(Received for publication, August 14, 1995)
From the
The structure of the nicotinic acetylcholine receptor (nAChR)
has been studied using a novel combination of hydrogen/deuterium
exchange and attenuated total reflectance Fourier transform infrared
spectroscopy. Fourier transform infrared spectra show marked changes in
both the amide I and amide II bands upon exposure of the nAChR to H
O. The substantial decrease in intensity of
the amide II band reflects the exchange of roughly 30% of the peptide
hydrogens within seconds of exposure to
H
O, 50%
after 30 min, 60% after 12 h, and 75% after prolonged exposure for
several days at room temperature or lower temperatures. The 30% of
peptide hydrogens that exchange within seconds is highly exposed to
solvent and likely involved in random and turn conformations, whereas
the 25% of exchange-resistant peptide hydrogens is relatively
inaccessible to solvent and likely located in the transmembrane domains
of the nAChR. Marked changes occur in the amide I contour within
seconds of exposure of the nAChR to
H
O as a
result of relatively large downshifts in the frequencies of amide I
component bands assigned to turns and random structures. In contrast,
only subtle changes occur in the amide I contour between 3 min and 12 h
after exposure to
H
O as a result of slight
downshifts in the frequencies of
-helix and
-sheet
vibrations. It is demonstrated that the time courses and relative
magnitudes of the amide I component band shifts can be used both as an
aid in the assignment of component bands to specific secondary
structures and as a probe of the exchange rates of different types of
secondary structures in the nAChR. Significantly, the intensities of
the band shifts reflecting the exchange of
-helical secondary
structures are relatively weak indicating that a large proportion of
the 25% exchange resistant peptides adopt an
-helical
conformation. Conversely, no evidence is found for the existence of a
large number of exchange-resistant
-strands. The exchange kinetics
suggest a predominantly
-helical secondary structure for the
transmembrane domains of the nAChR.
The nicotinic acetylcholine receptor (nAChR) ()from
the electric organ of Torpedo is a large integral membrane
protein (
290,000 daltons) that responds to the binding of
cholinergic agonists by transiently opening a cation-selective ion
channel across the postsynaptic membrane (for recent reviews see (1, 2, 3) ). The nAChR is composed of four
distinct subunits arranged as a pentamer
(
), pseudosymmetrically around a
central pore that functions as the ion channel. The four subunits all
share a high degree of sequence homology including four conserved
25-amino acid residue-long hydrophobic segments (designated
M1-M4) that likely form four transmembrane
-helices(4, 5) , although transmembrane
-strands are possible(6, 7, 8) . The
transmembrane M2 segment from each subunit lines the ion channel pore
and controls channel selectivity(9, 10) . All four may
play an important role in both channel gating and the inactivation of
channel gating that occurs upon either prolonged exposure to
acetylcholine (desensitization) or reconstitution into lipid membranes
lacking both cholesterol and anionic
lipids(11, 12, 13) .
The secondary
structure of the four putative transmembrane segments identified by
hydrophobicity plots has been the subject of considerable
controversy(36) . Each of the four segments was originally
assigned an -helical secondary structure based largely on the
-helical transmembrane segments identified in other integral
membrane proteins. An
-helical secondary structure of M2 is
suggested by site-directed mutagenesis and photoaffinity labeling with
noncompetitive channel blockers, which both reveal an
-helical
periodicity in the exposure of M2 to the ion channel pore (14-16;
however, see (17) ). Similarly, the labeling pattern by
hydrophobic photoaffinity probes suggests an
-helical periodicity
in the exposure of both M3 and M4 to the hydrophobic core of the lipid
bilayer. The labeling of M1 is consistent with an
-helix that is
slightly distorted due to the presence of a proline residue in the
C-terminal half of the hydrophobic region(18, 19) .
The presence of five cylindrical rods of electron density in the
structure of the nAChR probed at 9-Å resolution by electron
microscopy has also been interpreted in terms of an
-helical
secondary structure of the pore-lining M2 segment from each nAChR
subunit(7) .
However, the lack of similar cylindrical
regions of electron density in the periphery of the transmembrane
domains of the nAChR in the 9-Å resolution electron density map
raises questions concerning the -helical secondary structures
assigned to M1, M3, and M4(7) . If these three transmembrane
segments do not adopt an
-helical conformation, between five and
seven transmembrane
-strands are required for each subunit to
account for the 25-30% of the nAChR located within the
hydrophobic region of the lipid bilayer. The presence of a large number
of transmembrane
-strands was recently suggested by the appearance
of bands characteristic of
-sheet structures in FTIR spectra of
the nAChR pretreated with proteinase K to remove the extramembranous
domains of the protein(8) . In addition, some studies of the
secondary structure of the nAChR suggest an
-helical content of
between 18 and 23%(12, 13, 20) . The roughly
20%
-helix is not sufficient to account for both the four putative
transmembrane
-helices proposed for each subunit (20-25% of
the total protein) and the
-helical structures detected by
electron microscopy in the extramembranous regions of the nAChR, and
thus implies the existence of non-
-helical transmembrane
segments(13, 20) .
The possibility of transmembrane
-strands in the nAChR has profound implications for the mechanisms
of both channel gating and desensitization and calls into question the
validity of hydrophobicity plots for predicting the transmembrane
topology of integral membrane proteins in general. In this paper, we
have examined the structure of the nAChR using a combination of FTIR
spectroscopy and hydrogen/deuterium exchange. Spectra recorded after
prolonged exposure of the nAChR to
H
O are
characteristic of a mixed
/
protein and suggest a
predominance of
-helical secondary structures. The
hydrogen/deuterium exchange spectra indicate that a large percentage of
the
-helical peptide hydrogens are resistant to exchange with
deuterium and likely exist within the hydrophobic, relatively solvent
inaccessible region of the lipid bilayer. The large percentage of
exchange-resistant
-helical peptides provides strong support for a
predominantly
-helical secondary structure of the nAChR
transmembrane domains. The results also demonstrate several novel
features in hydrogen/deuterium exchange spectra that can be exploited
for probing integral membrane protein structure and function.
