(Received for publication, December 20, 1994; and in revised form, May 15, 1995)
From the
We reconstituted purified plasma membrane
H P-type ATPases form a family of ATPases constituted of one or
two polypeptide chains which use the free energy of ATP to generate ion
gradients across a variety of biological membranes. All of these
ATPases share a common mechanism that involves a phosphorylated
intermediate and inhibition by vanadate at µM
concentrations. One of the best characterized members of the P-type
ATPases is Neurospora crassa plasma membrane
H In a previous paper (Vigneron et
al., 1995), we described the efficient reconstitution of purified
plasma membrane H
All other
reagents were of the highest purity grade commercially available.
H
ATPase
was reconstituted by gel filtration as described elsewhere (Vigneron et al., 1995). Liposomes were prepared in the same way but in
the absence of ATPase.
Figure 6:
Infrared
polarized parallel (90°) and antiparallel (0°) (with respect to
the plane of the membrane) absorption spectrum of reconstituted
H
For
analysis of the deconvolved spectra during the time course of exchange,
each spectrum was self-deconvolved with a 30 cm
Plasma membrane H
The ATPase/lipid ratio after hydrolysis was estimated from the
infrared spectra as described under ``Experimental
Procedures.'' Fig. 1shows the spectra of the
proteoliposomes untreated (control) or treated for 30 min or 1 h with
proteinase K at a protease/ATPase ratio of 1:5 w/w. Evaluation of the
amount of protein associated with the proteoliposomes is reported in Fig. 2as a function of the incubation time in the presence of
proteinase K. It can be estimated that after 45 min of treatment with
proteinase K (protease/ATPase ratio of 1:5 w/w), 48-50%
Figure 1:
Infrared spectra
of reconstituted ATPase incubated at 30 °C with proteinase K
(proteinase K/ATPase ratio of 1:5 w/w). A, 0 min (control); B, 30 min; C, 1 h. The spectra have been rescaled so
that the
Figure 2:
Amount of protein associated with the
proteoliposomes during the time course of proteinase K treatment
(protease/ATPase ratio of 1:5 w/w). The percentage of protein is
estimated by calibration of the protein amide I absorption peak
(1,600-1,700 cm
To ascertain
that in our incubation conditions, no ATPase fragments produced by
proteolytic treatment stuck on the lipid vesicles, we performed the
following control. After incubation of ATPase-containing lipid vesicles
with an equal amount of ATPase-free vesicles together with proteinase K
(ATPase/protease ratio of 1:5 w/w), the two populations were separated
by gradient density glycerol centrifugation. A lipid dosage performed
on the collected fractions showed that the distribution of the
ATPase-free population was unchanged after proteolytic treatment,
ruling out that any of these vesicles were denser because of peptide
binding. Furthermore, the infrared spectra of both populations showed
that less than 0.001 mg of protein/mg of lipid was present in the
ATPase-free lipid vesicles population after proteinase K treatment,
demonstrating that ATPase fragments produced by the proteolytic
treatment did not bind unspecifically to lipid vesicles. The gradient
density ultracentrifugation used to separate vesicles whose density
differed in the third or fourth decimal place is described elsewhere
(Goormaghtigh and Scarborough, 1986; Goormaghtigh et al.,
1986; Vigneron et al., 1995).
Figure 3:
Infrared spectra between 1,800 and 1,500
cm
To provide evidence
that the unexpected
Figure 4:
Infrared spectra between 1,800 and 1,500
cm
The FTIR spectra in the 1,600-1,700
cm
Figure 5:
A and B, infrared spectra of
deuterated samples containing intact (A) and digested (B) ATPase. After ATPase treatment with proteinase K for 1 h
(protease/ATPase ratio of 1:5 w/w), removal of protease and the
released peptides was performed as described under ``Experimental
Procedures.'' C, released peptides after proteinase K
treatment for 1 h. Curve C is the difference between the
intact (A) reconstituted ATPase and the digested (B)
ATPase spectra. The dotted lines correspond to 1,654
(
Analysis of the shape of the amide I
absorption band in the ATPase spectra was carried out according to the
method routinely used in our laboratory (Goormaghtigh et al.,
1990). It indicates that the intact ATPase contains 38% An identical
analysis of the shape of the bacteriorhodopsin spectra reveals that the
1,636 cm
As
for the bacteriorhodopsin spectra recorded for polarized 90° and
0° incident light (not shown), the dichroic spectrum displays, for
intact or digested bacteriorhodopsin, a maximum at 1,662
cm
Peptide hydrogen exchange of the
protein was followed by monitoring the amide II absorption peak
(
Figure 7:
Infrared spectra between 1,800 and 1,400
cm
Figure 8:
Percentage of deuteration reported as a
function of the deuteration time for reconstituted
H
For
comparison, the hydrogen/deuterium exchange measurement for
reconstituted bacteriorhodopsin, treated or not with proteinase K under
the same conditions as ATPase, was also performed. Whereas the control
bacteriorhodopsin exchanges 30% of its amide protons in 2 h, this
proportion falls to 25% for the protease-treated sample. The aim of this work was to verify experimentally the models
proposed to describe the mode of insertion of N. crassa H The protein segments present in the vesicle inner water phase are
not accessible to proteinase K. Indeed, proteinase K cannot enter the
lipid vesicles; it has been shown convincingly on acetylcholine
receptor-containing vesicles (Grne-Tschelnokow et al., 1994) and gastric tubulovesicles Critical assumptions in the present work are that 1) proteolytic
cleavage is efficient outside of the membrane but does not reach the
membrane part of the protein, and 2) the structure of the membrane part
of the protein is not affected by removal of the extramembrane loops.
