Production and characterization of recombinant polypeptides
Expression in E. coli usually faces a problem of obtaining the recombinant proteins as water-soluble and correctly folded products. Quite often, recombinant proteins are accumulated in E. coli within the so-called inclusion bodies, which entails their poor solubility. One way to achieve correct folding in E. coli is to use vectors that code for signal sequences of proteins that are efficiently secreted into E. coli periplasm: the leader sequence should be selectively cleaved off during translocation through the ER membrane while the protein wanted would acquire the correct folding and disulfide bond formation. Another possibility is to use cells which facilitate formation of correct disulfide bridges in vivo. We tried these two approaches in order to obtain the N-terminal domain 1–209 of the T. californicaα-subunit, using namely (a) pET22b vector containing the leader sequence of the pelB protein, and (b) pQE31 vector and expression in the AD494 cells. Both of them code for a (His)6 tag, which is situated close to the C-terminus of polypeptides produced in pET22b vector, and at the N-terminus in the pQE31 constructs. We also used the pET22b vector in an attempt to obtain a polypeptide comprising residues 125–265 of the α-subunit, that is roughly a half of the N-terminal extracellular domain plus the transmembrane sequences M1 and M2.
Although it was anticipated that the protein corresponding to the N-terminal extracellular domain of the AChR α-subunit would be soluble in aqueous media, the α1–209 pET polypeptide was found mainly in the inclusion bodies. Not surprisingly, the same was found with the α125–265 pET polypeptide that comprises two transmembrane fragments. However, the protein expressed in the AD494 cells with the aid of pQE31 vector, designated α1-209pQE, had much better solubility in aqueous media: about 15–20% of the total expressed protein was found in the supernatant, not associated with the inclusion bodies. However, for practical reasons, a high concentration of urea or guanidinium chloride was used at the first stage to solubilize all recombinant polypeptides (α1-209pET, α125-265pET and α1-209pQE).
As can be seen from Fig. 1A, the (His)6 tag allowed us to isolate in one step the products α1-209pET and α1-209pQE, which appeared to be electrophoretically almost homogeneous (when analyzed in reducing or nonreducing conditions, cf. lanes 3, 4 and 6 in Fig 1A). The expression of the 125–265 fragment was considerably weaker than that of the 1–209 fragment. In addition to the main band of expected molecular mass (≈ 19 kDa), a band of higher molecular mass (≈ 33 kDa) could also be detected by Coomassie staining (Fig. 1B). Autoradiography after overlay of nitrocellulose blots (Fig. 1C), demonstrated that [125I]αBgt bound to a single band both in the case of α1-209pET and α1-209pQE fragments. With the α125-265pET protein it bound to the main band and, although less efficiently, to the band of ≈ 33 kDa.
Figure 1. SDS/PAGE analysis of the Torpedo AChR α-subunit domains expressed in E. coli. (A) (1) Standards; (2) cell lysate, (3 and 4) purified α1-209pQE protein under reducing and nonreducing conditions, respectively; 5- and 6-cell lysate and purified α1-209pET protein. (B) (1) Standards, (2) α125–265pET polypeptide eluted from the Ni2+-NTA column, (3) fraction obtained on subsequent purification of this protein by reverse-phase HPLC (see Fig. 2). (C) Autoradiography of the α1-209pQE (1) and α125–265pET (2) proteins after SDS/PAGE and incubation with [125I]αBgt.
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Figure 2 shows the results of the reverse-phase HPLC analysis of the recombinant proteins. In the case of the α1-209pET and α1-209pQE proteins, obtained after removal of urea and spontaneous air oxidation of cysteine SH-groups, one relatively broad peak is observed. The broadness of peaks (Fig. 2A,B) may be explained by the presence of different forms of a similar molecular mass (as follows from the dominance of one band on SDS/PAGE) that have similar hydrophobic properties. Noteworthy, when the α1-209pQE was applied on to a reverse-phase HPLC column immediately after elution from the Ni2+-NTA-agarose column or after reduction in denaturing conditions, the main peak was considerably narrower (see a dotted profile in Fig. 2B).
