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Originally published In Press as doi:10.1074/jbc.M310422200 on December 29, 2003

J. Biol. Chem., Vol. 279, Issue 12, 10955-10961, March 19, 2004
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Role of the Transmembrane Domain in the Stability of TrwB, an Integral Protein Involved in Bacterial Conjugation*

Itsaso Hormaeche{ddagger}, Ibón Iloro{ddagger}, José L. R. Arrondo{ddagger}, Félix M. Goñi{ddagger}§, Fernando de la Cruz¶, and Itziar Alkorta{ddagger}

From the {ddagger}Unidad de Biofísica (Centro Mixto Consejo Superior de Investigaciones Científicas-Universidad del País Vasco), and Departamento de Bioquímica, Universidad del País Vasco, Aptdo. 644, 48080 Bilbao, Spain and Departamento de Biología Molecular, Universidad de Cantabria, 39011 Santander, Spain

Received for publication, September 22, 2003 , and in revised form, December 23, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TrwB is an integral membrane protein encoded by the conjugative plasmid R388. TrwB binds ATP and is essential for R388-directed bacterial conjugation. The protein consists of a cytosolic domain, which contains an ATP-binding site, and a transmembrane domain. The complete protein has been purified in the presence of detergents, and in addition, the cytosolic domain has also been isolated in the form of a soluble truncated protein, TrwB{Delta}N70. The availability of intact and truncated forms of the protein provides a convenient system to study the role of the transmembrane domain in the stability of TrwB. Protein denaturation was achieved by heat, in the presence of guanidinium HCl, or under low salt conditions. In all three cases TrwB was significantly more stable than TrwB{Delta}N70 with other conditions being the same. IR spectroscopy of the native and truncated forms revealed significant differences between them. In addition, it was found that TrwB{Delta}N70 was stabilized in dispersions of non-ionic detergent, suggesting the presence of hydrophobic patches on the surface of the truncated protein. IR spectroscopy also confirmed the conformational stability provided by the detergent. These results suggest that in integral membrane proteins consisting of a transmembrane and a cytosolic domain, the transmembrane portion may have a role beyond the mere anchoring of the protein to the cell membrane. In addition, this study indicates that the truncated soluble parts of two-domain membrane proteins may not reflect the physiological conformation of their native counterparts.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The purification and study of integral membrane proteins present serious problems, derived from the requirements of a partly aqueous, partly hydrophobic environment for the maintenance of a native-like conformation and activity by those proteins (1–4). Membrane protein stability problems have been circumvented by the use of reconstitution techniques (5) or the development of novel, protein-friendly detergents (6, 7) or amphiphilic polymers (8). For integral membrane proteins consisting of a transmembrane and an extramembranous domain, the latter harboring the biochemical activity, truncated mutants devoid of the transmembrane region have been used (9–11).

Transport of DNA across cell membranes occurs in different biological processes. Among them, bacterial conjugation is one of the major routes by which bacterial pathogens acquire antibiotic resistance. Conjugative plasmid R388 has the shortest known mobilization region (12), and TrwB is the conjugative coupling protein encoded by this region (13). TrwB amino acid sequence analysis predicts an integral membrane protein of 507 residues (13, 14), where the transmembrane domain predicted by sequence analysis comprises the 70 N-proximal residues, and includes two transmembrane helices and a small periplasmic segment in between. The cytosolic domain contains a "Walker box," and in fact it binds ATP (11, 13). Because of the difficulty in handling the entire membrane protein, a soluble form of TrwB lacking the N-terminal transmembrane segments (TrwB{Delta}N70) was designed, purified, and partially characterized (13). This mutant protein shed some light on the role of TrwB in the conjugation process (13, 15, 16). More recently, purification of whole TrwB in detergent has been reported (17). The intact protein binds ATP with significantly higher affinity than the truncated soluble form.