These assumptions are best tested on a protein of known structure. We
have chosen here bacteriorhodopsin. Results reported by Dumont et
al.(1985) on bacteriorhodopsin indicated that proteinase K is able
to hydrolyze intramembrane segments of bacteriorhodopsin after several
days of incubation. Under our experimental conditions, the amount of
digested protein (about 10%) corresponds to the short extramembrane
loops present on one side of the membrane and connecting the
transmembrane The fact
that proteinase K completely cleaves the part of the protein protruding
from the vesicle in the case of ATPase is crucial for the conclusions
of this paper. Proteinase K has been shown to be one of the most
effective proteases used to remove the extramembrane part of proteins (e.g. Dumont et al., 1985; Fimmel et al.,
1989; Goormaghtigh et al., 1993; Challou et al.,
1994; Grne-Tschelnokow et al., 1994;
Sonveaux et al., 1994). Furthermore, as some specificity of
proteinase K for selected amino acids or secondary structures could
eventually hinder proteolytic action, the association of different
proteases is likely to overcome this barrier. We show here that
previous incubation with trypsin or a combination of different
proteases does not improve the extent of digestion. Moreover, the
sequencing of the bacteriorhodopsin fragments obtained after proteinase
K treatment under the same conditions demonstrates the efficient
cleavage of very short connecting loops of the protein protruding out
of the membrane. It must be stressed here that in agreement with the
current models, trypsin alone had been known to cut this ATPase at a
number of sites in the small and large cytoplasmic loops of the protein
as well as between transmembrane segments M2 and M3, and M6 and M7
(numbered according to the model of Rao et al., 1991),
indicating that no large protein region is completely resistant to the
proteases. In conclusion, we cannot definitively reject the possibility
of an extramembrane domain resistant to all of the proteases tested,
alone or in combination, but we consider this possibility unlikely. Proteolysis of Neurospora H Since
colorimetric protein assays are sensitive to selected amino acids whose
distribution between the membrane and solution-located protein segments
might not be random, they cannot be used to monitor the percentage of
protein removed from the membrane assembly by the proteases. Using the
ratio of the protein amide I area to the lipid The
membrane domain of the ATPase and the connecting loops located inside
the vesicle therefore consist of about 43% of the intact protein (i.e. 396 amino acid residues). Dithiothreitol pretreatment of
the ATPase did not allow us to increase the extent of digestion,
indicating that the only disulfide bridge identified in the Neurospora H Quantitative evaluation of the
structure of the reconstituted H Only a few data exist on the amide hydrogen/deuterium exchange
rate in membrane proteins (for a review, see Goormaghtigh et
al., 1994c). For bacteriorhodopsin, the exchange reaches a plateau
when 30% of the amide protons have been exchanged (Downer et
al., 1986; Earnest et al., 1990). This value corresponds
roughly to the fraction of the protein protruding from both sides of
the membrane. The results obtained here on bacteriorhodopsin
reconstituted into lipid vesicles exactly confirm these data and
suggest that the transmembrane helices are exchanged at an extremely
slow rate. Removal of the extramembrane connecting loops on one side of
the membrane decreases this value to 25%, indicating that this cleavage
does not significantly change the hydrogen/deuterium exchange
properties of the membrane-embedded amide protons. Similarly, the
single transmembrane In a recent paper, Corbalan-Garcia et al.(1994) applied a similar proteolytic approach to
reticular Ca In conclusion, our
results show the presence, in the membrane-associated domain of the N. crassa plasma membrane H
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-ATPase from Neurospora crassa into soybean
phospholipid vesicles (lipid/ATPase ratio of 5:1 w/w). The
proteoliposomes contained an active ATPase, oriented inside-out. They
were subjected to proteolysis by using Pronase, proteinase K, trypsin,
and carboxypeptidase Y. Fourier transform infrared attenuated total
reflection spectroscopy indicates that the amount of protein remaining
after hydrolysis and elimination of the extramembrane domain of ATPase
represents about 43% of the intact protein. The secondary structure of
intact ATPase and of the membrane-associated domain of ATPase was
determined by infrared spectroscopy. The membrane domain shows a
typical
-helix and
-sheet absorption. Polarized infrared
spectroscopy reveals that the orientation of the helices is about
perpendicular to the membrane. Amide hydrogen/deuterium exchange
kinetics performed for the intact H
-ATPase and for the
membrane-associated domain demonstrate that this part of ATPase shows
less accessibility to the solvent than the entire protein but remains
much more accessible to the solvent than bacteriorhodopsin membrane
segments.
-ATPase (Scarborough, 1976). It is composed of a
single polypeptide chain (Scarborough and Addison, 1984) whose monomer
is active (Goormaghtigh et al., 1986) and undergoes a major
tertiary structure change in the course of its catalytic cycle
(Goormaghtigh et al., 1994a). The ATP binding site and the
carboxyl- and amino-terminal ends of the polypeptide chain are located
on the cytoplasmic side of the membrane (Mandala and Slayman, 1989;
Hennessey and Scarborough, 1990). Development of protein chemistry
techniques associated with proteolytic approaches (Hennessey and
Scarborough, 1989) provided direct experimental evidence for the
presence of 497 amino acids out of a total of 920 on the cytoplasmic
side of the membrane (Scarborough and Hennessey, 1990; Rao et
al., 1992) and the identification of three peptides associated
with the membrane (Rao et al., 1991). Topological models
describing the polypeptide chain insertion in the membrane are all
based on the hypothesis that transmembrane segments would adopt an
-helix structure (Addison, 1986; Hager et al., 1986).
Recent discoveries that the transmembrane region of the porins is
composed of
-sheets (Weiss et al., 1991), that the
erythrocyte glucose transporter might fold as membrane
-barrels
(Fischbarg et al., 1993), and that the acetylcholine receptor
transmembrane domain contains
-sheets where
-helices were
expected (Unwin, 1993; Grne-Tschelnokow et
al., 1994) indicate that an
-helix could not be the sole
secondary structure present in the transmembrane region of the ATPase
molecule. Until now, no experimental evidence was available to confirm
an all-
-helical model for the transmembrane segments of
H
-ATPase.
-ATPase of Neurospora into
lipid vesicles. More than 99% of the ATPase molecules were oriented
inside-out, i.e. with the ATP binding site oriented toward the
outside of the vesicles, and at least 90% of them were fully active.
Proteolysis of the reconstituted ATPase by different proteases
(trypsin, Pronase, carboxypeptidase Y, and proteinase K) was performed
here to remove the extramembrane part of ATPase. Fourier transform
infrared attenuated total reflection spectroscopy (FTIR-ATR
spectroscopy)
(
)is an accurate technique for
determining the secondary structure and orientation of membrane
proteins (Goormaghtigh et al., 1989, 1991, 1993; Cabiaux et al., 1989). It is used here to evaluate the secondary
structure of the intact and of the membrane part of the N. crassa ATPase molecule isolated after proteolysis of the part of the
protein protruding outside the vesicle. Analysis of the deconvolved
FTIR-ATR spectra reveals that the membrane-associated domain of ATPase
contains both
-helix and
-sheet structures. Amide
hydrogen/deuterium exchange kinetics recorded on the entire and
membrane part of H
-ATPase indicate a slower exchange
rate for the membrane part of ATPase. However, this exchange is
significantly more rapid than the exchange rates of transmembrane
helices of bacteriorhodopsin measured under the same experimental
conditions.
Materials
ATP, L--phosphatidylcholine type IIS from
soybean, trypsin, chymotrypsin, bromelain, proteinase K, and
bacteriorhodopsin were purchased from Sigma, St. Louis, MO, U. S.A.
Pronase, leucine aminopeptidase, and carboxypeptidase Y were from
Boehringer Diagnostics Mannheim, FRG. Sephadex G-50 medium gel,
Sepharose CL-6B, and the G-25 M (PD-10) column were from Pharmacia
Biotech Inc. The hollow fiber bundle, used for concentration of the
samples, was from Bio-molecular Dynamics (Houston, TX).
-ATPase was purified from Neurospora cell
wall less mutant cells, designated fz;sg;os-1 V (Fungal Genetics Stock
Center, University of Kansas Medical Center, Kansas City).
Purification and Reconstitution of
H
H-ATPase
-ATPase purification and the ATPase
activity assay were carried out as described by Smith and
Scarborough(1984). Soybean lipids, used for all of the reconstitutions,
were purified by the procedure of Kagawa and Racker(1971).