The FTIR samples were prepared by spreading one aliquot of the
reconstituted nAChR membranes on the surface of a 50 20
2-mm germanium attenuated total reflectance (ATR) crystal (Harrick,
Ossining, NY). After evaporating the bulk solvent with a gentle stream
of N
gas, the ATR crystal was immediately installed in an
ATR liquid sample cell (also from Harrick) and the nAChR film
rehydrated with excess Torpedo Ringer buffer (250 mM NaCl, 5 mM KCl, 2 mM MgCl
, 3 mM CaCl
, and 5 mM Na
HPO
,
pH 7.0). Some samples were resuspended twice in 2 mM phosphate
buffer, pH 6.6, in
H
O and were left for 72 h at
4 °C (or as described in the legend to Fig. 2) to effect
extensive exchange of peptide hydrogens for deuterium before deposition
on the ATR crystal and subsequent rehydration with
H
O Torpedo Ringer buffer. Previous
studies have shown that nAChR samples prepared in this manner retain
the ability to reversibly bind agonists and noncompetitive antagonists
and to undergo the resting-to-desensitized state
transition(23, 24) .
Figure 2:
FTIR spectra of the nAChR recorded after
extensive exchange of the peptide hydrogens for deuterium. Spectra were
recorded at pH 7 (A) or pH 2 (B) in either H
O (dashed line) or
H
O buffer after 3 days in
H
O at 4 °C (solid line), 6 h in
H
O at 60 °C (short/long dashed
line), 1 h at pH 11.0, 95 °C (long dashed line). All
spectra were base line-corrected between 1800 and 1350 cm
and were normalized by either comparing the intensity of the
tyrosine vibration near 1516 cm
or qualitatively
assessing the intensity of the amide I
band.
The ``extended'' (3 min to 12 h) hydrogen
exchange experiments were performed by first equilibrating an nAChR
film on the ATR crystal with H
O. After
recording a 100-scan spectrum, the
H
O buffer
was removed from the sample compartment and replaced with
H
O buffer. Several spectra were recorded at
2-cm
resolution over the next 12 h with increasing
numbers of scans varying from 50 to 1500 scans/spectrum. The extended
exchange experiments were recorded using the deuterated triglycine
sulphate detector, which appeared more stable over the course of the
experiment. The fast exchange experiments were performed by rapidly
adding
H
O buffer to a dry nAChR film on the ATR
crystal and then recording spectra (4 scans each) using the fast
mercury cadmium telluride detector at 8 cm
resolution. The dry film was utilized to avoid mixing of
H
O and
H
O in the ATR
sample compartment, which hinders the acquisition of spectra at fast
time points after immersion in
H
O. All
experiments were performed at 22.5 °C.
FTIR spectra of affinity-purified nAChR reconstituted into
membranes composed of DOPC/DOPA/cholesterol were recorded as a function
of time from 5 s to 12 h after exposure to H
O
buffer and reveal a number of spectral changes that reflect the
exchange of peptide N-
H for N-
H (Fig. 1). These include a marked change in the shape of the
amide I band between 1600 and 1700 cm
and a
substantial decrease in the intensity of the amide II band, which is
centered near 1547 cm
in
H
O
but shifts down in frequency in
H
O to near 1450
cm
(amide II` band). The rates of the spectral
changes reflect the rates at which the various peptide hydrogens of the
nAChR exchange for deuterium in the bulk solvent and provide a
sensitive measure of nAChR structure and conformational change.
Figure 1:
FTIR spectra of the nAChR recorded as a
function of time after exposure to H
O. A, extended exchange spectra recorded at 2
cm
resolution after 3, 6, 9, 12, 22, 50, 80, 170,
360, 600, and 780 min of
H
O exposure (solid
line from top to bottom at 1547
cm
). The dashed line spectrum was recorded
in
H
O. The overlapping
H
O vibrations have been removed by spectral
subtraction (see ``Results and Discussion''). Spectra are not
normalized. B, rapid exchange spectra recorded at 8
cm
resolution after the addition of
H
O to a dry nAChR film (dashed line).
The first spectrum was recorded between 5 and 8 s after the addition of
H
O (solid line, top spectrum at 1547 cm
). Others were recorded after 38, 94,
196, 298, and 364 s (solid lines from second top to bottom at 1547 cm
). Spectra are normalized,
as the addition of
H
O to a dry film leads to
the expansion of the nAChR film on the surface of the ATR crystal and
thus a reduction in the absolute intensity of the protein signal. C, the time course of the peptide
N-
H/N-
H exchange plotted as a
change in the amide II:amide I ratio versus time.
The
percentage of nAChR peptides that have exchanged at any time can be
calculated by comparing the residual amide II band intensity at 1547
cm with the corresponding intensities in spectra
recorded under conditions of complete or no peptide
H/
H exchange. The amide II band intensity
corresponding to the fully protiated nAChR (no exchange) was estimated
from spectra recorded in
H
O buffer ( Fig. 1and 2, dashed lines) after subtraction of the
broad, overlapping
H-O-
H bending
vibration centered near 1640 cm
. The end point of
the subtraction was judged by a flat base line in the
2000-2500-cm
region (see also
``Discussion''). The amide II intensity at 1547
cm
, corresponding to 100% exchange of peptide
hydrogens for deuterium was determined by incubating the nAChR in
H
O buffer for extended periods of time (Fig. 2). Complete peptide
H/
H exchange
was achieved after 1 h at pH 11.0 and 95 °C, which resulted in a
significant structural alteration of the nAChR, as judged by a marked
change in the shape of the resulting amide I` band contour. The spectra
of the structurally altered or possibly denatured receptor were
recorded after readjusting the pH back to 7.0 (Fig. 2A, long dashed line). Spectra were then recorded at pH =
2.0 in order to shift the vibrations of acidic side chains from near
1580 cm
(asymmetric COO
stretch,
pH = 7.0) up to near 1700 cm
(C = O
stretch of a deuteriated carboxylic acid, pH = 2.0) and thus
provide an unobstructed view of any residual amide II band intensity.