Figure 2. Reverse-phase HPLC of the Torpedo AChR α-subunit domains expressed in E. coli. (A) α1-209pET protein, Nucleosil 300–5 C4 MPN column; the sample volume is 25 µL (4.5 mg·mL–1) in 50 mm Tris/HCl buffer, pH 8.0, 100 µm EDTA. The column was operated in gradient of solvent B [0.1% TFA in AcCN/H2O mixture (4 : 1)] concentration in solvent A (0.1% TFA in H2O), flow rate 0.5 mL·min–1. (B) α1-209pQE protein, a Nucleosil 300-7 Protein RP column; protein samples of 20 µL (4.6 mg·mL–1) in 50 mm Tris/HCl buffer pH 8.0, 6 m urea and 1 m imidazole immediately after elution from Ni2+-NTA-agarose column (dotted line) and of 100 µL (1.8 mg·mL–1) in 10 mm sodium phosphate buffer pH 7.3, 0.4 m urea, 1 mm EDTA after incubation in this solution for several weeks (solid line); the absorbances of the former chromatogram were multiplied by 1.32 for better comparison. The column was operated in gradient of solvent B [0.1% TFA in AcCN/H2O mixture (19 : 1)] concentration in solvent A (0.1% TFA in H2O), flow rate 0.5 mL·min–1. (C) α125–265pET protein, a Nucleosil 300–7 Protein RP column; protein sample is 820 µL (0.3 mg·mL–1) in 50 mm Tris/HCl buffer pH 8.0, 7 m urea, and 10% SDS. The column was operated in gradient of solvent B (0.1% TFA in AcCN) concentration in solvent A (0.1% TFA in H2O) at flow rate of 0.5 mL·min–1.
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We also used reverse-phase HPLC to monitor various reoxidation-refolding procedures. When disulfides in the α1-209pQE or α1-209pET proteins were reduced in denaturing conditions followed by a decrease in the denaturant concentration by dialysis or gel filtration, over 80% of the protein precipitated. The protein remaining in solution after reoxidation, gave a reverse-phase HPLC profile very close to that of the starting (spontaneously oxidized) protein (data not shown).
In another experiment, the α1-209pQE protein was treated with protein-disulfide isomerase in the presence of 0.5–1 mm cysteine or reduced glutathione. Again, such a treatment brought about only small changes in the reverse-phase HPLC characteristics (not shown). As these approaches did not change dramatically the properties of the recombinant proteins, binding studies and CD analysis (see below) were performed with the proteins obtained after spontaneous oxidation.
The reverse-phase HPLC profile for the α125-265pET protein shows the presence of several components (Fig. 2C). SDS/PAGE analysis of the main peak (marked with a bar on the chromatogram) demonstrates (see Fig. 1B) the enrichment in component with Mr≈ 19 kDa, although forms of higher molecular mass (≈ 33 and 43 kDa) could also be detected in some experiments.
The molecular masses of the expressed AChR α-subunit fragments were determined with the aid of MALDI mass spectrometry (Fig. 3). The spectrum of the α1-209pET protein confirms its homogeneity: all the observed peaks appear to be derived from one peak of 29568 (M + H+), peaks 14791, and 9869 having two and three positive charges, respectively, while a low-intensity 59158 peak indicates the presence of a dimer of the α1-209pET protein. Although the molecular ion with a maximum intensity at 21154 m/z and a respective mass with two positive charges (10581) are observed for the α125-265pET protein (Fig. 3B), the broadness of the signals and the presence of other masses reveal the heterogeneity of the preparation. As can be seen from Table 1, the measured molecular masses of α1-209pET and α125–265pET proteins exceed by about 2200 the calculated ones [on account of the (His)6 tag and polylinker fragments]. Edman degradation of both proteins revealed the same sequence MKYLLPTAA corresponding to the leader peptide of the pelB protein. Taking into account these additional sequences (see Table 1), the calculated molecular masses for the α1-209pET and α125-265pET proteins are 29380 and 21106, respectively. These values are in reasonable agreement with the experimentally determined masses (Table 1). In summary, MALDI mass spectrometry and Edman degradation show that the presence of the pelB leader sequence in the pET constructs failed to ensure the secretion of the proteins, and resulted in an undesirable lengthening of the expressed sequences.
Figure 3. MALDI mass spectra of the Torpedo AChR α-subunit domains. (A–C) α1-209pET, α125–265pET and α1-209pQE proteins, respectively.