Bacteriorhodopsin Reconstitution
1.25 mg of bacteriorhodopsin was dissolved in 268 µl of
20 mM Mes, pH 6.0, containing 2% (w/v) octyl glucopyranoside,
0.15 M KCl, and 3 mM NaN (Heyn et
al., 1981). This mixture was added to 5 mg of purified soybean
lipids in suspension in 268 µl of the same buffer. The solution was
dialyzed for 72 h at 4 °C against 2 liters of 20 mM Mes,
pH 6.0, with four changes. The proteoliposomes were then layered on a
17-50% discontinuous glycerol gradient, and centrifugation was
run at 35,000 rpm overnight (SW 60 rotor on a Beckman L7
ultracentrifuge). Lipid and protein assays performed on the gradient
fractions showed that all of the bacteriorhodopsin was reconstituted
into the soybean lipid vesicles.
Incubation of the Lipid Vesicles with Different
Proteases
Soybean lipid vesicles without ATPase at a concentration of
25 mg/ml in a 10 mM Mes, 50 mM CHCOOK (pH
adjusted to 6.8 with Tris) buffer were incubated overnight at 30 °C
alone or with Pronase, carboxypeptidase Y, leucine aminopeptidase,
proteinase K, trypsin, chymotrypsin, or bromelain. The lipid/protease
ratio was 25:1 w/w. CaCl
and MgCl
were added at
a final concentration of 2 mM in the incubation medium for all
samples, except incubation with bromelain, where EDTA 5 mM
final was present. After incubation, the samples were eluted on a
Sepharose CL-6B column with a 0.5 mM phosphate buffer, pH 6.8.
The lipid vesicles eluted in the excluded volume of the column, well
separated from the included volume fractions, which contained
water-soluble proteins and peptides. The samples were then concentrated
on a hollow fiber bundle and examined by FTIR-ATR spectroscopy.
Proteolysis of ATPase (or Bacteriorhodopsin) and
Isolation of the Membrane-bound Part of the Protein
The proteoliposomes were concentrated in reconstitution
buffer (10 mM Mes, 50 mM CHCOOK (pH
adjusted to 6.8 with Tris)) at a concentration of about 2.6 mg/ml with
the hollow fiber bundle. KCl was added to reach a 150 mM final
concentration for the incubation in the presence of the proteases. A
freshly prepared stock solution of protease (2 mg/ml) was then added to
the proteoliposome preparation (the protease/ATPase ratio (w/w) was of
1:100-1:5 for Pronase, 1:10-1:4 for trypsin, 1:10 for
carboxypeptidase Y, and 1:10-1:1 for proteinase K). The
proteoliposome suspension was then incubated at 30 °C for
increasing periods of time from 10 min to 50 h. A protease mixture
treatment was performed as follows. The sample was first incubated for
80 min with trypsin (trypsin/ATPase ratio of 1:10 w/w) and then for 15
min with carboxypeptidase Y (carboxypeptidase/ATPase ratio of 1:10 w/w)
or 30 min with proteinase K (proteinase K/ATPase ratio of 1:5 w/w). In
all cases the proteolytic digestion was stopped by adding
phenylmethylsulfonyl fluoride (5 mM final) and EDTA (10 mM final), and the samples were eluted at room temperature on a 1
30-cm Sepharose CL-6B column (0.5 ml/min flow with a 0.5 mM phosphate buffer, pH 6.8, containing 150 mM KCl). The
high KCl content is aimed at eliminating electrostatically bound
peptides. The lipid vesicles eluting with the excluded volume of the
column were concentrated on hollow fibers and eluted at room
temperature on a 1.5
5-cm G-25 M column equilibrated with a 0.5
mM phosphate buffer, pH 6.8, at a flow of 1 ml/min to remove
the KCl. The vesicles eluting in the excluded volume of the latter
column were concentrated once more on a hollow fiber bundle and kept on
ice until SDS-PAGE or infrared spectroscopy analysis. Controls were
incubated as the other samples but without protease; they were treated
further in the same way.
Incubation of Lipid Vesicles with Proteoliposomes and
Proteinase K
1.5 ml of a preparation of soybean lipid vesicles without
ATPase at a concentration of 12 mg/ml in reconstitution buffer was
mixed with 3 ml of a proteoliposome (lipid/ATPase ratio of 5:1 w/w)
preparation at a lipid concentration of 6 mg/ml, and the preparation
was adjusted to 150 mM KCl. The mixture was then incubated
with or without proteinase K (protease/ATPase ratio of 1:5 w/w) at 30
°C for 1 h. After incubation, phenylmethylsulfonyl fluoride (5
mM final) was added to stop the proteolysis, and the samples
were eluted on the Sepharose CL-6B column as described above. The lipid
vesicles were collected and layered on a 17% glycerol gradient and
centrifuged overnight as described above and elsewhere (Vigneron et
al., 1995). The lipid content of the collected fractions was
measured and revealed that the ATPase-free lipid vesicles population,
remaining at the top of the gradient, shows the same distribution
before and after the proteolytic treatment. The two vesicle
preparations were eluted on Sephadex G-50 (fine) with water to
eliminate glycerol and then analyzed by FTIR spectroscopy to estimate
the content of lipid-associated protein.SDS-PAGE Analysis
After concentration, the samples were mixed with 0.25 volume
of sample buffer (88 mM Tris, 4% (w/v) SDS, 3.6% (v/v)
2-mercaptoethanol, 3.5 mM EDTA, 36% (v/v) glycerol, 18
µg/ml chymostatin, 180 µg/ml bromphenol blue, pH adjusted to
6.8 with HPO
). They were then processed on a 7
9
0.15-cm gel containing 7.5% (ATPase) or 15%
(bacteriorhodopsin) polyacrylamide in the resolving gel and 4%
polyacrylamide in the stacking gel. The gels were stained with
Coomassie Blue 250 (Wilson, 1979) or with silver nitrate (Nielsen and
Brown, 1984).
Infrared Spectroscopy Analysis
FTIR-ATR spectra were recorded on a Perkin-Elmer infrared
spectrophotometer 1720X equipped with a liquid nitrogen-cooled
mercury-cadmium-telluride detector. The internal reflection element was
a germanium ATR plate (50 20
2 mm) with an aperture
angle of 45°. 128 scans were accumulated for each spectrum. Spectra
were recorded at a nominal resolution of 4 cm
. The
spectrophotometer was purged continuously with air dried on a silica
gel column (5
130 cm) at a flow rate of 7 liters/min. Every
four spectra, reference spectra of a clean germanium plate were
recorded automatically by a sample shuttle accessory and used to ratio
the recently run sample spectra. At the end of the scan the spectra
were transferred from the memory of the spectrophotometer to a computer
for subsequent treatments.
Preparation of Samples
Thin films were obtained
as described by Fringeli and Gnthard(1981) by
slowly evaporating 40-100 µl of the protein-containing
liposomes under a N stream on one side of a germanium
plate. This results in the formation of oriented multilayers at the
surface of the plate. Liposomes prepared in the absence of protein were
subjected to an identical treatment. The plate was then sealed in a
universal sample holder and rehydrated by flushing
D
O-saturated N
at room temperature. In this
period of time, only the readily accessible peptides bonds are
exchanged. Importantly, the random structure shifts from about 1,655
cm
to about 1,640 cm
upon
hydrogen/deuterium exchange, allowing differentiation of the
-helix from the random structures.
