AChR). Peptides containing the M2-M3
and M1-M2-M3 transmembrane segments of the AChR
-subunit and the M4
segment of the
- and
-subunits were isolated from proteolytic
digests of receptor subunits, purified, and reconstituted into lipid
vesicles. For each peptide, an amide I vibrational frequency centered
between 1650 and 1656 cm
1 and negative CD absorption
bands at 208 and 222 nm indicate that the peptide is largely
-helical. In addition, the CD spectrum of a tryptic peptide of the
-subunit containing the M1 segment is also consistent with a largely
-helical structure. However, secondary structure analysis of the
-M1 CD spectrum indicates the presence of other structures,
suggesting that the M1 segment may represent either a distorted
-helix, likely the consequence of several proline residues, or may
not be entirely
-helical. Overall, these findings are consistent
with studies that indicate that the transmembrane region of the AChR
comprises predominantly, if not exclusively, membrane-spanning
-helices.
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INTRODUCTION |
The nicotinic acetylcholine receptor
(AChR)1 from the electric
organ of Torpedo californica is a pentameric glycoprotein
composed of four homologous, transmembrane subunits in a stoichiometry of
2,
,
,
(1, 2). Each of the subunits associate
about a central axis to form a cation-selective ion channel (for recent reviews, see Refs. 3 and 4). The AChR is the best characterized member
of a family of ligand-gated ion channels that includes the
-aminobutyric acid type A, the glycine, and the 5-hydroxytryptamine (serotonin) receptors (recent reviews include Refs. 5-7).
In the postsynaptic membrane, about half of the mass of the AChR
projects extracellularly beyond the lipid bilayer. This projection, which is made up of the hydrophilic, amino-terminal half of each AChR
subunit, contains the agonist binding sites and sites of N-linked glycosylation, whereas about 30% of the AChR is
within the bilayer and the remaining portion is in the cytoplasmic
domain (8-10). Hydropathy profiles indicate that each subunit contains four hydrophobic segments 20-30 amino acids in length, designated M1-M4, that were proposed to be membrane-spanning
-helices
(11-14). Although the transmembrane disposition of each of these
segments has been fairly well established (reviewed in Ref. 15), the nature of their secondary structure is at present controversial. Based
on recent electron microscopy results (Refs. 10 and 16; 9-Å
resolution), the transmembrane structure of the AChR is proposed to
consist of a ring of five
-helices surrounded by a rim of
-strands. The five helices, which are associated at the central axis, lining the pore of the ion channel, are presumed to be made up of
M2 segments from each subunit, based upon results of photoaffinity labeling studies done with non-competitive antagonists and functional studies of AChRs with mutations within M2 (reviewed in Refs. 17 and
18). Since no additional
-helices were apparent in the rim of
electron density flanking the M2 helices, the remaining transmembrane
structure, which would be made up of the M1, M3, and M4 hydrophobic
segments, was assumed to be organized as
-sheets (see also Ref.
19).
In contrast, studies using several different photoactivatable
hydrophobic probes have identified amino acid residues in contact with
the lipid bilayer in the M3 and M4 segments of each of the AChR
subunits (20-23); from the periodicity of labeled residues, it was
concluded that each of these segments is organized as a transmembrane
-helix. The structure of the AChR was also examined using a
combination of hydrogen-deuterium exchange and FTIR spectroscopy (24).
The presence of an exchange-resistant pool of
-helical peptides was
interpreted as providing evidence for a predominantly
-helical
secondary structure of the AChR transmembrane domains. The simplest
interpretation of these studies is that the hydrophobic segments M1-M4
in each of the AChR subunits are organized as a bundle of four
membrane-spanning
-helices.