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Table 1. Molecular masses of expressed proteins.
| || ||Molecular mass|
|α1-209pET||MDIGINSDP-(1–209AChR)-KLAAALA-H6||27 374|| |
|MKYLLPTAAAGLLLLAAQPAMA-MDIGINSDP -(1–209AChR)-KLAAALE-H6||29 380||29 568|
|α125–265pET||MDIGINSDP-(125–265AChR)-KLAAALE-H6||18 895|| |
|MKYLLPTAAAGLLLLAAQPAMA-MDIGINSDP -(125–265AChR)-KLAAALE-H6||21 106||21 154|
|α1-209pQE||MRSP H6-TDP-(1–209 AChR)-KLN||26 617||26 651|
The molecular mass of the α1-209pQE protein, determined by MALDI mass spectrometry (Fig. 3C), was in satisfactory agreement with the calculated one (Table 1), and Edman degradation showed the presence of residues M-R-X-X-H6-T-D-P (from the vector) followed by the expected N-terminal sequence (SEHE) of the T. californica AChR α-subunit. Again, MALDI spectra provided the evidence for the chemical homogeneity of the α1-209pQE protein: there are no other peaks except the molecular ion (26651), its double-charged fragment (13335), and a low-intensity peak (53331), probably corresponding to a dimer.
As MALDI spectra provided evidence for the presence of a dimer, in addition to a monomer (see Fig. 3), we examined the α1-209pQE protein by gel-permeation chromatography. Figure 4 shows the predominance of the monomer (marked with an arrow at 26 kDa), whereas peaks that could be ascribed to oligomers are also present. These oligomers do not contain intramolecular disulfide bridges, since, as seen from Fig. 1A, the SDS/PAGE patterns obtained under reducing and nonreducing conditions do not differ.
Figure 4. Size-exclusion HPLC of the α1-209pQE protein on a Spherogel TSK 4000SW column. A sample volume is 25 µL (4 mg protein·mL–1) in 50 mm Tris/HCl buffer pH 8.0, 100 mm LiCl, 100 µm EDTA. Isocratic elution was performed at a flow rate of 0.5 mL·min–1 using the same buffer.
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Binding of [125I]αBgt, low-molecular mass antagonists and monoclonal antibodies to α1-209pET and α1-209pQE proteins
All expressed α-subunit fragments, as mentioned above, were found to bind [125I]αBgt on blots, but it was important to investigate the efficacy of binding in solution. αBgt binding to α1-209pET and α1-209pQE proteins (Fig. 5) was performed in aqueous solution of 1% β-octylglucoside. These conditions we have previously shown  to allow investigation of binding to isolated α-subunits of T. californica AChR after purification by SDS/PAGE and HPLC. KD values for the α1-209pET and α1-209pQE proteins were 141.4 ± 10.6 and 127.0 ± 11.3 nm, respectively, being only about fourfold higher than the value (30 nm) previously found for the isolated α-subunits under the same conditions . Bmax values were 9.5 ± 0.2 and 10.3 ± 0.2 nmol·mg–1, compared with theoretical maxima of 34 and 38 nmol·mg–1, respectively. Apparently, the pelB sequence in the α1-209pET protein, and the position of the His6 tag either at the C-terminus or N-terminus exerted no significant effects on the binding parameters. The KD values obtained are in the range of values reported for partially renatured α-subunit from Torpedo, and for α-subunit N-terminal fragments expressed as fusion proteins (see references [20–22,35,36]). However, with most of the expressed α-subunit fragments described previously, the fraction of the protein that bound [125I]αBgt, as calculated from the Bmax was a few percent, whereas in our case the values are 27–28%.
Figure 5. Specific binding of the [125I]αBgt to the α1-209pQE (closed circles) and α1-209pET (open circles) proteins. The total concentration of both proteins dissolved in 50 mm Tris/HCl buffer pH 8.0, 1% β-octylglucoside, was 400 nm. Nonspecific binding was determined in the presence of αBgt taken in 100-fold excess with respect to each concentration of the radioactive ligand. Each point is an average of triplicate measurements. KD and Bmax were calculated by means of Enzfitter (Elsevier Biosoft). Inset: Scatchard plot.
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The fact that even this value is far from 100% should not be discouraging. Noteworthy, even the whole α-subunit had no more than 50% of the theoretical αBgt-binding capacity, unless associated with the γ- and δ-subunits . The activity of the SDS/PAGE-isolated α-subunit measured, in terms of αBgt-binding sites, in the presence of various detergents, was reported by different authors to span the range of 35–95% [35,36,38]. In our hands, SDS/PAGE of the Torpedo membranes followed by reverse-phase HPLC and transfer to β-octylglucoside gave an α-subunit preparation that bound αBgt with KD≈ 28 nm and Bmax 0.5 nmol·mg–1 (≈ 2.5% activity) [34,39].