The low pH spectra (Fig. 2B, long dashed line)
demonstrate that complete exchange of the peptide hydrogens has been
achieved after 1 h at pH 11.0, 95 °C. Incubation of the receptor
for additional lengths of time under the same conditions had no further
effects on the intensity in the amide II band region (data not shown).
Comparison of either the intensity or area of the residual amide II
band in the various spectra presented in Fig. 1and Fig. 2indicates that roughly 30% of the nAChR peptides exchange
with deuterium within 5 s of exposure to H
O at
22.5 °C. Roughly 50% have exchanged after 30 min and 60% after 12
h. Prolonged exposure of the nAChR to
H
O for
several days at either 4 °C or room temperature leads to the
exchange of 75% of the peptide hydrogens, whereas 95% exchange after
subsequent incubation for up to 24 h at 60 °C (Fig. 2). The
30% of peptides that exchange after 5 s of exposure to
H
O includes a large proportion that are
hydrogen-bonded to bulk solvent and is consistent with the roughly 20%
random coil and 6% turn structures predicted for the nAChR by spectral
deconvolution and curve fitting the amide I` band (21) . The
25% of exchange-resistant peptides is similar to both the 25-30%
of the nAChR found within the lipid bilayer by electron microscopy (7) and proteinase K digestion (8) and the 20-25%
of peptides predicted using hydrophobicity plots to form transmembrane
-helices(4, 5) . A large proportion of the
exchange-resistant peptides is likely located within the hydrophobic,
relatively solvent inaccessible region of the lipid bilayer (see below
and ``Discussion''). Note that the 5% of peptides that remain
protiated after incubation of the nAChR for 24 h at 60 °C are
unusually resistant to exchange, even for transmembrane
-helices.
In comparison, complete exchange of all
-helical transmembrane
peptide hydrogens occurs after a 6-h incubation of the integral
membrane protein rhodopsin in
H
O at 60
°C(28, 29) . The 5% of extremely
exchange-resistant peptide hydrogens is sufficient to form five
transmembrane
-helices and may reflect five highly ordered
-helices lining the ion channel pore. The unusually high degree of
order of these pore-lining
-helices suggested by Blanton and Cohen (19) may be the cause of their high resistance to peptide
H/
H exchange and may be required to maintain a
consistently closed conformation of the ion channel in the absence of
bound acetylcholine.
Figure 3:
Resolution-enhanced amide I band from
spectra of the nAChR in DOPC/DOPA/cholesterol recorded in either H
O (A) or
H
O (B) buffer. Each panel shows the absorbance (top
curve), deconvolved (middle curve), and fourth derivative (bottom curve) spectra. The spectra were recorded at 2
cm
resolution and represent the average of 16,000
scans each. Spectral parameters are described under ``Experimental
Procedures.''
The deconvolved and fourth derivative
spectra reveal a relatively intense component band centered at 1655
cm in spectra recorded in both
H
O and
H
O buffer (Fig. 3). The relative intensity of the band decreases upon
peptide
H/
H exchange, which is the main cause
of the change in the shape of the amide I band contour in
H
O. The majority of the loss of intensity can
be attributed to a characteristic shift in the frequency of vibrations
due to polypeptide segments in random coil conformations from between
1650 cm
and 1660 cm
in
H
O down to near 1640 cm
in
H
O. The residual intensity remaining between
1650 and 1655 cm
is due to polypeptide chain in the
-helical conformation. This assignment is based on both normal
mode calculations(30) , which predict a band near this
frequency for the
-helix, and empirical observations, which show
that an intense band is observed near 1655 cm
in the
resolution enhanced spectra of numerous proteins exhibiting
predominantly
-helical conformations (31, 32) .
-Helical vibrations downshift in frequency by only a few wave
numbers upon peptide
H/
H exchange ((33) ; see Fig. 4). The strong intensity of the band
remaining at 1655 cm
is likely due to unexchanged
-helical peptides.
Figure 4:
Resolution-enhanced and exchange
difference spectra showing the amide I component shifts that occur upon
exposure of the nAChR to H
O. Deconvolved
spectra of the amide I` band upon
H/
H exchange
are presented for the nAChR in (panel A). The spectra were
recorded, from top to bottom at 1655
cm
, during the 1st, 2nd, 3rd, 6th, and 12th hour
after exposure to
H
O. Exchange difference
spectra are presented for the nAChR in panel B. The spectrum
recorded after 3 min of exposure to
H
O was
subtracted from spectra recorded after 6, 9, 16, 40, 80, 600, and 780
min of exposure to
H
O. Spectra were smoothed
using a Savitsky-Golay algorithm (GRAMS) with a polynomial of 3 and 15
smoothed points and were base line-corrected between 1713 and 1604
cm
.
Resolution enhancement resolves the high
frequency shoulder of the amide I band into three bands at 1691
cm, 1680 cm
, and 1672
cm
. Bands near 1670 cm
are often
attributed to
-sheet and those near 1690 cm
and
1680 cm
to turn structures. The relatively intense
shoulder near 1635 cm
in both the amide I and amide
I` bands is resolved into two main component bands centered near 1630
cm
and 1625 cm
. These bands have
been detected in numerous proteins and are highly diagnostic of
-sheet structures. In
H
O, the
1690-cm
band partially shifts down in frequency to
near 1680 cm
contributing to the marked change in
shape of the amide I contour upon exposure of the nAChR to
H
O. Several other relatively minor band shifts
upon exposure of the nAChR to
H
O are discussed
in more detail below.