Determining the structure of individual transmembrane segments in each
of the AChR subunit remains an important goal. This is particularly
true if, as the former set of studies suggest, the AChR
membrane-spanning domains comprise both
-helical and
-sheet type
structures. In the present study, we isolated peptides that contained
one or more transmembrane segments of the AChR and determined the
secondary structure of the lipid reconstituted peptide using both CD
and FTIR spectroscopy. The largely
-helical nature of each of these
peptides provides additional support for a model of the transmembrane
region of the AChR that comprises membrane-spanning
-helices.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Staphylococcus aureus V8 protease was
purchased from ICN Biochemicals and
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin from Worthington Biochemical Corp. Genapol C-100 (10%) was
purchased from Calbiochem. Asolectin, a crude soybean lipid extract,
was from Avanti Polar Lipids. Spectra/Por Dispo Dialyzers (molecular
weight cut-off = 2000) were from Spectrum.
AChR-rich Membranes--
AChR-rich membranes were isolated from
the electric organ of T. californica (Aquatic Research
Consultants, San Pedro, CA) according to the procedure of Sobel
et al. (25) with the modifications described previously
(26). The final membrane suspensions in 36% sucrose, 0.02%
NaN3 were stored at
80 °C under argon.
Preparation of Proteolytic Fragments of AChR Subunits Which
Contain One or More Transmembrane Segments--
AChR subunits were
resolved by SDS-PAGE (27) using 1.5-mm-thick 8% polyacrylamide gels
with 0.33% bis(acrylamide). Typically, 10-12 mg of AChR membranes
were resolved on a single 1.5-mm gel, with 40-50 mg processed in a
single experiment. Polypeptides were visualized by staining with
Coomassie Blue R-250 (0.25% w/v in 45% methanol and 10% acetic acid)
and destaining in 25% methanol, 10% acetic acid. Gels were soaked in
distilled water overnight and individual AChR subunit bands excised.
For the AChR
-subunit, the excised gel pieces were transferred to
the wells of individual 15% mapping gels (26, 28). The
-subunit was
proteolytically digested in the gel with S. aureus V8 protease and following staining and destaining the V8
fragments
-V8-10 (Asn339-Gly437) and
-V8-20 (Ser173-Glu338) were excised (21).
AChR subunits and V8 proteolytic fragments were isolated from the
excised gel pieces by passive elution (21, 29). The eluate was filtered
and the protein concentrated using a Centriprep-10 (Amicon). Excess SDS
was removed by acetone precipitation (overnight at
20 °C).
Receptor subunits,
-V8-10, and
-V8-20 were proteolytically
digested and peptides purified using protocols similar to those reported previously (21). For trypsin digestion, acetone-precipitated AChR subunits and V8 protease fragments were resuspended in a small
volume (~100 µl) of buffer (100 mM
NH4HCO3, 0.1% SDS, pH 7.8). The SDS
concentration was then reduced by diluting with buffer without SDS, and
Genapol C-100 (Calbiochem) was added, resulting in final concentrations
of 0.02% SDS, 0.5% Genapol C-100, and 1-2 mg/ml protein. Trypsin was
added up to a total of 1:5 (w/w) enzyme to substrate ratio for intact
subunits (~1 mg of protein) and to 1:1 (w/w) for
-V8-10 and
-V8-20. The digests were incubated at room temperature for 3-4
days and the resulting peptides separated on 1.5-mm-thick small pore
Tricine SDS-PAGE gels (30). Following staining and destaining of a
strip of each Tricine gel, selected fragments were excised based upon
previous results (21) and with the aid of prestained low molecular
weight standards (Life Technologies, Inc.).
Isolated fragments were further purified by reverse-phase HPLC using a
Brownlee Aquapore C4 column (100 × 2.1 mm). Solvent A
was 0.08% trifluoroacetic acid in water, and Solvent B was 0.05% trifluoroacetic acid in 60% acetonitrile, 40% 2-propanol. The flow
rate was maintained at 0.2 ml/min and 0.5-ml fractions collected. Prior
to injection, material isolated from excised gel pieces was filtered,
the protein concentrated using a Centricon-3 (Amicon), and the material
then spun briefly (15,000 rpm for ~10 s) in a table-top
microcentrifuge to sediment any insoluble material. Peptides were
eluted with a nonlinear gradient from 25% to 100% Solvent B in 80 min. The elution of peptides was monitored by the absorbance at 210 nm.