The lower affinity of that preparation as compared with native AChR was apparently due to incomplete renaturation after SDS/PAGE/HPLC, as well as to a loss of interactions with the neighboring γ- and δ-subunits. It is not clear which other factors further reduce the affinity of the expressed α-subunit domain 1–209, as compared with that of the complete α-subunit. One possibility is that, unlike the subunits obtained from purified Torpedo AChR, the proteins expressed in E. coli are not glycosylated. However, the data on the role of carbohydrates in the interaction of Torpedo AChR with αBgt are conflicting [40–42].
We attempted to increase the activity of the 1–209 proteins with the aid of affinity chromatography with immobilized α-neurotoxins. In fact, this step previously allowed Fraenkel et al.  to increase the toxin-binding capacity of the fusion protein encompassing the α-subunit fragment 184–200 from 3.3 to 16.5% and to use it for NMR analysis of ligand binding. Indeed, between 10 and 60% of the total protein in different preparations of the α1-209pET and α1-209pQE could be bound to the Sepharose columns with the attached neurotoxin II Naja naja oxiana or α-cobratoxin Naja naja siamensis. However, high concentrations of carbachol required to elute the specifically bound protein were found to precipitate it from aqueous solutions. Elution could be done by 1 m carbachol in 1% CHAPS. However, the binding parameters of the proteins thus eluted did not improve considerably as compared with the starting preparation.
To test the ability of the expressed proteins to interact with low molecular weight antagonists whose binding is more sensitive to the structure of the ligand binding site, we examined the interaction of [125I]αBgt with α1-209pQE in the presence of varying concentrations of d-tubocurarine and decamethonium (Fig. 6). These studies were performed in purely aqueous solution: the absence of β-octylglucoside did not affect the Bmax value (9.7 ± 0.3 nmol·mg–1), while the affinity was found to be about twofold higher (KD 75.0 ± 6.8 nm). The IC50 values determined, 200 µm for d-tubocurarine and 8 mm for decamethonium, are very close to those obtained earlier [36,44] for isolated α-subunits. It means that the expressed protein, retains, at least partially, the structure of the ligand-binding site.
Figure 6. Inhibition of [125I]αBgt specific binding to α1-209pQE by d-tubocurarine (squares) and decamethonium bromide (circles). Concentration of the protein in 50 mm Tris/HCl, pH 8.0 was 3 µm. Nonspecific binding was determined as indicated in the legend to Fig. 5. Each point is an average of duplicate measurements. C = concentration of the competitor. IC50 were calculated by means of Origin 2.94 (Microcal Software, Inc.).
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One characteristic of the intact AChR is binding of conformation-dependent antibodies. Of the mAbs previously described by Whiting et al.  raised against native Torpedo AChR, only two, M2D11 and M4D11, bound appreciably to the α-subunit on Western blots of purified Torpedo AChR , and M4D11 bound to both α1-209pQE and α1-209pET, although M2D11 did not (Fig. 7). However, both mAb M2C2, and to a lesser extent M5B5, bound to [125I]αBgt-labeled α1-209pQE and α1-209pET. Binding of these mAbs is conformation-dependent and directed at the αBgt-binding site that exhibits lower affinity for d-tubocurarine and other competitive ligands ; they inhibit [125I]αBgt binding to one site on the AChR, and do not precipitate Torpedo AChR that is fully saturated with [125I]αBgt. Yet, both α1-209pET and α1-209pQE apparently bind to a site that is separate from the αBgt-binding site, because they immunoprecipitate [125I]αBgt-labeled proteins (Fig. 7) and do not inhibit [125I]αBgt binding (data not shown). Thus, these proteins express at least some of the highly conformational antigenic sites that were previously described on Torpedo AChR , but they may be arranged slightly differently, perhaps because of the lack of interaction with the adjacent subunits that clearly influence the binding sites [2,3].