Lipid/ATPase Ratio
The lipid/protein
(w/w) ratio is correlated to the (C=O)
lipid/
(C=O) protein absorption band ratio in FTIR
spectroscopy. The ATPase/lipid ratio after hydrolysis was estimated
from the protein absorption peak (amide I (1,600-1,700
cm
))/lipid
(C=O) absorption peak
(1,700-1,800 cm
) ratio as established by
Goormaghtigh et al.(1990). Evaluation of the ATPase/lipid
ratio was also carried out after deuteration of the samples and yielded
identical results, demonstrating that H
O does not
significantly contribute to the intensity of amide I.
Secondary Structure Determination
Fourier
self-deconvolution was applied to increase the resolution of the
spectra in the amide I region which is the most sensitive to the
secondary structure of proteins. The self-deconvolution was carried out
using a Lorentzian line shape for the deconvolution and a Gaussian line
shape for the apodization. To quantify the area of the different
components of amide I revealed by the self-deconvolution, a least
squares iterative curve fitting was performed to fit Lorentzian line
shapes to the spectrum between 1,700 and 1,600 cm.
Prior to curve fitting, a straight base line passing through the
ordinates at 1,700 cm
and 1,600 cm
had been subtracted. The spectrum arising from the lipid part of
the system was found to be completely flat between 1,700
cm
and 1,600 cm
and was therefore
not subtracted. To avoid introducing artifacts caused by the
self-deconvolution procedure, the fitting was performed on the
nondeconvolved spectrum. The proportion of a particular structure is
computed to be the sum of the area of all of the fitted Lorentzian
bands having their maximum in the frequency region where that structure
occurs divided by the total area of amide I. In these conditions, the
difference with the x-ray determination amounts to no more than 8%
(standard deviation) for a large variety of proteins tested
(Goormaghtigh et al., 1990).
Orientation of the Secondary
Structures
The determination of molecular orientations by
infrared ATR spectroscopy was performed as described by Goormaghtigh
and Ruysschaert(1990). When orientation was to be evaluated, additional
spectra were recorded with incident light which is polarized parallel
(0°) and perpendicular (90°) relative to the plane of the
membranes. The dichroism spectrum was computed by subtracting the
0° polarized spectrum from the 90° polarized spectrum as
described in the legend of Fig. 6. A larger absorbance at
90° (upward deviation on the dichroism spectrum) indicates a dipole
oriented preferentially near a normal to the ATR plate. Conversely, a
larger absorbance at 0° (downward deviation on the dichroism
spectrum) indicates a dipole oriented closer to the plane of the ATR
plate. In the -sheet structure, the dipole associated with amide I
lies in the plane of the sheet, essentially parallel to the amide
C=O axis. In the
-helical structure, the amide I dipole is
oriented at about 26° with respect to the helix axis (for a review,
see Goormaghtigh et al., 1994b).
-ATPase of N. crassa between 1,800 and 1,400
cm
. The spectra were recorded after 2 h of
deuteration. The dichroic spectrum (top of the figure) is the
difference between the 90° and 0° spectra. The 0° spectrum
is enlarged by a factor 1.41 with respect to the 90° spectrum, and
1.41 is then the subtraction factor used to obtain the
90°-0° dichroic spectrum to take into account the
differences in the relative power of the evanescent field. The
dichroism spectrum is enlarged five times as compared with the two
other spectra and smoothed by apodization of its Fourier transform by a
4 cm
FWHH Gaussian line
shape.
Hydrogen/Deuterium Exchange
Films
containing 200-300 µg of protein (1-1.5 mg of lipid)
were prepared on a germanium plate as described above. Nitrogen gas was
saturated with DO (by bubbling in a series of five vials
containing D
O) at a flow rate of 51 ml/min (controlled by a
Brooks flow meter). Bubbling was started at least 1 h before starting
the experiment. At zero time, the tubing was connected to the cavity of
the sealed chamber surrounding the film. For each kinetic time point,
12 spectra were recorded and averaged at a resolution of 4
cm
. At the beginning of the kinetics, spectra were
recorded every 15 s. After the first 2 min, the time interval was
increased exponentially. After 16 min, the interval between the scans
was large enough to allow the interdigitation of a second kinetic. A
second sample placed on another ATR setup of the Perkin-Elmer sample
shuttle was then analyzed with the same time sampling with a 16-min
offset by connecting the D
O-saturated N
flow in
series with the first sample. From this time on, our program changed
the shuttle position to follow the two kinetics. Before starting the
deuteration, 10 spectra of each sample were recorded to test the
stability of the measurements and the reproducibility of area
determination. In our usual way of working, one of the samples placed
on the shuttle had been incubated in the absence of protease, and the
other one had been incubated with proteinase K. This procedure allowed
us to test the reproducibility of the experiment under identical
conditions. A background deuteration kinetic recorded with the same
germanium plate at the same position in the sample shuttle but in the
absence of the sample was recorded and subtracted from the kinetic
recorded in the presence of the sample. This allowed us to take into
account the unavoidable variations in the atmospheric water content
inside the spectrophotometer. Indeed, even though the spectrophotometer
was purged with dry air for 20 min before starting the experiment,
further removal of traces of water vapor took place for several hours,
superimposing distinct sharp bands from the water vapor onto the
protein spectra (Goormaghtigh and Ruysschaert, 1994). The subtraction
of the background kinetic was improved by adopting the following
automated procedure. A subtraction coefficient was first computed as
the ratio of the area of the atmospheric water band integrated between
1,565 and 1,551 cm
on the sample spectrum and on the
corresponding background spectrum. The subtraction coefficients
obtained for each kinetic time point (i.e. each spectrum) were
then plotted against the time, and the resulting curve was fitted with
a fourth-order polynomial. Values of the subtraction coefficients
obtained from the polynomial were finally used. The areas of amide I,
II, and II` were obtained by integration between 1,702 and 1,596; 1,596
and 1,502; and 1,492 and 1,412, respectively. For each spectrum, the
area of amide II and amide II` was divided by the area of amide I. This
permitted us to take into account small but significant variations of
the overall spectral intensity due in part to the presence of
D
O which, by virtue of its presence, induced the swelling
of the sample layer and therefore increased the average distance
between the protein sample and the germanium crystal surface. Since the
ATR spectrum intensity depends on this distance (Harrick, 1967), this
resulted in a loss of a few percent of the band intensity for all
measured bands. Undeuterated spectra were recorded before the kinetic
experiment as explained above, and 100% deuterated sample values were
extrapolated by assuming a value of zero for the amide II surface. The
area of amide II and II` (reported to the area of amide I) was finally
expressed between 0 and 100% for each kinetic time point.
FWHH
Lorentzian line shape and apodized with a Gaussian line shape with a
FWHH of 15 cm
, yielding to a resolution enhancement
factor of 2 (Kauppinen et al., 1981).
Other Procedures
ATPase activity was measured as described previously
(Vigneron et al., 1995). The protein content of the samples
was determined by the method of Lowry et al.(1951) as modified
by Bensadoun and Weinstein(1976) with bovine serum albumin as a
standard. The lipid content of the samples was determined by choline
content determination with a phospholipid enzymatic colorimetric test
(Boehringer Mannheim).