Peptide containing HPLC fractions were pooled and dried by vacuum
centrifugation. Peptides were resuspended in 1 ml of 2% octyl-
-glucoside and asolectin lipid in 2% sodium cholate added to
achieve an estimated lipid to peptide molar ratio of ~15:1. The
octyl-
-glucoside and sodium cholate were removed by dialysis using
Spectra/Por CE Dispo Dialyzers (molecular weight cut-off, 2000) for 2 days against phosphate buffer (10 mM phosphate, 5 mM NaCl, pH 7.0). Each sample was concentrated to 200 µl
using a Centricon-3 and stored at
80 °C. A small aliquot of each
sample was subjected to NH2-terminal amino acid sequence
analysis to confirm the identity of the peptide and estimate its
concentration. An additional aliquot was labeled with the hydrophobic
photoreagent [125I]TID. The labeled peptide was resolved
on a 1.0-mm-thick Tricine gel and the dried gel subjected to
autoradiography. Labeling with [125I]TID served as an
additional test of peptide purity and to confirm that no significant
peptide aggregation (irreversible) had occurred.
Circular Dichroism--
CD spectra were measured with a AVIV
60DS spectropolarimeter in a rectangular quartz cuvette of 0.1 cm path
length. Repetitive scans (5-10 scans) were recorded and averaged at
25 °C with a 1-s integration time, a 0.1-nm step size, and a 1.5-nm
bandwidth. The spectra were collected in phosphate buffer (10 mM phosphate, 5 mM NaCl, pH 7.0). The protein
concentration in the samples was determined by NH2-terminal
amino acid sequence analysis. Spectra of peptide-free lipid vesicles
prepared in parallel with the lipid-peptide samples and were used to
correct for background lipid absorbance and scattering. Secondary
structure estimations were obtained by spectral deconvolution using the
Prosec program (AVIV Instruments) using the four-basis spectra set
(
-helix,
-sheet,
-turn, and other) of Chang et al.
(31).
FTIR Spectroscopy--
FTIR spectra were acquired using the
attenuated total reflectance (ATR) technique on an FTS-40 spectrometer
equipped with a mercury cadium telluride detector. The spectrometer was
purged with dry air (dew point
100 °C) from a Balston air dryer
(Balston, Haverhill, MA). FTIR spectra were obtained at a resolution of 2 cm
1 with a minimum of 256 scans/spectrum. Samples were
prepared by spreading an aliquot of the reconstituted peptide on the
surface of a 50 mm × 20 mm × 2-mm germanium ATR crystal
(Harrick, Ossining, NY). Bulk solvent was evaporated with a gentle
stream of N2 gas and the ATR crystal immediately installed
in an ATR liquid sample cell. Evaporation of the bulk
1H2O solution was required to obtain a protein
signal of reasonable strength relative to the overlapping absorbance of
1H2O. After data acquisition, samples were
exposed to 2H20 buffer (5 mM
phosphate, 250 mM NaCl, 5 mM KCl, 2 mM MgCl2, 3 mM CaCl2,
pH 6.6). The bulk solvent was evaporated by flowing N2 gas
through the ATR sample compartment, and spectra of the deuterated
peptides were recorded.
Spectral deconvolution was performed using GRAMS/386 version 1 (Galactic Industries, Salem, NH) and a
= 3.5 and a smoothing factor
of 0.2. Prior to deconvolution, all spectra were examined for the
presence of water vapor as described by Reid and Baenziger (32).
Sequence Analysis--
Amino-terminal sequence analysis was
performed on a Beckman Instruments (Porton) model 20/20 protein
sequencer using gas phase cycles (Texas Tech Biotechnology Core
Facility). Peptide aliquots (~5-10 µl) were immobilized on
chemically modified glass fiber discs (Beckman Instruments), which were
used to improve the sequencing yields of hydrophobic peptides (33).