Note that the symmetric amide I` band contour
with a relatively broad maximum between 1630 and 1660 cm observed in spectra acquired after several days' exposure
to
H
O (Fig. 3B, top
trace) contrasts the typical amide I` band contours observed for
both predominantly
-helical and predominantly
-sheet proteins
and is consistent with a mixed
/
protein. A mixed
/
structure is supported by the strong intensities of the amide I`
component bands due to peptides in
-helix and
-sheet
conformations in the resolution-enhanced spectra, although the greater
intensity of the
-helix band near 1655 cm
suggests a predominance of
-helical secondary structures in the
nAChR. The
-helix amide I` component band is relatively less
intense in spectra recorded here using ATR than in spectra recorded
previously using transmission techniques(21) . The reduction in
intensity is likely due to a predominant orientation of the
-helices perpendicular to the bilayer surface(7) , and
thus the plane of the intrinsically dichroic ATR crystal(27) .
The spectra recorded using ATR slightly underestimate the relative
contribution of
-helices to the secondary structure of the nAChR.
The qualitative interpretation of the FTIR spectra is in agreement
with recent secondary structure estimates from both FTIR and CD that
suggest roughly 40% -helix and between 20 and 35%
-sheet (21, 35) but contrasts with other reports, which have
suggested a predominantly
-sheet protein with only 20%
-helix
and closer to 42%
-sheet (13, 20) .
Significantly, the roughly 40%
-helix confirmed here in the
qualitative analysis of the spectra is sufficient to account for the
four putative transmembrane
-helices predicted for each nAChR
subunit (20-25% of the protein) by hydrophobicity plots (4, 5) as well as a substantial portion of the
extramembranous domains (see (21) for a detailed discussion).
The rapidity of the major
spectral changes that occur after the addition of H
O indicates that the
H/
H exchange-sensitive peptide carbonyls whose
downshifts in frequency are responsible for the majority of the
spectral changes in
H
O must be highly
solvent-accessible, and likely hydrogen bonded to bulk solvent rather
than to peptide hydrogens in ordered secondary structures. The solvent
accessibility of the peptide carbonyls suggests that the bands near
1680 and 1640 cm
in
H
O (1690
and 1655 cm
in
H
O,
respectively) are involved in either turn or random coil conformations,
in agreement with the frequency based assignment of these two component
bands discussed above. The magnitude of the rapid spectral changes also
suggests that the downshifts in frequency of solvent exposed peptide
carbonyls upon exposure to
H
O are much larger
than the band shifts observed for
-helix and
-sheet secondary
structures. The lack of a dramatic change in frequency of
-helical
peptides upon exposure to
H
O may be due to
compensating effects of
H
O on the amide I
vibration coupling to the nearest neighbors across hydrogen
bonds(33) . The differences in the magnitudes of the band
shifts of peptides in ordered secondary structures relative to those
exposed to aqueous solvent accounts for the apparent differences
between the time courses of the changes in the amide I and amide II
bands.
The relatively minor amide I component band shifts that occur
upon the exchange of -helix and
-sheet secondary structures
are seen more clearly upon resolution enhancement of the spectra
recorded over the time course of the experiment (Fig. 4A)
and in ``exchange difference spectra'' calculated by
subtracting the spectrum recorded after 3 min of exposure to
H
O from those recorded at the various time
points indicated in Fig. 4B. Note that the downshift in
frequency of vibrations due to random coil structures hydrogen-bonded
to bulk solvent should be complete within 1 s of exposure of the nAChR
to
H
O (see above) and therefore do not
contribute to the changes observed in spectra recorded over this time
period(26) .
The effects of peptide H/
H exchange on the vibrational frequencies of
-sheet structures are revealed in the resolution-enhanced spectra
by a very slight downshift in intensity of bands above 1660
cm
and near 1635 cm
and in the
exchange difference spectra by a negative and a positive band near 1680
and 1630 cm
(Fig. 5A). The weak negative
band near 1680 cm
in the exchange difference spectra
could reflect a slight down shift of a high frequency
-sheet
component band with the corresponding positive band masked by a
negative
-helical band near 1660 cm
(see
below). The positive maximum near 1630 cm
could
reflect the downshift of the low frequency
-sheet vibration with
the corresponding negative band masked by a positive
-helical band
near 1645 cm
. Alternatively, the
-sheet minimum
and maximum near 1680 and 1630 cm
could conceivably
reflect a change in the intensities of either
-sheet vibrations
upon exposure to
H
O.
The resolution-enhanced
spectra reveal a gradual decrease in the intensity of the -helical
amide I component band near 1655 cm
over the first
12 (Fig. 4A) and up to 72 (Fig. 3B)
hours of peptide
H/
H exchange that is due to a
slight downshift in frequency of the
-helix vibration. The
downshift in frequency is reflected in the exchange difference spectra
by a negative band near 1660 cm
coupled with a
positive band near 1645 cm
. The latter band is
partially obscured by the bands to the exchange of
-sheet
structures but is more evident in exchange difference spectra recorded
from the nAChR reconstituted into DOPC, where there is an enhanced
exchange of
-helical secondary structures. (
)Spectral
simulations show that a slight downshift in frequency of a broad amide
I band centered near 1655 cm
can give rise to
exchange difference spectra with a negative and a positive band at
frequencies near 1660 cm
and 1645
cm
, respectively (data not shown). Exchange
difference spectra recently reported for the water-soluble protein,
cytochrome c, also exhibit similar bands characteristic of the
exchange of
-helix and
-sheet secondary
structures(41) .
The time course of the spectral changes in
both the resolution-enhanced and exchange difference spectra suggests
that most of the -sheet peptides exchange within the first 2 h of
exposure of the nAChR to
H
O, whereas a large
percentage of the
-helical structures exchange between 2 and 12 h
after the addition of
H
O. The
resolution-enhanced spectra also show that the decrease in intensity of
the
-helix vibration near 1655 cm
is not
complete even after several days of exposure of the nAChR to
H
O (Fig. 3B and 4A).