-ATPase of N.
crassa, purified by the procedure of Smith and Scarborough(1984),
was reconstituted into soybean lipids vesicles by gel filtration as
described under ``Experimental Procedures.'' This method
permits reconstitution of H
-ATPase with a lipid/ATPase
ratio of 5:1 w/w. More than 90% of the ATPase molecules are
catalytically active, and 99% of the ATPase molecules are oriented
inside-out, with the large cytoplasmic portion (containing the ATP
binding site) facing outward.
Choice of Protease for Proteolytic Cleavage
The
first criterion for choosing a protease was our ability to separate the
protease and the potential protease fragments from the lipid vesicles.
We incubated various proteases (leucine aminopeptidase, chymotrypsin,
Pronase, proteinase K, trypsin, bromelain, and carboxypeptidase Y) with
lipid vesicles at 1 mg of protease for 25 mg of lipid as described
under ``Experimental Procedures.'' The removal of the
proteases from the vesicle preparation after incubation was tested as
follows. After an overnight incubation at 30 °C of pure lipid
vesicles with the different proteases, alone or in combination (see
``Experimental Procedures''), the samples were eluted through
a Sepharose CL-6B column and concentrated; then the vesicles were
examined by FTIR spectroscopy. The ratio of the integrated intensities
in the protein absorption region (amide I: 1,600-1,700
cm) and in the lipid
(C=O) region
(1,700-1,800 cm
) allowed us to evaluate the
amount of self-digested enzyme fragments associated with the lipids
(Goormaghtigh et al., 1990) (not shown). We discarded leucine
aminopeptidase, chymotrypsin, and bromelain for which 0.014-0.06
mg of protein/mg of lipid was found sticking to the vesicles. For
Pronase, proteinase K, and trypsin + carboxypeptidase Y (in
combination), no significant signal was visible in the amide I region,
indicating that less than 0.01 mg of protein was present per mg of
lipid. Therefore, only these last four proteases were used in the rest
of the work.
Digestion of ATPase
Hydrolysis of
reconstituted ATPase was started by adding freshly prepared protease
(Pronase, trypsin, carboxypeptidase Y, or proteinase K) to the
reconstituted vesicles at various protease/ATPase ratios (see
``Experimental Procedures'' for details). After an incubation
at 30 °C for time periods ranging from 10 min to 50 h, the reaction
was stopped and the vesicles isolated and concentrated as described
under ``Experimental Procedures.'' For each sample, one
aliquot was analyzed by SDS-PAGE and another by infrared spectroscopy.
SDS-PAGE analysis (not shown) reveals that the ATPase band completely
disappears upon treatment with trypsin, Pronase, or proteinase K in all
of the above conditions. This is in agreement with a previous study
where we show that trypsin treatment carried out at similar
trypsin/protein ratios eliminates 99% of the ATPase band at 105 kDa on
the gel after 2 min of incubation (Vigneron et al., 1995). (
)of the protein remains associated with the vesicles.
Increasing the incubation time (up to 50 h) or adding more protease
only slightly augments the amount of digested protein. Preincubation of
the samples with 5 mM dithiothreitol (Sonveaux et
al., 1994) did not improve the extent of digestion. On the other
hand, incubation of the proteoliposomes with protease and elution of
the protease-treated vesicles in a buffer containing 150 mM KCl were sufficient to eliminate electrostatically bound ATPase
peptides. As compared with the 58-60% of the protein remaining
associated with the vesicles after incubation with proteinase K in the
absence of KCl, a further 5-10% of the vesicle-associated
proteolytic fragments was eliminated by adding KCl to the buffer. After
maximal digestion with the other proteases chosen in the presence of
KCl, we estimated that about 51%
(trypsin + proteinase
K), 55%
(Pronase) to 68%
(trypsin +
carboxypeptidase Y) of the peptide groups of the ATPase molecule remain
associated with the vesicles after the proteolysis (not shown). In
turn, we present below the data obtained with proteinase K at an
ATPase/proteinase K ratio of 5:1 or 1:1 (w/w), which was the most
efficient condition tested. No difference was detected between the 1:1
and 5:1 ratios. The hydrolysis rate reported in Fig. 2was
analyzed further as the sum of two components by a curve fitting
approach (see the legend of Fig. 2). The fast component could
represent the easily accessible part of the protein and the slow one
the hardly accessible segments, presumably the membrane-embedded
segments. This results in a reasonably good and unique fit. The fast
component (t
= 13 min) represents 52% of
the protein, and the slow component (t
=
216 h) represents the remaining 48% of the protein.
(C=O) lipid absorption peaks are
equal.
) compared with the lipid
(C=O) absorption peak (1,700-1,780
cm
), as established by Goormaghtigh et
al.(1990). The values are reported for samples before (white
circles) and after 2 h of deuteration (black circles).
These experimental data were fitted as a sum of two exponential
components (solid line) by nonlinear regression (algorithm of
Marquardt-Levenberg) of a function f =
(a
e
+
a
e
)
where a
and a
are
the proportions of each of the two populations, and k
and k
the time constants of these two
components.
Digestion of Bacteriorhodopsin
To
evaluate the efficiency of the proteinase K treatment on a well
characterized system, we treated reconstituted bacteriorhodopsin with
proteinase K under the same conditions as the
H-ATPase-containing vesicles. Bacteriorhodopsin was
reconstituted into soybean lipid vesicles at a lipid/bacteriorhodopsin
ratio of 5:1 w/w as described under ``Experimental
Procedures.'' Estimation of the amount of protein remaining
associated with the vesicles by infrared spectroscopy was performed as
for ATPase and revealed that, in agreement with the fraction of the
protein protruding on one side of the membrane (Henderson and Unwin,
1975; Henderson et al., 1990; Fimmel et al., 1989),
10% of the protein was eliminated after 2 h of digestion. Increasing
the incubation time (up to 8 h) did not allow us to eliminate more
protein. The presence of a SDS-PAGE band pattern (not shown), stable as
a function of the digestion time, and the limited digestion of the
bacteriorhodopsin as observed by infrared spectroscopy indicate that
the proteinase K treatment was restricted to the protein regions
protruding from the membrane and that no intramembrane cleavage (see
Dumont et al., 1985) occurred after increasing the incubation
time. Moreover, sequencing the four major bands visible on a SDS-PAGE
15% acrylamide gel (23, 20, 19, and 16 kDa) showed that the fragments
obtained corresponded to cleavages at the amino- and carboxyl-terminal
ends as well as at two sites situated in the extramembrane loops
connecting, respectively, helices 2 and 3, and 4 and 5 of
bacteriorhodopsin, demonstrating the efficiency of our proteolysis
conditions to cleave very short extramembrane domains.