Peptides were subjected to 10 sequencing cycles and Initial yield
(Io) and repetitive yield (R) were
calculated by nonlinear least squares regression of the observed
release (M) for each cycle (n): M = IoRn
(phenylthiohydantoin-derivatives of Ser, Thr, Cys, and His were omitted
from the fit).
 |
RESULTS |
Isolation and Reconstitution of Proteolytic Fragments Containing
AChR Transmembrane Segments--
Peptides containing the transmembrane
segments M2-M3 (Met249-Arg307), M1-M2-M3
(Lys216-Arg307) of the AChR
-subunit, the
M4 segment of the
- and
-subunits (Tyr401-Gly437,
Val446-Arg485, respectively), and the M1
segment (Ile210-Lys242) of the
-subunit
were isolated from proteolytic digests of receptor subunits. Subunit
digests were resolved by Tricine SDS-PAGE, and bands corresponding to
these selected fragments were excised on the basis on their known
electrophoretic migration (20, 21, 34). Figs.
1 and 3A show the
reverse-phase HPLC elution profile of each fragment. Peak HPLC
fractions (arrow) were pooled, the solvent removed, the
peptide resuspended in detergent (octyl-
-glucoside), and
reconstituted into asolectin (a crude soybean lipid extract) vesicles.
The identity and concentration of each reconstituted peptide was
assessed by NH2-terminal amino acid sequence analysis. For
each peptide, any secondary sequences (peptides) that were detected
were present at 1/10 to 1/20 the amount of the primary sequence. An
aliquot of each peptide was labeled with the hydrophobic photoreagent
[125I]TID and the labeled peptide resolved by Tricine
SDS-PAGE. For each peptide, a single band was apparent in the
corresponding autoradiograph and migrating with the appropriate
apparent molecular weight. [125I]TID-labeled peptide also
provided a means of verifying that the peptide had not undergone any
significant (irreversible) aggregation.

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Fig. 1.
Reverse-phase HPLC purification of
transmembrane fragments from tryptic digests of -V8-10 and the -
and -subunit. AChR-rich membranes (~50 mg of protein) were
resolved by SDS-PAGE and each of the AChR subunits isolated. For the
-subunit, -V8-10 (Asn339-Gly437) was
isolated from a limited "in gel" digest of the intact subunit (see
"Experimental Procedures"). Receptor subunits as well as -V8-10
were then digested in solution with trypsin, and the digests resolved
on 1.5-mm -hick 16.5% T, 6% C Tricine SDS-PAGE gels. Tryptic gel
fragments -T-4K, -T-10K, -T-8K, and -T-5K were isolated
from -V8-10, -subunit, and -subunit, respectively. The
fragments were then further purified by reverse-phase HPLC on a
Brownlee Aquapore C4 column (100 × 2.1 mm); the elution gradient is shown by a dotted line (solvent A, 0.08% trifluoroacetic
acid in water; solvent B, 0.05% trifluoroacetic acid in 60%
acetonitrile, 40% 2-propanol). The elution of each peptide was
monitored by absorbance at 210 nm (solid line). Panels
A-D contain the HPLC elution profiles for gel fragments -T-8K,
-T-10K, -T-4K, and -T-5K, respectively. In each panel, an
arrow shows where each fragment elutes and the identity of
each fragment was subsequently confirmed by NH2-terminal
amino acid sequence analysis.
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CD Spectroscopy--
CD measurements provide a convenient
method of looking at the overall secondary structure of a given
protein or peptide, from which the relative contributions of different
secondary structures can be estimated (35, 36). However, the CD spectra
of membrane proteins is complicated by the fact that the spectra often
exhibit various degrees of distortion in shapes, intensities, etc.,
that result from optical artifacts of differential light scattering and
adsorption flattening (reviewed in Ref. 37). It is therefore important
that secondary structure estimations be interpreted qualitatively.