Availability of both the native protein (TrwB) and the truncated mutant lacking the transmembrane domain (TrwB{Delta}N70) prompted us to compare the stability of both protein forms. Native and truncated TrwB were subjected to different stability challenges, namely guanidinium hydrochloride (GdmHCl),1 heat, and low ionic strength. In all cases the whole protein was more stable than the truncated form. Moreover, truncated TrwB was found to be stabilized by detergent, but even in this case stability was lower than for whole TrwB. These results provide direct evidence for the role of the transmembrane anchor in maintaining the functional conformation of an extramembranous domain in an integral membrane protein.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Plasmids—Escherichia coli strains and plasmids are listed in Tables I and II. For conjugation experiments, the recA strains DH5{alpha} and UB1637 were used as donor and recipient, respectively.


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  		TABLE I
E. coli strains used in this work

 


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  		TABLE II
Bacterial plasmids used in this work

 
Oligonucleotides and Construction of Plasmid pBU3—The oligonucleotides used were as follows. The forward primer was oligonucleotide 1, 5'-GGAGATATACATATGCATCCAGA-3', with an NdeI site (underlined), and the reverse primer was oligonucleotide 2, 5'-AAGAGTCAGctcgagGATAGTCCC-3', which creates an XhoI site (underlined). Lowercase letters show the variation from the wild type sequence introduced by oligonucleotide (2) at the 3'-terminus of the trwB gene to eliminate the stop codon to accommodate an in-frame C-terminal His tag in the TrwB protein.

To overproduce TrwBH6 protein, plasmid pBU3 was constructed as follows. Plasmid pUB1, which carries the trwB gene with a silent mutation that creates a KpnI site (17), was used as template for a PCR reaction with oligonucleotides 1 and 2. The resulting 1.5-kbp fragment was digested with NdeI and XhoI. Subsequently, this fragment was cloned in NdeI-XhoI-digested pET-22b(+) (Novagen, Madison, WI). This construction places a His6 tag at the C terminus of TrwB. The resulting construction directs expression of TrwB protein under control of the T7 promoter. Plasmid pBU3 was transformed to and stored in strain D1210. For protein overproduction, plasmid pBU3 was introduced in E. coli BL21 C41(DE3), a mutant strain of E. coli BL21(DE3) (18).

Genetic Techniques—Bacterial transformation with plasmid DNA was carried out by the CaCl2 method (19). Genetic complementation analysis of the conjugal ability of TrwB was carried out as described by Llosa et al. (14). Donor cells were selected on Tp + Ap and transconjugants on Su + Tp.

Purification of TrwB{Delta}N70 —TrwB{Delta}N70 was produced by overexpression and purified as described by Moncalián et al. (13).

Purification of His-tagged TrwBH6—TrwBH6 protein was overproduced and solubilized in dodecylmaltoside (DDM) as described by Hormaeche et al. (17). The membrane fraction was loaded onto a cellulose phosphate P-11 column (Whatman), and the elution pattern was as described by Hormaeche et al. (17), so that fractions II (eluted at 500 mM NaCl) and II' (eluted at 1 M NaCl) were obtained. From this point onward the two fractions were purified separately.

Fraction II was supplemented with 5 mM imidazole and loaded onto a 5-ml HiTrap chelating column (Amersham Biosciences) connected to a Pharmacia FPLC system equilibrated with a buffer containing 200 mM NaCl, 0.05% (w/v) DDM, 0.1 mM EDTA, 50 mM Tris-HCl, pH 7.8. Proteins were eluted from the column with a 0–175 mM imidazole gradient in the above described buffer at a flow rate of 2.5 ml/min. Fractions containing TrwB protein were pooled (Fraction III), concentrated, and loaded onto a Superdex HR-200 column. TrwB eluted from this column in a monomeric state (Fraction IV).

Fraction II' was loaded onto a HiTrap chelating column as described previously. The resulting Fraction III' was concentrated and loaded onto a Superose 6 column. A TrwB sample was obtained that corresponded to the hexameric form. All protein determinations were performed by the Bradford method (20).

Intrinsic Fluorescence Measurements—The intrinsic tryptophanyl fluorescence of TrwB and Trw{Delta}N70 was measured in a PerkinElmer LS50 spectrofluorometer. Excitation wavelength was 295 nm, and emission wavelength was 336 or 340 nm. Slits were 10 nm. Protein concentration was in the 0.8 to 1 µM range.