In addition, considering the predominant intensity of the
-helix
vibration in resolution enhanced spectra of the nAChR (Fig. 3),
the bands reflecting the exchange of
-helical peptides near 1660
and 1645 cm
in the exchange difference spectra are
very weak relative to those due to the exchange of peptides involved in
-sheet structures. For example, the
-helical amide I
component band near 1655 cm
dominates the
resolution-enhanced spectrum of the nAChR and is several times more
intense than the
-sheet component band near 1670 cm
(see (21) ), whereas the ratio of the corresponding
-helix and
-sheet difference bands near 1660 and 1685
cm
, respectively, (referred to as
I
/I
) is only 1.2 after 12 h and 1.4
after 3 days of exposure to
H
O (data not
shown). In contrast, the I
/I
of the
same two bands in the exchange difference spectra recorded from
cytochrome c is roughly 10, which is similar to the ratio of
the corresponding amide I component bands in the resolution-enhanced
spectra of cytochrome c(41) . Both the
resolution-enhanced and exchange difference spectra strongly suggest
that a large majority of the 25% of peptides suggested above to be
resistant to exchange and to exist within the hydrophobic environment
of the lipid bilayer adopt an
-helical conformation. Conversely,
no evidence is detected for the existence of a large number of
exchange-resistant
-sheets. These results suggest that at least a
majority of the transmembrane domains of the nAChR adopt an
-helical secondary structure.
The rates at which the different peptide hydrogens in a
protein tertiary structure exchange with protons (or in this case
deuterium) in the bulk solvent vary over several orders of magnitude,
depending on hydrogen bonding and solvent accessibility. Peptide
hydrogen exchange rates are thus very sensitive to protein secondary
structure and conformational change. They are also strongly influenced
by the transmembrane topology of an integral membrane protein that
governs the proportion of peptides that reside within the hydrophobic,
relatively solvent inaccessible region of the lipid bilayer. We have
for the first time monitored the rates of exchange of affinity-purified
nAChR peptide hydrogens by following changes in ATR FTIR spectra
recorded as a function of time of exposure of the nAChR to H
O. The resulting hydrogen/deuterium exchange
spectra provide new insight into the exchange kinetics and thus
structure of the nAChR. The important novel features of the exchange
data are described in the following paragraphs.
(i) The
hydrogen/deuterium exchange spectra reveal a rapid decrease in the
intensity of the amide II band corresponding to the exchange of 30% of
the peptide hydrogens for deuterium within 5 s after exposure to H
O, 50% after 30 min, 60% after 12 h, and 75%
after days at room temperature or lower temperatures. The roughly
biphasic nature of the changes in amide II band intensity are
indicative of the existence of both fast and slow exchanging
populations of peptide hydrogens similar to the at least two
populations of exchanging peptide hydrogens that have been observed for
numerous water-soluble and integral membrane proteins, including native
nAChR membranes(35) . However, for the nAChR the percentage of
slowly exchanging peptides is larger, and the exchange times are longer
than the exchange times of many water-soluble proteins, despite the
location of a substantial number (
75%) of the nAChR peptides in
the extramembranous domains of the nAChR. In addition, a relatively
large percentage (
25%) of the peptide hydrogens are resistant to
exchange even after prolonged exposure of the nAChR to
H
O. It appears that the close association
and/or anchoring of the nAChR within the lipid bilayer attenuates the
internal motions and/or reduces the accessibility of the peptide
hydrogens to
H
O.
(ii) The rapid decrease in
amide II band intensity within seconds of exposure to H
O is accompanied by a marked change in shape
of the amide I contour. The rapidity of both spectral changes indicates
that the majority of the affected peptide N-H (amide II) and
C=O (amide I) groups are hydrogen-bonded to bulk solvent, rather
than to other peptides in ordered secondary structures, and are thus
involved in random and turn conformations. The 30% extremely fast
exchanging peptide hydrogens is consistent with the roughly 20% random
coil and 6% turns suggested by a previous curve-fitting analysis of the
amide I` band contour(21) . Similarly, the rapid changes in the
amide I contour, which are due predominantly to shifts in frequency of
an intense band near 1655 cm
in
H
O down to near 1640 cm
in
H
O and a relatively weak band from near 1690
cm
down to near 1680 cm
, are
consistent with the previous frequency-based assignment of these
H/
H exchange-sensitive component bands to
random and turn structures, respectively(21) . The relatively
large magnitude of the rapid spectral changes in the amide I band
confirm that the downshifts in frequency of random and turn vibrations
upon
H/
H exchange are much larger than those
due to the exchange of peptide hydrogens involved in ordered secondary
structures (see below and (33) ). The rapidity of the band
shifts provides the first direct experimental evidence that bands
typically assigned to random and turn structures exhibit the exchange
kinetics expected for highly solvent exposed peptides.
(iii) The
very subtle changes in amide I band contour between 3 min and 12 h
after exposure of the nAChR to H
O reflect the
slight amide I component band shifts that occur as a result of the
exchange of ordered secondary structures and provide insight into the
individual exchange kinetics of
-helices and
-sheet. The
relatively small downshifts in frequency of the
-helical amide I
vibrations are reflected in the resolution-enhanced amide I band by a
gradual decrease in the intensity near 1655 cm
,
whereas the exchange of
-sheet leads to slight downshifts in
intensity of bands above 1660 cm
and near 1635
cm
. The downshifts in frequency of
-helix and
-sheet component bands can also be followed in exchange difference
spectra and yield bands near 1660 and 1645 cm
, and
near 1680 and 1630 cm
, respectively, of both
positive and negative intensity. Similar bands due to the exchange of
-helix and
-sheet structures have been reported in exchange
difference spectra recorded from the water-soluble protein, cytochrome c(41) .