Fig. 2 shows the CD spectra of peptides
-M2-M3,
-M1-M2-M3,
-M4, and
-M4 reconstituted into
asolectin lipid vesicles (lipid:protein ratio, ~15:1). In each
sample, minima are observed at approximately 208 and 222 nm that are
characteristic of
-helical structure. Similar CD spectra (data not
shown) were obtained for these same peptides reconstituted into
octyl-
-glucoside (2% w/v) or sodium dodecyl sulfate (10 mM) micelles. Micellar SDS is considered to be a good
membrane-mimetic solvent (38, 39). For example, synthetic peptides of
transmembrane segments of bacteriorhodopsin retain their
-helical
structure in SDS (40, 41). However, it should also be said that there
exists considerable uncertainty about the accuracy of predicting the
correct structure of a peptide in any single solvent environment (42).
To qualitatively estimate the amount of peptide present in the
-helical state, the CD spectra were deconvolved using the basis
spectra of Chang et al. (31). The Chang et al.
basis spectra were derived from linear analysis of the CD spectra from
16 globular proteins with high resolution crystal structures. The
spectral deconvolution results are summarized in Table
I. For each peptide,
-helix was the
predominant secondary structure conformation (e.g.
-M2-M3, 85%
-helix, 15%
-turn).

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Fig. 2.
CD spectra of peptide fragments -T-8K,
-T-10K, -T-4K, and -T-5K. HPLC-purified fragments
-T-8K, -T-10K, -T-4K, and -T-5K were reconstituted into
lipid (asolectin) vesicles as described under "Experimental
Procedures." CD spectra were measured for each reconstituted peptide
(in 10 mM sodium phosphate, 5 mM sodium
chloride buffer, pH 7.0). Panel A, CD spectrum of the
-subunit tryptic peptide -T-8K
(Met249-Arg307), which contains the
transmembrane segments M2 and M3 (~5 µM). Panel
B, CD spectrum of -T-10K
(Lys216-Arg307), which contains the
transmembrane segments M1, M2, and M3 (~0.35 µM).
Panel C, CD spectrum of -T-4K
(Tyr401-Gly437), which contains the
transmembrane segment M4 (~5 µM). Panel D,
CD spectrum of -T-5K (Val446-Arg485), which
contains the transmembrane segment M4 (~ 5 µM). Spectra were obtained by averaging over 5-10 scans with background
subtracted.
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Table I
Summary of secondary structure results derived from CD spectra
Values for CD analysis were obtained by spectral deconvolution using
the basis spectra of Chang et al. (31). Other indicates unordered/random structure and structures otherwise not assigned. Dash
indicates the absence of a particular conformation in the secondary
structure estimation.
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Fig. 3B shows the CD spectrum
of the reconstituted
-M1 peptide
(Ile210-Lys242). Although the spectrum
exhibits minima at approximately 208 and 222 nm characteristic of
-helical structure, the minimum at 208 nm is substantially reduced
in magnitude. The results of spectral deconvolution (Table
II) indicate that the peptide is largely
-helical (56%) but that there is also a substantial amount of
non-helical structure present (44%).

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Fig. 3.
Reverse-phase HPLC purification and CD
spectra of the transmembrane fragment -M1. Panel A,
AChR-rich membranes (~50 mg of protein) were resolved by SDS-PAGE and
each of the AChR subunits isolated. For the -subunit, -V8-20
(Ser173-Glu338) was isolated from a limited
"in gel" digest of the intact subunit (see "Experimental
Procedures"). The -V8-20 fragment was then digested in solution
with trypsin, and the digest resolved on 1.5-mm-thick 16.5% T, 6% C
Tricine SDS-PAGE gels. The tryptic gel fragment -T-4K was isolated
and further purified by reverse-phase HPLC (see Fig. 1 legend) with the
elution of the peptide monitored by absorbance at 210 nm (solid
line). The -T-4K fragment elutes at about 85 min (see
arrow), and the fragment identity
(Ile210-Lys216, -M1) was subsequently
confirmed by NH2-terminal amino acid sequence analysis.
Panel B, HPLC-purified fragment -T-4K ( -M1) was
reconstituted into lipid (asolectin) vesicles as described under
"Experimental Procedures" and the CD spectrum measured (in 10 mM sodium phosphate, 5 mM sodium chloride
buffer, pH 7.0). The -T-4K (10 µM) peptide contains
the transmembrane segment M1 of the -subunit, and the final CD
spectrum was obtained by averaging over five scans with background
subtracted.