Treatment of Enzyme with GdmHCl—Enzyme (0.5 µM) was incubated with different concentrations of GdmHCl ranging from 0 to 8 M at 25 °C for 1 h in a volume of 100 µl. Emission spectra of the enzyme in the presence of GdmHCl were recorded in a 100 µl volume in 50 mM Tris-HCl, pH 7.8, 0.1 mM EDTA, 20% glycerol, either 150 mM KCl or 200 mM NaCl, and 0.05% DDM.

Infrared Spectroscopy—The protein samples were measured typically at 4 mg/ml in 50 mM Tris-HCl, 150 mM NaCl buffer, pH 7.8. The H-D exchange was carried out by lyophilization. The spectra were recorded in a Nicolet Magna II 550 spectrometer equipped with a mercury-cadmium-telluride detector using a demountable liquid cell (Harrick Scientific, Ossining, NY) with calcium fluoride windows and 50-µm spacers. A tungsten-copper thermocouple was placed directly onto the window, and the cell was placed into a thermostatted cell mount. Typically 1000 scans for each, background and sample, were collected, and the spectra were obtained with a nominal resolution of 2 cm–1. The data treatment and band decomposition of the original amide I have been described elsewhere (21–23). The mathematical solution to the decomposition may not be unique, but if restrictions are imposed such as maintenance of the initial band positions in an interval of ±1 cm–1, preservation of bandwidth within the expected limits, or agreement with theoretical boundaries or predictions, the result becomes, in practice, unique.

Thermal analysis was performed by heating continuously in the range of 25 to 80 °C with a heating rate of 1 °C/min. Spectra were collected by using a rapid scan software running under OMNIC (Nicolet). Typically 305 interferograms were collected/spectrum and then referred to a background, the spectra being obtained with a nominal resolution of 2 cm–1. Thermal profiles were obtained as described elsewhere (24).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Denaturation by Guanidinium Hydrochloride—To compare the stability of TrwB with that of TrwB{Delta}N70, the proteins (0.5 µM) were equilibrated in buffer (50 mM Tris-HCl (pH 7.8), 0.1 mM EDTA, 20% (v/v) glycerol, 200 mM NaCl, 0.05% DDM (w/v)) with different GdmHCl concentrations. Protein unfolding was detected by measuring fluorescence emission due to aromatic residues, likely dominated by the tryptophan residues. Unfolding is accompanied by a shift of the maximum fluorescence emission wavelength toward higher values, because of exposure of Trp residues to a more polar (aqueous) environment. Fig. 1A shows that the proteins followed different denaturation patterns. For the intact protein in detergent, denaturation started above 3 M GdmHCl and increased linearly with denaturant agent concentration, up to about 7 M GdmHCl. The truncated form displayed a higher maximum fluorescence emission wavelength ({lambda}em) even in the absence of GdmHCl. This may be a sign of partial unfolding or else because two Trp residues are missing in the truncated protein, so that its fluorescence emission properties may be different. There was no significant change in TrwB{Delta}N70 fluorescence until GdmHCl reached 4 M. However, complete denaturation of TrwB{Delta}N70 took place at concentrations as low as about 5 M GdmHCl. Note that in Fig. 1A, TrwB{Delta}N70 was suspended in the same detergent concentration as TrwB, so that the difference in stability can be solely attributed to the transmembrane domain.



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  		FIG. 1.
Changes in the maximum wavelength of emission of TrwB intrinsic Trp fluorescence as a function of GdmHCl concentration. Proteins (0.5 µM) were equilibrated in 50 mM Tris-HCl (pH 7.8), 0.1 mM EDTA, 20% (v/v) glycerol, 200 mM NaCl, 0.05% (w/v) DDM with different GdmHCl concentrations. A, effect of truncation: ({circ}) TrwB{Delta}N70, () TrwB. B, effect of detergent: TrwB{Delta}N70 in the presence ({circ}) or absence ({triangleup}) of 0.05% (w/v) DDM.