Secondary Structure of Inserted
ATPase
Assigning different amide I components revealed by
Fourier self-deconvolution to different secondary structures are best
substantiated by monitoring their shift upon hydrogen/deuterium
exchange. Fig. 3reports the spectra of ATPase-containing
vesicles isolated after incubation for 24 h at 30 °C in the absence (Fig. 3A) and in the presence (Fig. 3B)
of proteinase K (protease/ATPase ratio of 1:1 w/w), respectively, for a
deuteration time from 0 to 160 min. Spectra have been self-deconvolved
with a 30-cm FWHH Lorentzian line shape and apodized
with a Gaussian line shape with a FWHH of 15 cm
to
yield a so-called resolution enhancement factor of 2 (Kauppinen et
al., 1981) as described elsewhere (Goormaghtigh and Ruysschaert,
1990). The lipid 117 (C=O) band is located at 1,741
cm
before or after protease treatment in Fig. 3. For ATPase incubated in the absence of protease (Fig. 3A), the amide I region before deuteration
presents two main features located at 1,656 and 1,634 cm
assigned, respectively, to the
-helix (and/or random coil
structure) and
-sheet structure. The 1,694 cm
band is tentatively assigned to the high frequency component of
the
-sheet structure. Remarkably, the exchange of the amide
hydrogen by deuterium (50% of exchange after 160 min, see below) does
not alter the frequency of the two main features resolved here,
indicating that little hydrogen/deuterium exchange has taken place for
the associated secondary structures. This confirms the assignment of
the 1,656 and 1,634 cm
bands to
-helix and
-sheet stable secondary structures. This observation is important
to confirm the assignments since several structures (loops, various
turns, extended chains; see Goormaghtigh et al. (1994d) for a
more exhaustive compilation) may absorb in the same frequency range but
are expected to exchange much faster because of weaker internal
hydrogen bonds. However, the intensity of the 1,656 cm
feature decreases as the hydrogen/deuterium exchange proceeds.
The random contribution of amide I absorbs in this region too but is
shifted near 1,645 cm
after deuteration. We could
therefore assign the intensity decrease at 1,656 cm
to the shift of random structure contributions to about 1,645
cm
. The random coil-associated band is probably too
broad to be resolved in the self-deconvolution conditions used here
(Byler and Susi, 1986) and is therefore hidden in the base line
underlying the resolved features described above. Surprisingly, the
same features are found in the membrane part of ATPase (Fig. 3B) except for their relative intensities.
Moreover, no significant intensity decrease at 1,656 cm
is observed, a possible indication that most of random component
has been removed by the proteinase K treatment.
of the reconstituted H
-ATPase
(lipid/ATPase ratio of 5:1 w/w) incubated for 24 h at 30 °C without (A) or with (B) proteinase K (protease/ATPase ratio
of 1:1 w/w). Spectra were recorded as a function of the time of
exposure to D
O-saturated N
flow, which is
indicated (in min) in the center of the figure. All spectra
are self-deconvolved with a resolution enhancement factor of 2 (see
``Experimental Procedures'').
-sheet component clearly present in the
membrane part of the ATPase is not an artifact brought by the protease,
by 30 °C incubation, or by any step of the subsequent treatment, we
report in Fig. 4the data obtained for bacteriorhodopsin handled
under the same conditions as ATPase. Clearly, a single peak located at
1,660 cm
and assigned to the
-helix structure
dominates the spectra. The reasons for the rather high frequency of
this helical component have been discussed elsewhere (Downer et
al., 1986; Rothschild and Clark, 1979). A weaker band at 1,684
cm
can be assigned to membranous
-turns
(Earnest et al., 1990). The even weaker band located at 1,636
cm
has been described before (Earnest et
al., 1990; Haris et al., 1989). As the maximum of this
component does not change upon deuteration, this can not be attributed
to the C=N of the Schiff base vibration (Rothschild et
al., 1984). This small feature at 1,636 cm
could arise from various sources, including membrane-embedded
turns or side chain contribution. The spectra of the bacteriorhodopsin
incubated in presence of protease are essentially identical, in
agreement with the cleavage of only 10% of its amino acids after
protease treatment.
of the reconstituted bacteriorhodopsin
(lipid/protein ratio of 5:1 w/w) incubated for 24 h at 30 °C
without (A) or with (B) proteinase K (protease/ATPase
ratio of 1:1 w/w). Spectra were recorded as a function of the time of
exposure to D
O-saturated N
flow, which is
indicated (in min) in the center of the figure. All spectra
are self-deconvolved with a resolution enhancement factor of 2 (see
``Experimental Procedures'').
region (amide I) of a deuterated film of
proteoliposomes incubated without (spectrum A) or with proteinase K
(spectrum B) at 30 °C for 1 h, are shown in Fig. 5. About
50% of the protein has been removed upon this treatment. The infrared
spectrum of the digested ATPase (B) reveals two shoulders
located at 1,654 and 1,636 cm
, corresponding to the
absorbance domains of
-helical and
-sheet structures. The
infrared spectrum of the part of the protein which was removed after
proteolytic digestion of the proteoliposomes was obtained by
subtracting the spectrum of the digested proteoliposomes from the
spectrum of control proteoliposomes (Fig. 5C). Spectrum
C presents a broad maximum centered around 1,646 cm
,
indicating the presence of a significant amount of random structure;
however, the deconvolved spectrum displays shoulders at
-helix and
-sheet frequencies (results not shown), confirming that most of
the random coil structure was present in the part of the protein which
has been removed by proteinase K treatment. It must be emphasized here
that spectrum C obtained by the difference between the intact ATPase
and the membrane part of the ATPase represents the spectrum of the
cleaved part of the protein as it was in the intact protein and gives
no information on the structure of the peptides released after
cleavage. The presence of a random structure in the extramembrane
domain of ATPase and the increase in
-helical and
-sheet
structure contribution in the amide I band of ATPase after protease
treatment can be observed for incubations with all of the proteases
tested (Pronase, proteinase K, trypsin + carboxypeptidase Y) (not
shown). Identical results are obtained for proteinase K whatever the
digestion time as soon as the plateau of digestion is reached (see Fig. 2), indicating the stability of the membrane fragments
during long incubation times.
-helical domain), 1,646 (random domain), and 1,636
cm
(
-sheet domain). Curves are rescaled so that
addition of the membrane part of the ATPase (spectrum B) and of the
cleaved part of the ATPase (spectrum C) yields intact ATPase (spectrum
A).
-helix,
26%
-sheet, 18% random, and 16%
-turn. For the isolated
membrane part of the protein, no random structure was allowed according
to the hydrogen/deuterium exchange data. It contained 59%
-helices, 25%
-sheets, and 15%
-turns.
component represents 23% of the total amide
I area before and after proteolytic treatment; this indicates that
proteolytic treatment and subsequent handling of the sample do not
induce an increased intensity of a low frequency component in amide I.
The major 1,658 cm
component, assigned to helices,
represents the majority of the peak.
Orientation of ATPase Secondary Structures with
Respect to the Membrane in the Films
The orientation of the
secondary structures in the intact and digested ATPase was determined
from the FTIR-ATR spectra recorded with parallel and antiparallel
polarized incident light (see ``Experimental Procedures'').