Significantly, the time courses of the
various amide I component band shifts reflected in the exchange
difference spectra indicate that most -sheet secondary structures
undergo a relatively rapid exchange within the first 2 h of exposure of
the nAChR to
H
O, whereas the
-helical
secondary structures exchange over a much slower time scale. In
contrast, the exchange difference spectra recorded from cytochrome C
suggest very similar exchange rates for both
-helix and
-sheet(41) . The relatively slow exchange of
-helical
secondary structures in the nAChR appears to be the cause of the
generally slow peptide
H/
H exchange rates that
are evident by following the spectral changes in the amide II band
intensity.
In addition, both the residual intensity remaining at
1655 cm in the resolution-enhanced spectra recorded
after 3 days' exposure of the nAChR to
H
O
and the weak intensities of bands in the exchange difference spectra
reflecting the
H/
H exchange of
-helical
peptide hydrogens relative to those reflecting the exchange of
-sheet strongly suggest the existence of a large population of
exchange-resistant
-helical peptide hydrogens (this interpretation
is discussed in more detail below). Conversely, no features suggesting
the existence of a large population of exchange-resistant
-strands
are detected in either the resolution-enhanced or exchange difference
spectra of the nAChR. A large proportion of the 25% of peptide
hydrogens that are resistant to exchange likely exist in an
-helical conformation.
The existence of 25% exchange-resistant
peptide hydrogens coupled with both the strong evidence for the
existence of a large number of exchange-resistant -helical peptide
hydrogens and lack of evidence for the existence of a corresponding
population of exchange-resistant
-strands suggests that the
majority of the peptides in the transmembrane domains of the nAChR
adopt an
-helical secondary structure. A predominantly
-helical secondary structure of the putative exchange-resistant
transmembrane domains of the nAChR is consistent with the four
hydrophobic transmembrane segments predicted for each nAChR subunit
using hydrophobicity plots (4, 5) and by the
-helical periodicity in chemical labeling of M2, M3, and M4 by
either chemically reactive noncompetitive channel blockers or
hydrophobic photoactive
probes(14, 16, 18, 19) . The
labeling pattern of M1 is consistent with a slightly distorted
-helix(19) . Five rods of density are observed lining the
ion channel pore in the 9-Å resolution electron density map of
the nAChR, consistent with five pore-lining M2
-helical segments
(one per subunit; (7) ). However, the lack of well defined
electron density at the periphery of the transmembrane domains of the
nAChR is suggestive of the existence of transmembrane
-strands(7) . Infrared bands characteristic of extended
-sheet structures are also observed in FTIR spectra recorded from
the nAChR after treatment with proteinase K to remove the
extramembranous domains of the nAChR(8) .
There is currently
a consensus that the five pore-lining transmembrane segments, which of
any of the transmembrane segments are likely to be accessible to
aqueous solvent and thus undergo relatively rapid H/
H exchange, are
-helical in nature. If
lipid-exposed M1, M3, and M4 are not
-helices, as possibly
suggested by the electron microscopy data, between five and seven
-strands are required to fulfill the 25-30% of nAChR
peptides that exist within the lipid membrane(7) . Although our
data do not completely rule out the existence of transmembrane
-strands, no spectral features indicative of a large population of
exchange-resistant transmembrane
-structures are observed in the
spectra recorded as a function of time after exposure of the nAChR to
H
O as would be expected if there were five or
seven transmembrane
-strands per nAChR subunit. However, a more
detailed analysis of the exchange data is warranted in order to clarify
the discussion.
The 25% of exchange-resistant peptide hydrogens was
determined by comparing the residual amide II band intensity remaining
after 3 days' exposure of the nAChR to H
O
with the amide II band intensity determined under conditions of both no
(100% N-
H) and complete exchange (100%
N-
H). The amide II band intensities corresponding to
100% N-
H were obtained from spectra recorded in
H
O after subtraction of the overlapping
H-O-
H bending vibration. Although
difficult, the subtraction is facilitated because the amide I and II
bands are generally more intense relative to the overlapping absorbance
of water in spectra recorded using ATR than in spectra recorded using
conventional transmission techniques(27) . The ATR technique
also offers an internal control in that both the
H
O and
H
O spectra are
recorded from the same sample, which is deposited on the surface of a
germanium ATR crystal. The resulting intensity of the amide I band
after
H
O subtraction is similar to the
intensity of the same band immediately after exchanging the
H
O buffer in the ATR sample compartment for
H
O, illustrating the accuracy of the spectral
subtraction (the
H-O-
H bending
vibration absorbs near 1200 cm
). In addition, the
shape and relative intensities of the amide I and amide II bands in the
H
O-subtracted spectra (Fig. 1A, dashed line) are similar to the shape and relative intensities
of the same bands in spectra of dry films of the nAChR, where no
subtraction of the
H-O-
H bending
vibration is required (Fig. 1B, dashed line).
The base line corresponding to the amide II band intensity after
complete exchange was determined after incubation of the nAChR at pH
11.0 and 95 °C in spectra recorded at both pH 7.0 and pH 2.0. At pH
2.0, the vibrations of acidic side chains are shifted out of the amide
II spectral region, thus providing an unobstructed view of any residual
amide II band intensity. Based on the rigorous methods used to
determine the amide II band intensities corresponding to 100%
N-H and 100% N-
H and an analysis of
the factors affecting the quantitative analysis of the data, we believe
that the percentage of exchange-resistant peptides in the nAChR is
accurate well within a ±10% limit (i.e. 25 ±
10%).