Figure 7. Binding of monoclonal antibodies to the expressed proteins. Immunoprecipitation of [125I]αBgt-prelabeled α1-209pET and α1-209pQE proteins by monoc1ona1antibodies against AChR [45,46], compared with precipitation of T. californica[125I]αBgt-prelabeled AChR. Non-site mAbs and anti-site mAbs refer to antibodies that do not or do compete with [125I]αBgt for its binding site (the one with lower affinity for d-tubocurarine [45,46]) on Torpedo AChR. The results are expressed as fmol of binding sites precipitated per assay and are the mean ± SD of at least two determinations.
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As the expressed proteins interact with a specific neurotoxin (αBgt), with low-molecular mass antagonists (d-tubocurarine and decamethonium) and with Torpedo AChR-specific antibodies (that do not cross-react well with AChR from other species ), they should be useful models for the examination of the spatial structure of the AChR extracellular domain. As a first step along these lines, we measured CD spectra of the expressed proteins (see Fig. 8 and Table 2).
Figure 8. CD spectra of the Torpedo AChR α-subunit domains expressed in E. coli. (1 and 2) α1-209pET protein (0.7 mg·mL–1) and α1-209pQE protein (1.0 mg·mL–1), respectively, in 50 mm Tris/HCl buffer (pH 8.0) with 100 µm EDTA [in order to measure the spectra of the α1-209pET protein in aqueous solution, the starting solution of the protein (3.5 mg·mL−1) containing 1% β-octylglucoside was first diluted fivefold, and then dialyzed extensively against the indicated buffer], (3) α1-209pQE protein (0.5 mg·mL–1) in 10 mm sodium phosphate buffer pH 7.3, 0.2% SDS and 500 µm EDTA, and (4) α1-209pQE protein (0.8 mg·mL–1) in 50% aqueous TFE.
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Table 2. Secondary structure of the Torpedo AChR α-subunit domains expressed in E. coli as calculated from the CD spectra of the respective proteins in different conditions.
|Proteins||Solvents||α-helix (%)||β-sheet (%)||β-turn (%)||Random coil|
|α1-209pET||50 mm Tris/HCl, pH 8.0||35||56||0||10|
|α1-209pQE||50 mm Tris/HCl, pH 8.0||32||50||8||10|
|α1-209pET||10 mm phosphate Na, pH 7.3, 0.2% SDS||36||32||20||11|
CD spectra of α1-209pET and α1-209pQE proteins
The far-ultraviolet CD spectra of α1-209pET and α1-209pQE proteins in Tris/HCl buffer (Fig. 8, curves 1 and 2) were characterized by a positive Cotton effect in the 190–200 nm region, and a negative one in the 200–240 nm region. Such a spectrum suggests a major contribution from β-sheet structure. However, quite a high dichroism intensity over a relatively wide region around 215 nm is indicative of the presence of bands at 208 and 222 nm (π–π* and n –π* transitions, respectively), characteristic of α-helices . The calculation by the CONTIN program [30,31] showed that 32% of amino acid residues in α1-209pQE protein have an α-helical conformation, 50% form a β-pleated sheet, while β-turns and random coil account for 8 and 10%, respectively (Table 2). The α1-209pET protein has a very similar structure: there is only a weak (3–6%) increase in the α-helix and β-structure content (at the expense of β-turns). Apparently, neither the pelB leader sequence, nor the location of the His6 tag closer to the N- or C-termini, greatly change the secondary structure. We also found that the presence of 0.5–1% CHAPS or β-octylglucoside as well as the treatment of proteins by the protein disulfide isomerase did not perturb the CD spectra (data not shown). Adding 0.2% SDS, an anionic surfactant, entails significant changes of secondary structure, with increase in the α-helix and β-turn contents and a concomitant decrease in β-structure (curve 3 in Fig. 8, Table 2).The most visible changes in the CD spectra were caused by 50% trifluoroethanol (TFE), which increased the helicity to 50% (Fig. 8). This is not surprising as the α-helix promoting activity of TFE is well known . Thus, the CD spectra show that the expressed N-terminal domain in aqueous solution has a defined secondary structure, which can be perturbed by changing the environment upon adding SDS or TFE.