Comparison of the amide I absorption of these two polarizations is made
more convincing from a dichroism spectrum obtained by subtracting the
0° polarized spectrum from the 90° polarized spectrum as
described (Goormaghtigh and Ruysschaert, 1990). The dichroic spectrum (Fig. 6) shows a maximum in the amide I region around 1,652
cm. This reveals a preferential orientation of the
-helices near the normal to the membrane. For protease-treated
ATPase, whatever the protease used, the dichroic spectrum shows the
same positive deviation of amide I (not shown) in the
-helix
region, indicating that the helices oriented in the transmembrane
direction are the helices that are located in the isolated membrane
part of ATPase. The persistence of the dichroism also indicates that no
major reorientation of the helical component occurs upon proteolytic
removal of the cytoplasmic region of the ATPase molecule. No dichroism
can be detected for the
-sheet component of the spectrum.
, corresponding to
-helices clearly oriented
perpendicularly to the lipid bilayer, as shown previously (Goormaghtigh et al., 1990).
Amide Hydrogen/Deuterium Exchange
Kinetics
At constant experimental conditions (pH and
temperature), the rate of hydrogen/deuterium exchange is related to the
solvent accessibility of the NH amide groups of the protein, which is
related to the tertiary structure of the proteins and to the stability
of the secondary structures.(N-H), maximum at 1,544 cm
) decrease or amide
II` absorption peak (
(N-D), maximum at 1,450
cm
) increase as a function of the time of exposure
to D
O-saturated N
flow (from 15 s to 3 h; for
details, see ``Experimental Procedures''). This is
illustrated in Fig. 7, where a series of spectra recorded as a
function of the deuteration time of
H
-ATPase-containing samples is reported. Data were
recorded in the course of the same experiment (see ``Experimental
Procedures''; not shown) for samples isolated after incubation for
24 h at 30 °C without (control) or with proteinase K
(protease/ATPase ratio of 1:1 w/w). The evolution of the area of amide
II for each sample was then computed between 0 and 100% as explained
under ``Experimental Procedures'' reported in Fig. 8.
It appears that the hydrogen/deuterium exchange is faster for the
control than for the protease-treated sample. After 2 h of deuteration,
about 50% of the peptide N-H is exchanged in the control, whereas for
the protease-treated sample only 40% is exchanged after 2 h.
of reconstituted H
-ATPase
(lipid/ATPase ratio of 5:1 w/w) incubated for 24 h at 30 °C
(without protease). Spectra were recorded as a function of the time of
exposure to D
O-saturated N
flow, which is
indicated (in min) in the right margin of the figure.
Integration of the areas of amide I, amide II, and amide II` is
performed using the base lines indicated by the dotted lines drawn between the intersection of the spectra with the vertical
line segments. Amide II and II` surfaces are hatched at 0 and 160
min.
-ATPase in a 0.5 mM phosphate buffer at pH
6.8 after a 24-h incubation at 30 °C without (control) or with
proteinase K at a proteinase K/ATPase ratio of 1:1 w/w. The percentage
of deuteration was estimated from the amide II surfaces evolution as
described under ``Experimental Procedures.'' Each curve represents the average for three independent exchange experiments. Error bars represent the maximal deviation from the
average.
-ATPase into the membrane. The approach chosen
was to use proteases to cleave the segments of ATPase protruding from
the membrane while preserving the integrity of the membrane domain.
(
)that proteinase K does not cross the vesicle membrane.
-helices (Henderson and Unwin, 1975; Henderson et al., 1990; Fimmel et al., 1989). Moreover, the
extent of digestion does not increase significantly when increasing the
proteolysis time as demonstrated by FTIR spectroscopy and SDS-PAGE.
These results confirm that 1) the extramembrane loops are cleaved close
to the membrane-buffer interface, and 2) proteolytic digestion of the
transmembrane peptides does not occur. The stable digestion pattern
seen on SDS-PAGE gels of digested bacteriorhodopsin is a supplementary
evidence for the stability of membrane peptides. Furthermore, the
strong amide I polarization indicating a transmembrane orientation of
the
-helices in the intact bacteriorhodopsin molecule and in its
membrane domain isolated after various incubation times in the presence
of proteinase K is an additional indication of the integrity of the
membrane domain structure of the protein under all of our experimental
conditions. Finally, the very slow hydrogen/deuterium exchange measured
by infrared spectroscopy before and after proteolytic digestion
confirms the presence of stable membrane structures in the intact
protein as well as in its isolated membrane fraction. These data are in
line with cleavage experiments performed by Draheim et al. (1991) which showed that the secondary structure and the
orientation of the isolated membrane domain of bacteriorhodopsin do not
change after proteolysis. Moreover, the integrity and stability of
membrane fragments of bacteriorhodopsin after proteolysis or after
separated reconstitution are well documented (Kahn and Engelman, 1992;
Kahn et al., 1992; Popot et al., 1987).
-ATPase
requires a highly oriented reconstitution. We showed before that the
proteoliposomes obtained by reconstitution of the purified ATPase in
the soybean lipid extract have more than 99% of the ATPase molecules
oriented with their cytoplasmic side facing the outside of the vesicles
(Vigneron et al., 1995). The bulky cytoplasmic side of the
ATPase molecule is therefore accessible to the protease, but the small
connecting loops located in the vesicles interior are not. The presence
of a very fast (t
= 13 min) and a very
slow component (t
= 216 h) in the course
of the proteolysis indicates that one part of the protein, presumably
exposed to the buffer, is hydrolyzed rapidly while the other part of
the protein is not hydrolyzed significantly within the time course of
our experiments. The similar polarization of the
-helical
component of amide I in the intact protein and in its isolated membrane
part indicates the stability of the orientation of this membrane
protein structure upon removal of the connecting segments.
(C=O) area
to evaluate the lipid/protein ratio change is based on the number of
peptide bonds present on the protein and is accurate as shown
previously (Goormaghtigh et al., 1990). It is, however, based
on the assumption that the amide-integrated extinction coefficients are
similar for all of the secondary structure types. It has been reported
that the amide I-integrated extinction coefficient of the random
structure is only 81% (hydrogen form) or 80% (deuterated form) of the
value determined for the
-helix or
-sheet structure (computed
from Venyaminov and Kalnin, 1991; Chirgadze and Brazhnikov, 1974). If
from the 52% of the protein removed by the protease, 18% is organized
as random structures, the measured 18% is underestimated because of the
lower extinction coefficient of the random structure, and 18%/0.8
= 23% is the true value. In turn, we can estimate that at most
57% of the protein is removed by proteinase K treatment.
-ATPase molecule (Rao and
Scarborough, 1990) is not essential to maintain parts of the
polypeptide chain embedded in the membrane. This 43% represents more
than expected from the hydropathy plots (about 20% of the polypeptide
chain in the membrane and 4% of connecting loops located at the
interior of the vesicles (Hager et al., 1986; Addison, 1986)),
which postulates that all transmembrane segments are
-helices.