The assignment of the 25% exchange-resistant peptides to those
in the transmembrane domains is based on a large body of evidence
indicating that transmembrane peptide hydrogens, in general, are
resistant to hydrogen exchange. The relative solvent inaccessibility of
the hydrophobic environment of the lipid bilayer should inhibit the
hydrogen exchange rates of the transmembrane domains. The transient
folding/unfolding motions of ordered secondary structures that are
necessary for rapid exchange are also likely to be restricted in
transmembrane structures because such motions would expose the highly
polar peptide N-H and C=O groups to the hydrophobic lipid
acyl chains. The exchange resistance of transmembrane peptide hydrogens
is borne out experimentally by the very large percentage of
exchange-resistant peptide hydrogens that appear to be related to the
number of peptides found within the hydrophobic core of the lipid
bilayer in both bacteriorhodopsin and
rhodopsin(28, 29, 37, 38) . In the
case of bacteriorhodopsin, the transmembrane secondary structures have
been shown directly to be resistant to exchange(38) . An
unusually large number of exchange-resistant peptides have also been
reported for the multisubunit photosynthetic reaction center, the
transmembrane -barrel pore, porin, as well as other integral
membrane proteins of less defined tertiary
structure(25, 34, 42, 43) . In
analogy to the exchange kinetics of the transmembrane peptide hydrogens
observed for other integral membrane proteins, it is concluded that the
exchange-resistant peptides detected here likely include those found
within the transmembrane domains of the nAChR. As the roughly 25% of
H/
H exchange-resistant peptides observed for
the nAChR is similar to the roughly 20-25% of peptides predicted
to exist as transmembrane
-helices based on the four transmembrane
-helices per nAChR subunit predicted by hydrophobicity plots (4, 5) and the 25-30% of the nAChR found within
the lipid bilayer by both electron microscopy (7) and
proteinase K digestion of the extramembranous portions of the
nAChR(8) , a majority of the 25% exchange-resistant peptides in
the nAChR are likely involved in transmembrane structures (however, see
below).
A more quantitative analysis of the possible numbers of
exchange resistant -helices and
-sheet can also be obtained
if it is assumed that the nAChR exhibits transmembrane
-strands
corresponding to roughly of the transmembrane domains (five to seven
-strands/subunit) and that 25% of the nAChR is located in the
membrane (see above). In this case, 19% of the total nAChR peptide
hydrogens would be found as
-strands and 6% as transmembrane
-helices (five pore-lining
-helices). If all of the
transmembrane structures are resistant to exchange, the 19%
exchange-resistant
-strands would still be greater in number than
the total number of exchange-resistant
-helical structures,
regardless of whether 25% (6% exchange-resistant
-helical
intramembranous peptides) or even 35% (16% exchange resistant intra-
plus extramembranous helical peptides) of the total number of nAChR
peptides are resistant to exchange. In either scenario, the relative
intensities of bands in the exchange difference spectra reflecting the
exchange of
-sheet structures should be reduced in intensity
relative to those due to the exchange of
-helices, when the
relative intensities are compared with the relative intensities of the
corresponding
-helix and
-sheet amide I component bands in
the resolution-enhanced spectra of the nAChR. In contrast, the exchange
difference spectra recorded for the nAChR reveal a dramatic enhancement
(as opposed to a reduction) in the intensities of the
-sheet bands
relative to those due to the exchange of
-helical secondary
structures indicating far fewer than 19%, if any, exchange-resistant
-strands in the nAChR. Note that for cytochrome c, the
exchange rates of both
-helix and
-sheet structures appear to
be similar, leading to exchange difference spectra where the relative
intensities of the
-helix and
-sheet bands in the exchange
difference spectra are similar to the relative intensities of the
corresponding bands in the resolution-enhanced amide I band. It is
clear that the nAChR does not have sufficient exchange-resistant
-strands, if any, to account for the 5-7 transmembrane
-strands suggested for each nAChR subunit. Therefore, either the
transmembrane structures of the nAChR are resistant to exchange and
adopt a predominantly
-helical conformation, as indicated above,
or the nAChR exhibits a substantial population of transmembrane
-strands that exchange rapidly within the first few hours of
exposure of the nAChR to
H
O (see above).
Given the well established inaccessibility of transmembrane
structures to H
O and their restricted ability
to undergo the transient folding/unfolding reactions necessary for
relatively rapid exchange, the possibility of fast exchanging
transmembrane
-strands in the nAChR seems unlikely. Transient
folding/unfolding of transmembrane
-strands would also require
compensatory motions of the extramembranous domains; yet the overall
exchange rates of the nAChR are relatively slow, indicating a
relatively ordered protein structure. In addition, it is difficult to
imagine how the transmembrane structures of the nAChR could undergo the
rapid folding/unfolding motions required for relatively fast exchange
while still maintaining a closed ion channel pore against a substantial
electrochemical gradient. While the possibility of relatively dynamic
solvent-accessible transmembrane structures has important structural
implications for the nAChR, given the points raised above it seems
unlikely that there exists a large number of relatively fast exchanging
transmembrane
-strands. Consequently, the most plausible
conclusion is that at least a majority of the transmembrane domains of
the nAChR are
-helical in nature. This interpretation of our data
questions the possible interpretation of the diffuse electron dense
bands in the low resolution electron density map of the nAChR in terms
of
-strands and supports an
-helical secondary structure of
all the transmembrane domains.
Note that the above analysis depends
entirely on both the exchange kinetics measured by following the
changes in intensity of the amide II vibration and the qualitative
analysis of the band shifts that occur in the amide I region upon
exposure of the nAChR to H
O. The changes in the
amide I band were monitored in both resolution-enhanced and exchange
difference spectra. Both methods of data analysis provide a consistent
picture of the shifts in band intensity that occur over the time course
of the exchange. Moreover, the spectra were stringently examined for
the presence of water vapor in order to eliminate potential artifacts
that can arise upon resolution enhancement.