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Table II
Secondary structure analysis of the CD spectrum of -M1 peptide
Values for CD analysis were obtained by spectral deconvolution using
the basis spectra of Chang et al. (31). Other indicates unordered/random structure and structures otherwise not assigned. Dash
indicates the absence of a particular conformation in the secondary
structure estimation.
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ATR-FTIR Spectroscopy--
Representative FTIR spectra for the
transmembrane peptide
-M4 (
-T-5K) recorded in both
1H2O and 2H2O are shown
in Fig. 4, A and B,
respectively. The amide I band (1600-1700 cm
1) is due
predominantly to the stretching vibration of the peptide carbonyl, the
frequency of which is highly sensitive to hydrogen bonding and thus
protein secondary structure (43). In 1H2O, the
amide I band for
-M4 is roughly symmetric with an intense peak
maximum centered near 1656 cm
1. Two weak shoulders are
also located near 1677 and 1627 cm
1. The spectral shape
and peak maximum are both highly characteristic of peptides in an
-helical conformation, which suggests that
-M4 adopts a
predominantly
-helical secondary structure. However, peptides in a
solvent exposed "random coil" conformation can also vibrate in the
1655 cm
1 region in 1H2O.

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Fig. 4.
Resolution-enhanced amide I band from FTIR
spectra of the peptide fragment -T-5K ( -M4) recorded in either
1H20 (A) or
2H20 (B) buffer. Each panel
shows the absorbance (top curve) and deconvolved
(lower curve) spectra. Spectra parameters are described
under "Experimental Procedures."
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To distinguish between the contributions of
-helical and random
structures to the 1655 cm
1 maximum, spectra were recorded
after exposure of the nAChR to 2H2O. The
exchange of peptide hydrogens for deuterium leads to a shift in
intensity from the 1656 cm
1 down to both 1653 cm
1 and roughly 1640 cm
1. The former shift
in intensity from near 1656 cm
1 down to 1653 cm
1 is highly characteristic of the up to 5 cm
1 downshifts in frequency that have been observed upon
exposure of other
-helical peptides to 2H2O
(24, 44). In contrast, the shift in frequency from near 1656 cm
1 down to near 1640 cm
1 is highly
characteristic of the downshifts in frequency of random coil. The
strong intensity at 1653 cm
1 in
2H2O further supports the predominantly
-helical nature of the
-M4 polypeptide. Similar spectral changes
were observed upon transfer of the peptide
-T-8K (
-M2-M3) from
1H2O to 2H2O.
To quantify the relative amounts of secondary structure in both the
transmembrane segments
-M4 and
-M2-M3, the spectra were curve fit
according to the procedure described by Méthot et al. (45). The resulting curve fit spectra are shown in Fig.
5 and the numerical secondary structure
estimates presented in Table III. The
curve fit data suggest a predominance of
-helical secondary structure (65% and 75% for
-M4 and
-M2-M3, respectively) in agreement with the qualitative analyses of the FTIR spectra. However, the curve fit may actually underestimate the percentage of
-helices for two reasons. 1) Residual intensity remains in the amide II band
near 1547 cm
1 in both sets of spectra recorded in
2H2O (data not shown), indicating that there
are unexchanged peptide hydrogens. Although it is standard practice to
fit the
-helical band as one peak, the broad absorbance centered
near 1653 cm
1 may actually contain unresolved vibrations
due to both protonated and deuterated
-helices. Curve fitting either
spectra of
-M4 or
-M2-M3 with two
-helical bands due
protonated and deuterated
-helices would likely increase the
estimated proportion of
-helices at the expense of random coil. 2)
Deposition of the peptide-lipid bilayers on the planar surface of the
ATR crystal should lead to planar orientation of the samples. The
intrinsic dichroism of the ATR crystal will reduce slightly the
intensity of vibrations oriented predominantly perpendicular to the ATR
surface relative to those vibrations that are randomly oriented. This
could lead to a slight reduction in the intensity of the
-helical
vibrations, as is observed in spectra of intact nAChR recorded using
the ATR versus the transmission techniques (45, 46). Note we
attributed the vibration near 1627 cm
1 to
-sheet.