 
Resistance to GdmHCl denaturation was also used to test the effect of the detergent on the soluble protein TrwB{Delta}N70. Fig. 1B shows the effect of GdmHCl (up to 8 M) on the fluorescence emission maximum wavelength in protein preparations incubated in buffer with or without 0.05% (w/v) DDM. Although similar red-shifts in the emission maximum were observed in both cases (11 nm), the detergent confers stability to the protein because in its presence the red-shift occurs only at concentrations above 4 M GdmHCl, whereas in the absence of detergent even at 2 M GdmHCl a change in fluorescence emission maximum is detectable. In consequence, the stability of the proteins under study increases in the order: TrwB{Delta}N70 in the absence of detergent < TrwB{Delta}N70 in 0.05% (w/v) DDM < TrwB in 0.05% (w/v) DDM.

Secondary Structure of TrwB and TrwB{Delta}N70 —Information on the secondary structure of the native and truncated proteins was obtained by analysis of the infrared amide I band, located between 1700 and 1600 cm–1 and arising mainly from the C=0 stretching vibration of the peptidic bond. This band is conformationally sensitive and can be used to monitor either the secondary structure composition or the changes induced in the protein by external agents (21, 23). Fig. 2 shows the curve-fitted spectra of TrwB and of TrwB{Delta}N70, both in the presence of DDM, i.e. the same initial conditions as in Fig. 1A. The original amide I envelope was curve-fitted using the band positions obtained from the deconvolved spectra, and the results are summarized in Table III. TrwB was an {alpha}/{beta} protein, in agreement with data from the crystal structure of the truncated form; 38% {alpha}-helix and 17% {beta}-sheet were found by x-ray versus 35.9% {alpha}-helix and 32.5% {beta} + unordered estimated from IR measurements of TrwB{Delta}N70 (15, 16). For the intact protein in detergent, {alpha}-helical structures clearly predominated. In TrwB{Delta}N70 removal of the transmembrane domain caused major changes in the distribution of secondary structure elements, mainly a decrease in the proportion of {alpha}-helix with a concomitant increase in the band components centered at 1639 and 1625 cm–1. The signal at 1639 in D2O corresponds to both {beta}-sheet and unordered structure, and thus truncation appeared most likely to increase the proportion of unordered components. The band at 1625 cm–1 in non-aggregated proteins has been associated with monomer-monomer contacts in oligomeric proteins (25), thus suggesting subunit aggregation under the conditions of the IR measurements. It should be noted in this respect that TrwB{Delta}N70 crystallized in hexamers (15). Finally, the band component around 1660 cm–1 has been attributed to turns but also to 310-helices (21). X-ray data indicated a 310-helix structure for helix E in the so-called all-alpha domain of TrwB{Delta}N70 (15).



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  		FIG. 2.
Amide I region of the infrared spectrum of intact and truncated TrwB. The spectra were curve-fitted to show the different secondary structure components as detailed in Table III. The proteins were freeze-dried and reconstituted in D2O buffer containing 50 mM Tris-HCl (pD 7.8), 0.1 mM EDTA, 200 mM KCl, and 0.05% (w/v) DDM. A, native TrwB. B, TrwB{Delta}N70.

 


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  		TABLE III
Secondary structure components of native and truncated TrwB as derived from the amide I band of infrared spectra recorded in D2O medium

Both native (TrwB) and truncated (TrwB{Delta}N70) proteins were suspended in the same buffer containing salts and 0.05% (w/v) DDM. The spectra are shown in Fig. 2. For details on the assignments, see text.

 
Thermal Denaturation Studies—The structural difference between the native and truncated forms of TrwB in the presence of detergent has been studied by infrared spectroscopy looking at the changes in secondary structure observed by decomposition of the amide I band and their response to temperature. The study of protein structural changes caused by an external perturbation such as temperature provides information on the protein conformation and organization during unfolding. Fig. 3, A and B, shows plots of the deconvolved amide I band in D2O of TrwB and TrwB{Delta}N70 in the 20–80 °C interval. At high temperatures, the infrared spectrum was characterized by the appearance of two bands around 1617 and 1685 cm–1, representative of protein aggregation. The temperature at which these bands appear is usually related to the compactness of the protein. A looser protein tends to melt at a lower temperature (23). This denaturation temperature could be obtained from the changes in the amide I; the widths at half-height (Fig. 3C) showed that there was a difference of around 15 °C in the denaturation temperature between the truncated and native proteins, the latter being more stable. Similar differences have been reported between cytochrome c and its apocytochrome (26) and in concanavalin A between the native and the Mn2+-depleted forms (27).