As low concentrations (0.02%) of SDS were reported to increase the affinity of the isolated α-subunit  and of synthetic peptides  for αBgt, we checked possible effects of 0.02% SDS on the CD spectra of the 1–209 proteins. No marked changes, as compared with the spectra taken in aqueous buffers, were observed (data not shown), indicating that possible alterations in the affinity for α-neurotoxins are not necessarily associated with reorganizations of the secondary structure. In fact, we could not detect marked effects of αBgt or d-tubocurarine (antagonists) or of acetylcholine and carbachol (agonists) on the CD spectra of the α1-209pET or α1-209pQE proteins either in the absence, or in the presence of 0.02% SDS. As the capacity of d-tubocurarine and decamethonium bromide to interact with the α1-209pQE protein was disclosed (via their competition with [125I]αBgt), it means that their binding is not accompanied by gross changes of the secondary structure (small changes would not have been visible because of the ≈ 25% activity of our preparations).
Secondary structure and functional properties of various AChR domains
It is interesting to compare the results obtained with previous data on the secondary structure of the whole AChR or its domains. From Fig. 1 in , based on the analysis of the sequences of all T. californica AChR subunits, we deduced the following secondary structure composition for the 1–209 region: α-helix 30%, β-structure 45%, β-turns 19%, and random coil 6%. These values are quite close to those determined by us experimentally, especially for the α1-209pQE protein in aqueous solution.
A recent attempt to envisage the spatial structure of the Torpedo AChR extracellular domain, involved modeling of the α-subunit sequence 31–200 according to the X-ray structures of plastocyanin and pseudoazurin with which it shares about 30% homology . The model postulated the predominance of β-structure (nine β-strands) and the presence of three α-helices, which in total would give 19% α-helical structure.
As far as direct experimental estimation of the secondary structure is concerned, CD and FTIR data are available for the whole Torpedo receptor, either free or in a liganded form [52–54]. Information on the secondary structure of the intramembrane domain was obtained by simultaneous chopping off the extracellular and intracellular domains by proteinase K, and subsequent FTIR analysis of the residual membranes . Our data are direct determinations of the secondary structure of polypeptides that represent the extracellular N-terminal domain of the Torpedo AChR α-subunit. The predominance of β-structure disclosed by CD spectra may explain the propensity of the expressed α1–209 fragment to form aggregates, but it can also be an important element in the assembly of the α-subunit and the other subunits into the intact pentaoligomeric AChR complex. On the other hand, the 32% percentage of α-helices is sufficient to form the three helices whose presence in the extracellular domain of the Torpedoα-subunits was inferred from cryo-electron microscopy data .
After our results were reported in an abstract , Lester and collaborators published the expression in the Chinese hamster ovary cells of the protein comprising residues 1–210 of the mouse muscle AChR α-subunit . The protein obtained is glycosylated, does not have a His6 tag and is reported to have a very high affinity for αBgt, although the binding capacity was only about 40%. The secondary structure calculated from the CD spectrum of this protein (which has a 73% sequence identity to the T. californicaα-subunit in the 1–209 region) was 12% α-helix, 51% β-structure, 18% β-turns, and 20% random coil. The common feature for the N-terminal domains of the α-subunits from these two different species, expressed either in eukaryotic or prokaryotic systems, is the prevalence of β-structure (50–65%) and the presence of appreciable amounts of α-helices (12–32%).
Thus, one can see that the secondary structure determined for the 1–209 proteins expressed in E. coli is in general agreement with the theoretical predictions and the experimental results for a related protein . As the 1–209 protein was found to bind αBgt and low-molecular mass antagonists, and also preserved some of the conformational antigenic sites (see above), we believe that this protein is similar to the respective domain of the intact receptor not only with respect to its secondary structure, but also with certain elements of the topology. The situation is reminiscent of the protein state known as ‘molten globule’, which has essential features of the native secondary structure and of the native overall architecture, but lacks the native tertiary structure characterized by well-packed side chains .
Other recent publications demonstrate that heterologously expressed extramembrane fragments of the oligomeric ligand-gated or voltage-gated ion channels are adequate models for elucidating the structure of the respective domains of the intact molecules [58–61]. Interestingly, the extramembrane fragment of the Shaker potassium channel expressed in E. coli gave exclusively tetramers (that are also characteristic for intact channels) whose three-dimensional structure was solved by X-ray crystallography . Expression in Xenopus oocytes of the N-terminal domain of the α7 subunit of the pentaoligomeric α7 AChR was also found to result in oligomers, most probably pentamers .
In view of these results we believe that expression of the N-terminal domain of Torpedo AChR α-subunit in E. coli, reported in the present communication provides large amounts of a protein which is, indeed, an appropriate model for elucidating important aspects of the AChR structural organization.