However, the
-helical protein myoglobin absorbs in this region of
the spectrum, despite a complete absence of
-strands. In addition,
bands near this region have also been attributed to denatured and
aggregated polypeptides (43). The conclusion that can thus be drawn
from the FTIR studies is that both transmembrane fragments have a
propensity to adopt an
-helical conformation.

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Fig. 5.
Curve fit analysis of FTIR spectra (amide I
region) recorded in 2H20 of peptide fragments
-T-5K (A) and -T-8K (B). In each panel, the solid line represents the experimental data. The
curve fit spectrum (dotted line) is superimposed onto the
experimental spectrum. The best fit of individual component bands
(dotted lines) are also presented.
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Table III
Summary of secondary structure results derived from FTIR spectra
Values for FTIR analysis were obtained using the curve-fitting routine
(GRAMS/386 version 1, Galactic Industries, Salem, NH) and according to
the procedure described by Méthot et al. (45). Dash
indicates the absence of a particular conformation in the secondary
structure estimation.
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 |
DISCUSSION |
The goal of the present work was to determine the secondary
structure of peptide fragments of the nicotinic AChR that contained one
or more membrane-spanning segments. The CD and FTIR results show that
the membrane-bound AChR peptides (
-M2-M3,
-M1-M2-M3,
-M4,
-M4, and
-M1) are all largely
-helical. In each of the peptides, the majority of amino acid residues are contained within the
hydrophobic stretch, which constitutes the transmembrane segment. For
example, in
-M4 23 out of 29 residues (80%) are contained within
the transmembrane segment M4; (
-M2-M3, 46/60, 77%;
-M1-M2-M3, 72/92, 77%;
-M4, 24/40, 60%;
-M1, 25/33, 75%). Therefore, the largely helical nature of each peptide is predominantly a reflection of
the secondary structure of the membrane-spanning domain. The polytopic
or multispanning membrane protein bacteriorhodopsin provides an example
in which the structure of isolated transmembrane segments/peptides has
been shown to retain the
-helical structure observed in the high
resolution three-dimensional structure of the intact protein (see,
e.g., Ref. 40). Nonetheless, it should be noted that the
secondary structure of isolated peptides containing transmembrane
segments of the AChR may not necessarily reflect the structure found in
the intact receptor. Although, as is discussed in more detail below,
the present spectroscopic results are consistent with the results of
structural studies done with the intact AChR, a definitive answer as to
whether isolated lipid-reconstituted AChR peptides retain the structure
found in the intact protein remains to be established.
A wealth of information has accumulated pointing to the
-helical
nature of the AChR M2 domain (reviewed in Ref. 47). These studies
include such diverse approaches as cyroelectron microscopy (10),
examining the labeling pattern of photoreactive noncompetitive antagonists (34), to CD and NMR spectroscopy of synthetic
channel-forming peptides (48, 49). In the present study, the largely
-helical structure of the peptides
-M2-M3 and
-M1-M2-M3
indicate that the M3 and M1 domains are also likely
-helical.
Although considerably fewer studies have focused on the structure of
the transmembrane segments M1, M3, and M4, for the most part those
studies are in agreement with the present spectroscopic results. For
example, the structure of the M3 segment in the intact AChR has been
examined using different lipophilic photoreactive probes (21, 22). For
the M3 segment of each AChR subunit, the pattern of incorporation was
consistent with that of an
-helix with the labeled residues situated
on a common face, extending three or four turns of the helix. In a
similar fashion, the M4 segment of each receptor subunit was also
determined to be
-helical (20-23). In contrast, as is discussed in
more detail below, the structure of the M1 domain was not readily
apparent from the few structural studies that have been conducted.
Although deconvolution of the CD spectrum of
-M1 (Fig.