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  		FIG. 3.
A and B, deconvolved IR spectra of TrwB (A) and TrwB{Delta}N70 (B) in buffer with DDM at increasing temperatures, showing thermal denaturation of the proteins. The samples were freeze-dried, resuspended in D2O buffer, and analyzed by IR spectroscopy at different temperatures. The bands appearing at high temperatures at 1617 and 1685 cm–1 are typical of protein aggregation. C, thermal denaturation of TrwB and TrwB{Delta}N70 as seen by IR spectroscopy. The widths at half-height (WHH) of the amide I bands in A and B are plotted as a function of temperature for TrwB () and TrwB{Delta}N70 ({circ}). Thermal denaturation is marked by an abrupt increase in bandwidth.

 
Ionic Strength and Protein Stability—Ionic strength and salt composition of the medium are well known factors that influence protein stability. In our case, experiments were designed to test the effect of salt concentration on the stability of TrwB and of the soluble mutant TrwB{Delta}N70. To study the effect of ionic strength, TrwB{Delta}N70 (0.8 µM) was incubated in different detergent-free, salt-containing buffers. Protein stability was detected by measuring the time course of decrease in fluorescence emission due to aromatic residues, likely dominated by the tryptophan residues. The decrease in fluorescence emission intensity is likely because of the transfer of Trp residues to a more polar (aqueous) environment. Fig. 4A shows the effect of salt concentration on TrwB{Delta}N70 intrinsic fluorescence. There is an important decrease in tryptophan fluorescence in the absence of salt. On the contrary, when salt concentration was 150 mM, the protein intrinsic fluorescence remained virtually unaltered with time, even when other buffer components, e.g. glycerol, were removed (Fig. 4A, plots 1 and 2). To establish a relationship between ionic strength and fluorescence stability, TrwB{Delta}N70 was incubated in buffers containing different salt concentrations. Fig. 4B illustrates the salt-dependent profile of the protein intrinsic fluorescence. As salt concentration increased, the difference between the initial intrinsic fluorescence value and the one measured after 90 min of incubation was smaller, indicating that salt had a stabilizing effect.



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  		FIG. 4.
Effect of ionic strength on TrwB{Delta}N70 intrinsic fluorescence emission. A, a protein preparation (0.8 µM) was incubated in different salt-containing buffers: 50 mM Tris-HCl (pH 7.8), 0.1 mM EDTA, 20% (v/v) glycerol, 150 mM NaCl (plot 1); 50 mM Tris-HCl (pH 7.8), 150 mM NaCl (plot 2); 50 mM Tris-HCl (pH 7.8) (plot 3). The intrinsic fluorescence decay was recorded versus time. B, dependence on salt concentration of the TrwB{Delta}N70 intrinsic fluorescence decay.

 
The relative stabilities of TrwB and TrwB{Delta}N70 under low salt conditions were studied, and the results are shown in Fig. 5A. In this case, because of technical difficulties in exchanging the buffer of TrwB, the salt concentration could not be decreased below 30 mM. Thus for both proteins in Fig. 5A the buffer composition was 50 mM Tris-HCl (pH 7.8), 3% (v/v) glycerol, 30 mM NaCl, 0.01% (w/v) DDM. As expected from the data in Fig. 4B, protein denaturation was not dramatic at 30 mM NaCl, but still TrwB was clearly more stable than TrwB{Delta}N70, in agreement with the GdmHCl and thermal denaturation studies.



