©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Fourier Transform Infrared Spectroscopy Study of the Secondary Structure of the Reconstituted Neurospora crassa Plasma Membrane H-ATPase and of Its Membrane-associated Proteolytic Peptides (*)

(Received for publication, December 20, 1994; and in revised form, May 15, 1995)

Laurence Vigneron (§) Jean-Marie Ruysschaert Erik Goormaghtigh (¶)

From the Laboratoire de Chimie Physique des Macromolecules aux Interfaces, CP206/2, Universit Libre de Bruxelles, Campus Plaine, B1050 Brussels, Belgium

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We reconstituted purified plasma membrane H-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.


INTRODUCTION

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-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.

In a previous paper (Vigneron et al., 1995), we described the efficient reconstitution of purified plasma membrane H-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.


EXPERIMENTAL PROCEDURES

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).

All other reagents were of the highest purity grade commercially available. H-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-ATPase

H-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).

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.

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 DO-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 HO 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).


Figure 6: Infrared polarized parallel (90°) and antiparallel (0°) (with respect to the plane of the membrane) absorption spectrum of reconstituted H-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 DO) 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 DO-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 DO 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.

For analysis of the deconvolved spectra during the time course of exchange, each spectrum was self-deconvolved with a 30 cm 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).


RESULTS

Plasma membrane H-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).

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%()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.


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 (C=O) lipid absorption peaks are equal.




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) 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(1)e + a(2)e) where a(1) and a(2) are the proportions of each of the two populations, and k(1) and k(2) the time constants of these two components.



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).

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.


Figure 3: Infrared spectra between 1,800 and 1,500 cm 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 DO-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'').



To provide evidence that the unexpected -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.


Figure 4: Infrared spectra between 1,800 and 1,500 cm 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 DO-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'').



The FTIR spectra in the 1,600-1,700 cm 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.


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 (-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).



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% -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.

An identical analysis of the shape of the bacteriorhodopsin spectra reveals that the 1,636 cm 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.

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, 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.

Peptide hydrogen exchange of the protein was followed by monitoring the amide II absorption peak ((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 DO-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.


Figure 7: Infrared spectra between 1,800 and 1,400 cm 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 DO-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.




Figure 8: Percentage of deuteration reported as a function of the deuteration time for reconstituted H-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.



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.


DISCUSSION

The aim of this work was to verify experimentally the models proposed to describe the mode of insertion of N. crassa H-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.

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()that proteinase K does not cross the vesicle membrane.

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 -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).

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-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.

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 (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.

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-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.

Quantitative evaluation of the structure of the reconstituted H-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.

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 -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.

In a recent paper, Corbalan-Garcia et al.(1994) applied a similar proteolytic approach to reticular Ca-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.

In conclusion, our results show the presence, in the membrane-associated domain of the N. crassa plasma membrane H-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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture Fellow. To whom correspondence should be addressed. Tel.: 32-2-650-53-86; Fax: 32-2-650-51-13.

Research Associate of the National Fund for Scientific Research (Belgium).

The abbreviations used are: FTIR-ATR spectroscopy, Fourier transform infrared attenuated total reflection spectroscopy; Mes, 2-(N-morpholino)ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; FWHH, full width at half height.

These values should be corrected by about 5% as explained under ``Discussion.''

V. Raussens, personal communication.

M. le Maire, unpublished data.


ACKNOWLEDGEMENTS

We acknowledge P. Duquenoy for drawing some of the figures.


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