However, our results are compatible with the model proposed by Rao et al.(1991, 1992) on the basis of sequencing membrane and
extramembrane peptides of the H
-ATPase isolated after
trypsin digestion. According to these authors, about 387 amino acids
out of 920 are membrane-embedded or membrane-associated, corresponding
to 42% of the protein. A hypothesis to explain the discrepancy between
experimental data and hydropathy plot-derived models is that the
extracytoplasmic domain of the ATPase is more important than presented
in the current models. However, this explanation seems improbable, as
this non-cytoplasmic extramembrane part of the ATPase has been
described by Hennessey and Scarborough(1990) as highly resistant to
tryptic digestion and therefore small, in agreement with crystal
analyses performed for Ca
-ATPase (Taylor et
al., 1986; Stokes and Green, 1990; Toyoshima et al.,
1993). It has also been shown for
Na
/K
-ATPase that models based on the
hydrophobicity of the amino acid sequence underestimate the proportion
of amino acids that are inserted in the bilayers, in comparison with
models based on chemical labeling experiments (Sweadner and
Arystarkhova, 1992). An alternative hypothesis to explain this
discrepancy is that only transmembrane
-helix secondary structures
have been considered so far.
-ATPase before
incubation with proteases (38%
-helix, 26%
-sheet, 16%
-turn and 18% random) is in good agreement with the secondary
structure determined by Hennessey and Scarborough (1988) by circular
dichroism for H
-ATPase in solution in a detergent
(lyso-PG): 36%
-helix structure, 20%
-sheet structure, 11%
-turns, and 26% random structure. Analysis of the infrared spectra
of the isolated membrane part of the ATPase molecule indicates the
presence of 59%
-helix (230 amino acid residues), 25%
-sheet
(99 amino acid residues), and 15%
-turns (59 amino acid residues).
It must be stressed that we eliminated the potential presence of random
coil structures located in the membrane domain of the ATPase molecule
on the basis of the spectral behavior upon hydrogen/deuterium exchange.
Particularly convincing as to the presence of
-sheet structures in
the membrane-associated proteolytic peptides of the ATPase are the
spectra reported in Fig. 3B, where one of the two main
resolved features in amide I is located at 1,634 cm
,
a frequency assigned to the
-sheet structure and which is never
overlapped by helical contribution (Goormaghtigh et al.,
1994b, 1994d). Extended polypeptide chains have been found at similar
frequencies in some protein (Byler and Susi, 1986), but this hypothesis
is unlikely here since only
-sheet structures can form the
internal hydrogen bonding required in the membrane environment. The
absence of a distinct dichroism from the
-sheet structure implies
that these
-sheets are either disordered or are oblique, as is the
case for porine (Weiss et al., 1991) in agreement with the
lack of distinct infrared linear dichroism (Nabedryk et al.,
1988). The presence of
-sheet structures in the
membrane-associated domain of ATPase implies that the current models
for these proteins need to be revised. Such an overhaul is also
dictated by the finding that the 272-291 segment of the protein,
identified previously as transmembrane (Rao et al., 1991), is
in fact located out of the membrane (Lin and Addison, 1994). In the
transmembrane structure of the porin monomer, 16 segments of 11
± 5 residue length form a
-pleated sheet structure and are
connected by turns (Paul and Rosenbusch, 1994), but segments as short
as 5 residues in length are sufficient to span the hydrophobic core of
the membrane (Cowan and Rosenbusch, 1994). It is premature to describe
how the
-sheet structure of ATPase is associated with the
membrane, but the presence of a rather large number of short stretches
spanning the membrane would explain the relatively large number of
amino acids involved in the turn structure. The presence of 230 amino
acid residues involved in
-helices is compatible with the presence
of about 10 transmembrane helices in ATPase. As for the analysis of the
dichroic spectra, it reveals a transmembrane orientation of the
-helices in agreement with hydropathy plot-derived models but does
not show significant dichroism for the
-sheet component of amide
I.
-helical segment of glycophorin is virtually
not exchangeable (Sami and Dempsey, 1988; Challou et al.,
1994). On the other hand, the human erythrocyte glucose transporter
exchanges about 75% of its amide protons by deuterium within the 1st h
(Alvarez et al., 1987) even though structure predictions
suggest the presence of 12
-helices (accounting for 50% of the
protein) in the membrane. It was concluded that an access to the
aqueous medium exists in the transmembrane part of the protein (Alvarez et al., 1987). It must be mentioned that the 12
-helix
model has been challenged recently (Fischbarg et al., 1993).
The channel-forming peptide of colicin E1, which has been shown to be
predominantly
-helical, also has a very fast exchange (80%
exchange in 5 min (Rath et al., 1991)). The results obtained
here for Neurospora H
-ATPase show that the
intact protein exchanges 50% of its N-H protons after 1 h (about 460
amino acids out of 920). For the membrane region of ATPase, the
40% of exchange observed after 1 h (corresponding to about 158
membrane amino acids out of 396) indicates that the transmembrane
region is much less tightly protected against hydrogen/deuterium
exchange than the membrane region of bacteriorhodopsin. These data
suggest that we cannot build a model of the transmembrane region of
ATPase based only on the structure and on the packing of the
transmembrane segments of bacteriorhodopsin, as is already evident from
secondary structure analysis.
-ATPase. Digestion by trypsin (30 min)
and proteinase K (30 min) on the cytoplasmic side of the membrane
resulted in the removal of 63% of the protein (according to lipid and
protein assays) in agreement with the model-derived value of 66%
(Clarke et al., 1989a, 1989b). Fourier self-deconvolution and
partial least squares analysis of amide I demonstrated the absence of
-sheet structure in the proteinaceous material associated with the
membrane after proteolytic digestion, in good agreement with the models
of Ca
-ATPase. We must stress two facts here. 1) The
amide I/amide II ratio close to 1:1 in solution spectra instead of 2:1
for usual proteins indicates that the area of amide I could have been
decreased artificially by oversubtraction of the water band. The amide
I/amide II ratio is normal in the ATR spectra. In the latter case,
spectra were recorded in the absence of liquid water, and water
subtraction was not necessary. 2) The ATR spectra display a clear
-sheet shoulder in amide I which does not exist in the solution
spectra when oversubtraction could have occurred. The marked
discrepancy between these data and the results accumulated in the
present paper for another P-ATPase prompted us to repeat the
proteolytic digestions on Neurospora ATPase according to the
experimental conditions described by Corbalan-Garcia et
al.(1994). We obtained results identical to those described in the
present paper. When our digestion conditions are applied to gastric
H
,K
-ATPase and sarcoplasmic reticular
Ca
-ATPase, identical results are observed for
H
,K
-ATPase, whereas
Ca
-ATPase shows a clear increase in the
-helix content and the disappearance of the
-sheet
content,
(
)suggesting that Neurospora H
-ATPase and gastric
H
,K
-ATPase show a similar resistance
to proteases, but Ca
-ATPase does not. Whether a
serious structural difference exists between H
and
H
,K
-ATPase on one hand and
Ca
-ATPase on the other is suggested by the present
work but needs to be controlled by other means.
-ATPase, of 230
amino acid residues involved in
-helical structures, a figure that
is in agreement with the presence of 8-10 transmembrane helices.
Linear dichroism data confirm their transmembrane orientation. In
addition, 99 amino acid residues are involved in
-sheet structures
associated with the membrane, and 59 are involved in turns. The
hydrogen/deuterium exchange rate of the membrane part of the protein
suggests an organization of these different secondary structures in
such a way that the solvent has access to a large fraction of the
protein, perhaps through a membrane hole or pore delimited by the
protein.
We acknowledge P. Duquenoy for drawing some of the
figures.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.