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  		FIG. 5.
Intrinsic fluorescence changes of TrwB and TrwB{Delta}N70. A, the proteins (0.8 µM) were equilibrated in 50 mM Tris-HCl (pH 7.8), 3% (v/v) glycerol, 30 mM NaCl, 0.01% (w/v) DDM, and the intrinsic fluorescence variation versus time was recorded. Native TrwB, plot 1; TrwB{Delta}N70, plot 2. B, effect of detergent on TrwB{Delta}N70. The protein (0.8 µM) was incubated in 50 mM Tris-HCl (pH 7.8), 0.05% DM (plot 1) or 50 mM Tris-HCl (pH 7.8) (plot 2), and the intrinsic fluorescence variation versus time was recorded.

 
Dodecylmaltoside and TrwB{Delta}N70 Stability—The protective effect of DDM against denaturation, which already had been observed in the presence of GdmHCl, was also seen against the low salt challenge for TrwB{Delta}N70, as shown in Fig. 5B. In the presence of 0.05% (w/v) DDM and the complete absence of NaCl, TrwB{Delta}N70 was perfectly stable judging from the constancy of the fluorescence emission signal.

The phenomenon of low salt denaturation and detergent protection was further studied by IR spectroscopy. The amide I bands of the IR spectra of TrwB{Delta}N70 in (a)50mM Tris HCl, pH 7.8, 0.1 mM EDTA, 150 mM NaCl buffer, (b)50mM Tris HCl, pH 7.8 buffer, and (c) 50 mM Tris HCl, pH 7.8, 0.05% (w/v) DDM buffer are shown in Fig. 6. The corresponding results of curve fitting and quantification of secondary structure components are summarized in Table IV.



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  		FIG. 6.
IR spectra of TrwB{Delta}N70 in different buffers. TrwB{Delta}N70 in 50 mM Tris-HCl (pH 7.8), 0.1 mM EDTA, 150 mM KCl was dialyzed overnight against the same buffer (A), 50 mM Tris-HCl (pH 7.8) (B), or 50 mM Tris-HCl (pH 7.8) plus 0.05% DDM (C). In the latter sample, 0.05% DDM was also added to the protein suspension prior to dialysis. After dialysis, all samples were freeze-dried, resuspended in D2O, and analyzed by IR spectroscopy.

 


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  		TABLE IV
Secondary structure components of truncated TrwB (TrwB{Delta}N70) in various buffers at 20 °C

Data are taken from the spectra shown in Fig. 6. For details on the assignments, see text.

 
The secondary structure of the control protein (Fig. 6A) is qualitatively similar to that recorded in the presence of both salt and detergent in Fig. 2B, except that in Fig. 6A (salt but no detergent) the proportion of {alpha}-helix appears decreased, particularly at the expense of turns and monomer-monomer contacts. Salt removal (Fig. 6B and Table IV) has as its main effect the apparent conversion of turns and {beta}-sheet + unordered structures into {alpha}-helix, the latter becoming the dominant feature of the spectrum. In contrast, when DDM is present instead of salt, the IR spectrum (Fig. 6C) is virtually indistinguishable from that of the control protein sample (Fig. 6A). Thus IR spectroscopy confirms the low salt denaturation data in presenting the detergent DDM as stabilizing the structure of the water-soluble TrwB{Delta}N70.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
As a part of an investigation on the conjugation proteins encoded by plasmid R388, the study of TrwB was undertaken in our laboratories. TrwB is an integral membrane protein that is essential in R388-directed bacterial conjugation (14). The specific role of TrwB consists of coupling conjugative DNA processing with DNA transfer (28). After some preliminary purification efforts (29), a strategy was devised to purify a truncated form of TrwB lacking the 70 N-terminal amino acids (TrwB{Delta}N70) (13). TrwB{Delta}N70 lacks the transmembrane domain of TrwB, and thus the truncated form is a soluble protein that can be purified in a convenient way. TrwB{Delta}N70 binds with high affinity fluorescent ATP derivatives, although no ATP hydrolase activity has been elicited to date. TrwB{Delta}N70 has been crystallized and the three-dimensional atomic structure solved (15, 16). More recently, the complete TrwB has been purified in detergent micelles and has been found to bind fluorescent ATP derivatives with higher affinity than TrwB{Delta}N70. TrwB also binds ATP, with an apparent Kd = 0.48 mM, in the same order of magnitude of other cytosolic ATP-binding proteins (17).