3B, Table II) indicates that the peptide is largely
-helical, there is also a substantial amount of non-helical
structure present (
-helix = 56%,
-turn = 15%,
random = 29%). Similar results were obtained from the CD spectrum
of a synthetic peptide containing the M1 domain of the AChR
-subunit
(
-helix = 60%,
-sheet = 19%,
-turn = 21%;
see Ref. 48). A unique feature of the M1 domain is that it contains
three proline residues including a highly conserved proline
(i.e.
-Pro221) in the middle of the domain.
Among globular proteins the proline residue is considered the classic
helix-breaker (50). The proline nitrogen lacks a proton so that the
peptide bond cannot participate in hydrogen bonding to a neighboring
carbonyl group within the helix. It is then somewhat surprising that
proline residues are found frequently in the putative transmembrane
domains of many integral membrane proteins, including the
-helical
segments of bacteriorhodopsin (51), the photosynthetic reaction center
(52), and aquaporin (53, 54). In contrast, proline is excluded from the
core of the
-barrel transmembrane region of bacterial porins, an
observation that is consistent with its ability to also act effectively
as a
-sheet breaker (55). A proline residue introduces a kink in the
backbone of an
-helix (56), and this distortion may account for the
deviation in the M1 CD spectrum from that of a more classical
-helix
(57). The presence of two proline residues in the extracellular half of
-M1 might also explain the irregular structure deduced for this
region in a recent report from Akabas & Karlin (58). The authors found
that several residues in the amino-terminal half of
-M1 (between
Pro211 and Pro221) when mutated to cysteine are
accessible to hydrophilic sulfhydryl reagents. The pattern of
accessibility suggested an unordered structure for this region (see
also Ref. 59). An alternative explanation is that distortions in the
helical backbone introduced by Pro211 and
Pro221 might alter the exposure of surrounding residues to
these hydrophilic sulfhydryl reagents. It is also important to point
out that the hydrophilic sulfhydryl reagents are presumed to access
residues in
-M1 from the aqueous lumen of the ion channel,
suggesting that these residues are exposed to the channel pore. In
contrast, from the pattern of labeling by the hydrophobic reagent
[125I]TID, Blanton and Cohen (21) concluded that residues
in the COOH-terminal half of
-M1 are exposed to the lipid bilayer.
Interestingly, the pattern of [125I]TID incorporation
into
-M1 was like the sulfhydryl reagent neither consistent with
that of an
-helix or a
-sheet. The hydrophobic labeling pattern
might also be a consequence of the presence of proline residues within
-M1 (Pro221 and Pro236). Alternatively, the
membrane-spanning segment (
-M1) might contain sections that adopt an
unordered or irregular secondary structure. Given the functional
importance of the M1 domain (60, 61), further structural examination is
clearly warranted.
Finally, the residual non-helical structures estimated from
deconvolution of the CD spectra of most of the AChR transmembrane peptides, the exception being
-M1, is largely estimated as being
-turn (Table I). Turn structure is consistent with the presence of
residues in each peptide that contribute to the loops connecting each
of the membrane-spanning segments, in particular the short loops
joining M1-M2-M3. The presence of
-turn is also consistent with what
appears to be a consensus loop structure connecting the transmembrane
segments of integral membrane proteins (62).
 |
CONCLUSION |
In this paper, we have combined CD and ATR-FTIR spectroscopy to
determine the structure of peptides that contain one or more transmembrane domains of the nicotinic AChR. The spectroscopic results
demonstrate that the isolated membrane-spanning domains M1, M2, M3, and
M4 all adopt a largely
-helical conformation. These studies provide
additional support for reports (20, 21, 24) that conclude that the
transmembrane region of the AChR comprises predominantly, if not
exclusively, of bundles of membrane-spanning
-helices. These studies
also represent the first step of a possible approach for examining in
detail the nature of the interactions between transmembrane segments
and specific lipids that are required for receptor function. Finally,
by further examining the structure of peptides containing one, two, and
three or more transmembrane domains, insight into the nature of
inter-domain interactions may be achieved.
We thank Dr. Anthony Fink for kindly allowing
us use of his AVIV 60DS CD spectropolarimeter.