Our conclusions
are not dependent upon the secondary structure of the nAChR that has
been estimated from the amide I band shape using curve-fitting
techniques. We have assumed that the molar absorptivities of the amide
I and II bands are the same for both
-helix and
-sheet
conformers. However, the close correlation for most proteins between
the relative areas of the
-helix and
-sheet amide I component
bands and the relative
-helix and
-sheet contents as
determined by x-ray crystallography suggest that potential variations
are relatively minor. Slight variations in the relative molar
absorptivities of different secondary structures should have no bearing
on the interpretations of our data.
Furthermore, a recent study in
our lab shows that upon reconstitution of the nAChR into a more fluid
lipid membrane composed solely of DOPC, there is an enhanced exchange
of the peptide hydrogens for deuterium that results predominantly from
an increased exchange of -helical secondary structures.
The enhanced exchange of
-helical secondary structures in
DOPC is suggested by a further decrease in intensity of the
-helical amide I component band near 1655 cm
in
resolution-enhanced spectra of the nAChR recorded after 3 days in
H
O and by an increased intensity of the two
bands reflecting the exchange of
-helices near 1660 and 1645
cm
in exchange difference spectra of the nAChR, but
it does not result from a change in the secondary structure (see
Footnote 3 for a detailed discussion). The increased exchange of the
nAChR
-helical peptides in DOPC may arise from an increase in the
overall dynamics of the nAChR or an increased permeability of the
bilayer and thus accessibility of the transmembrane structures to
H
O, both as a result of a more fluid lipid
bilayer(39, 40) . As one would expect changes in
membrane fluidity to have a predominant affect on the transmembrane
domains of the nAChR, the enhanced exchange of
-helical secondary
structures in DOPC provides additional support not only for the
existence of exchange-resistant
-helices but also for the
existence of a significant proportion of the exchange-resistant
-helical structures within the lipid bilayer. In this regard, it
is interesting to note that the photosynthetic reaction center
exchanges to a greater extent in the presence of detergents, which
likely increase the ``fluidity'' of the surrounding lipid
bilayer (compare the residual amide II band in the FTIR spectra of (42) versus(43) ).
Blanton and Cohen have
suggested that the electron dense cylinders interpreted by Unwin as
transmembrane -helices may reflect highly ordered
-helices,
whereas the lack of well defined electron density profiles at the nAChR
periphery may be due to less ``highly ordered''
-helical
conformations at the lipid-protein interface(19) . The roughly
5% of the nAChR peptide hydrogens that are extremely
H/
H exchange-resistant, even after treatment
for 24 h at 60 °C (Fig. 2), is consistent with this
interpretation. The high order of the pore-lining
-helical
structures may be the cause of the high resistance of these peptides to
exchange relative to the other transmembrane
-helices in the nAChR
and to transmembrane
-helices in other integral membrane proteins,
such as rhodopsin. The electron density maps may also have been
influenced by the assumption of pentahedryl symmetry used in the
averaging of the data. While one might expect the pore-lining
structures to adopt a well defined and reproducible position relative
to the pore axis in each subunit, the location of the transmembrane
structures distal to the pore-lining
-helices may vary slightly
from subunit to subunit, resulting in a smearing of electron density
upon averaging of the data. In addition, the observed infrared bands
assigned to transmembrane
-like structures in FTIR spectra of
proteinase K-treated nAChR could reflect short unordered polypeptides
that remain at the surface of the membrane after incomplete enzymatic
digestion of the extramembranous domains. The bands could also reflect
a disruption in the structure of the transmembrane domains upon
extensive proteinase K treatment(36) .
(iv) Finally, the
hydrogen exchange spectra presented here reveal several novel features
that can be exploited for probing integral membrane protein structure
and function. FTIR techniques, in general, offer important advantages
over conventional H/
H exchange measurements in
that the exchange of peptide hydrogens for deuterium can be monitored
directly without interference from the exchange of labile side chain
protons and without prior exchange-in of
H by incubation of
the sample in
H
O. This is an important
consideration for integral membrane proteins that generally exhibit a
large number of exchange-resistant peptide hydrogens whose exchange
kinetics would not be monitored using the
H/
H
exchange technique.
We demonstrate that ATR FTIR is particularly
advantageous in that it allows a very accurate quantitative
interpretation of the exchange data in terms of the number of exchanged
protons at a given time after exposure to H
O.
The ATR technique can be used to acquire exchange spectra within
seconds of exposure of a protein to
H
O, which
may be the fastest that hydrogen exchange data have ever been obtained
for any protein. The result illustrates the potential of the ATR
technique for examining the exchange kinetics of highly solvent exposed
peptide hydrogens that are directly involved in ligand binding and/or
protein action, especially if data are recorded at lower temperatures
and pH where exchange times are markedly reduced. In addition, the
ability to monitor the exchange kinetics of individual secondary
structures is unique to FTIR spectroscopy but has not been exploited
previously for monitoring the exchange kinetics of integral membrane
proteins. We demonstrate for the first time that both the time scales
and relative magnitudes of the downshifts in frequency of amide I
component bands can be used as an aid in the assignment of amide I
component bands to specific secondary structures. The ability to probe
the exchange kinetics of different secondary structure can provide
unique insight into the structure and function of integral membrane
proteins.
FTIR spectra have been recorded as a function of time of
exposure of the nAChR to H
O. The resulting
hydrogen/deuterium exchange spectra reveal a number of changes that
reflect the exchange of peptide hydrogens for deuterium and provide
insight into the secondary structure of the transmembrane domains of
the nAChR. Roughly 25% of the peptide hydrogens are found to be
resistant to peptide
H/
H exchange. A large
majority of the exchange-resistant peptides exist in an
-helical
conformation, likely within the hydrophobic, relatively solvent
inaccessible region of the lipid bilayer. The results provide strong
evidence for an exchange-resistant core of
-helical transmembrane
peptide hydrogens in the nAChR and illustrate the utility of FTIR
spectroscopy coupled with hydrogen/deuterium exchange for probing
integral membrane protein structure and function.