In keeping with the above results, the data presented in this paper are relevant not only for bacterial conjugation but also in the wider context of the variety of integral membrane proteins that consist of a cytosolic domain in which the biochemical activity resides and a transmembrane domain that anchors the protein to the cell membrane. From this perspective, the experiments described above demonstrate that (a) the transmembrane domain may be instrumental in ensuring the maintenance of the native conformation and activity of the cytosolic domain, and (b) the soluble cytosolic domain may be stabilized by interaction with amphiphilic molecules, detergents, and perhaps lipids.

The Transmembrane Domain Stabilizes the Cytosolic Domain—Heat and GdmHCl are two conventional methods for inducing protein denaturation. Low salt denaturation is perhaps less widely used but is also well known. These three methods are based on rather different physical principles, and thus it is conceivable that TrwB{Delta}N70 will give rise to different denatured forms when subjected to the above treatments. The fact that with all three denaturing agents TrwB happens to be more stable than TrwB{Delta}N70 (Figs. 1A, 3C, and 5A) indicates that the observation that the transmembrane domain stabilizes the cytosolic domain is certainly significant. The inherent structural instability of the truncated protein also has a functional correlation, because TrwB has a significantly higher affinity for ATP than TrwB{Delta}N70 (17). In turn, ATP binding by and/or ATPase activity of TrwB is essential in bacterial conjugation, because a point mutant in the Walker box of TrwB inhibited conjugation with a negative dominant character (13). The structural studies by Gomis-Rüth et al. (16) reveal an analogy between the hexameric structure of TrwB{Delta}N70 and that of mitochondrial H+-ATPase. Hormaeche et al. (17) also showed that, under certain conditions, TrwB would form hexamers with a protruding hydrophobic stalk. The present study indicated an important difference between TrwB and H+-ATPase in that, in the former, transmembrane and cytosolic domains are covalently bound. In the case of H+-ATPase, the hexameric F1 domain can be easily solubilized without the use of detergents, and soluble F1 is a stable protein that readily binds and hydrolyzes ATP. For TrwB, however, the fact that both domains are covalently bound appears to have important structural and functional implications. For similar two-domain membrane proteins, the present data are an indication that studies involving the truncated, water-soluble moieties must be interpreted with care.

Amphiphiles Stabilize the Water-soluble Domain—The observation that the non-ionic surfactant DDM was able to stabilize TrwB{Delta}N70 was made only (Fig. 1) as a consequence of our interest in subjecting both TrwB and TrwB{Delta}N70 to Gdm-HCl under the same initial conditions. A detailed IR study (Fig. 6) confirmed that the detergent has an obvious stabilizing effect on the truncated protein. This can be interpreted in the light of the three-dimensional structure of TrwB{Delta}N70. In fact, {beta}-strands 9 and 10 are not defined in the TrwB{Delta}N70 crystal structure, probably because they form the internal lining of the inner membrane pore covered by the absent transmembrane {alpha}-helices (15, 16). If this were the case, the detergent would cover the hydrophobic surfaces in these {beta}-strands that are apposing the transmembrane helices in the complete protein. This observation would imply tertiary structure interactions between the cytoplasmic and transmembrane domains that are not immediately obvious from the sequence. Further studies involving other two-domain (cytoplasmic + transmembrane) integral membrane proteins will be required to determine the more or less general validity of the observations in this paper concerning the structural implications of the transmembrane domain for protein stability.


   FOOTNOTES
 
* This study was supported in part by grants from the Ministerio de Ciencia y Tecnología (BMC 2001-0791) and Universidad del País Vasco (13552/2001) (to F. M. G.) and from the Ministerio de Ciencia y Tecnología (BMC 2002-00379 to F. de la C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed. E-mail: gbpgourf{at}lg.ehu.es.

1 The abbreviations used are: GdmHCl, guanidinium hydrochloride; DDM, {beta}-D-dodecylmaltoside; TrwB{Delta}N70, a truncated form of the TrwB protein lacking amino acid residues 1–70. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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