The sequence-related chaperonin 10 (cpn10, ()hsp10, or 10-kDa antigen) class of proteins assists the noncovalent assembly of other protein-containing structures in vivo(1) . This biological activity requires aggregation of cpn10 into a heptameric structure(2) . In addition to this activity, several cpn10 proteins such as the Mycobacterium tuberculosis and Mycobacterium leprae molecules are among the most potent stimulators of the immune system known(3, 4, 5, 6, 7, 8) . For example, in a leprosy patient, one in three T lymphocytes that respond to M. leprae may react to M. leprae cpn10(4) . Similarly, M. tuberculosis cpn10 induces T cell proliferation in healthy tuberculin reactors to an extent that is greater than that elicited by any other mycobacterial protein(5) . Intriguingly, there is a dramatic difference in antigenicity between cpn10 proteins from different species: human cpn10 and Escherichia coli cpn10, for instance, are very poor immunogens(9) .

In addition to antigenicity, chaperonins, at low concentrations, have other biological properties. For example, several micrograms or less of mycobacterial chaperonins/milliliter will immunize animals, stimulate T lymphocyte proliferation in vitro, and induce cytokine secretion from a human monocyte line and from human monocytes(3, 4, 5, 6, 7, 8, 10, 11) . They also induce apoptosis of human p19 cells. ()Furthermore, there is evidence to suggest that human cpn10 may be involved in control over cell growth and development(9) . These data indicate that cpn10 proteins have several distinct biological activities, namely as molecular chaperones and non-chaperone activities.

This study concerns the aggregation behavior of the M. tuberculosis cpn10 protein under a variety of different experimental conditions, in particular, low concentrations of cpn10 at which non-chaperone activities occur. Aggregation of both the full-length protein and truncated cpn10 peptides was examined. Furthermore, the secondary structure of the molecule was analyzed by CD spectroscopy. Most of the work was carried out using chemically synthesized full-length protein including the N-terminal and C-terminal fragments. Recombinant material was used for comparison.

Preliminary accounts on both the synthesis of the protein and its structure have been reported previously(13, 14) . Here, we demonstrate that M. tuberculosis cpn10 exists, surprisingly, as a tetrameric aggregate with -type structure in dilute solutions and in whole M. tuberculosis lysate. In the presence of a large molar excess of divalent ions, the protein has the expected heptameric structure and, together with cpn60, is functional in a refolding assay. In contrast, we show that E. coli cpn10 is heptameric under all conditions tested, while human cpn10 is predominately found as a heptamer, although dissociation into smaller oligomers takes place under certain solution conditions. These differences in structural assembly between species may help to explain differences in biological activity such as antigenicity.

MATERIALS AND METHODS

Chemical Synthesis of M. tuberculosis cpn10 and Fragments

()

()

M. tuberculosis Lysate

Recombinant M. tuberculosis, E. coli, and Human cpn10 Proteins

Size-exclusion Chromatography

Ultracentrifugation

CD Spectroscopy

pH titration experiments were performed by preparing a stock solution of peptide (2 mg/ml in either distilled water or slightly acidified water) and mixing aliquots of this solution with sufficient phosphate solution buffered at the pH of choice so as to yield a final solution containing 0.1 mg/ml peptide in 0.1 M phosphate. The exact pH was then monitored with a pH-meter equipped with a microelectrode. Prior to use, the final solution was equilibrated for 1 h at 22 °C in the CD chamber. During temperature studies, the cuvette containing the peptide solution and the CD chamber were equilibrated for 1 h prior to collection of data.

RESULTS

Fig. 1shows the sequences of the three proteins considered in this work. Two of the M. tuberculosis cpn10 fragments (i.e. peptides 1-58 and 59-99) were selected because they include or exclude, respectively, a sequence (amino acids 46-59) predicted to be a loop region (Fig. 1). This loop contains the M. tuberculosis cpn10 monoclonal antibody (SA12)-binding site(5, 26, 27) . SA12 is, within the cpn10 family, specific for the mycobacterial molecule. There were no special reasons for the choice of the other two fragments other than the fact that peptide 51-99 cuts almost in the middle the loop antibody-binding region. This could provide information about which residues are necessary for antibody recognition. Peptide 26-99 begins with a conserved Gly, which follows a long sequence predicted to be another loop region (Fig. 1).

Figure 1: Alignment and secondary structure prediction of the M. tuberculosis (mt), E. coli (ec), and human (hu) cpn10 proteins discussed under ``Results.'' Numbering of residues refers to the M. tuberculosis protein. Footnote a, shown is the cpn10 motif described in the SWISS-PROT data base (release 30.0, October 1994). h, hydrophobic residues; q, mostly charged residues (Lys, Arg, Glu) or Gln. Footnote b, the secondary structure composition of cpn10 proteins was predicted by first aligning 27 sequences of cpn10 proteins and then considering the secondary structure of each section (separated by gaps) using two different algorithms: Chou-Fasman (12) and GORII (18) . II Str.Pred., secondary structure prediction; h, -helix; b, -strand; t, turn; l, loop. Boldface residues denote identity.

Chemical Synthesis

The M. tuberculosis protein was synthesized and purified according to these protocols. The final purified product had the correct mass, amino acid composition, and sequence. Furthermore, samples of recombinant material that became available during the final stages of this study were found to have the same physicochemical characteristics as synthetic M. tuberculosis protein.

Aggregation Studies

Figure 2: Size-exclusion chromatography of M. tuberculosis cpn10 (0.2 mg/ml) in PBS, pH 7.4, and in PBS plus 7 mM Mg. The addition of the latter changed the aggregation state of the protein from 4 to 7. BSA, bovine serum albumin; OVA, ovalbumin.

Figure 3: Enzyme-linked immunosorbent assays on size-exclusion chromatography fractions of recombinant M. tuberculosis cpn10 () and M. tuberculosis lysate (). Fractions (0.5 ml) were collected every minute after 13 min from injection. 100 µl of each fraction were coated on a 96-well microtiter plate, and the presence of M. tuberculosis cpn10 was revealed with the M. tuberculosis cpn10 monoclonal antibody SA12 using standard enzyme-linked immunosorbent assay techniques. BSA, bovine serum albumin; OVA, ovalbumin.

The two larger fragments, i.e. peptides 26-99 and 51-99, also had retention times that, in a calibration curve, corresponded to an aggregation state of 4. A similar conclusion was reached when peptide 1-58 was tested, while peptide 59-99 eluted as either a trimer or dimer. The apparent molecular mass of the polypeptides did not change in the pH range 5-8.5, at which a calibration curve was still reliable using standard proteins (data not shown).

Given the unexpected nature of the oligomeric state of the protein, a second independent measurement of the aggregation properties of the cpn10 molecule was carried out by analytical ultracentrifugation (AUC). This confirmed that in PBS, pH 7.4, M. tuberculosis cpn10 was a tetramer (Table 1). However, the fragments gave the following results. At concentrations varying between 0.05 and 1 mg/ml, peptide 1-58 was always a dimer, while all other C-terminal fragments were, in the same concentration range, monomeric (Table 1). Given that, in some cases, the results from the two techniques (SEC and AUC) differed, and due to the superior reliability of AUC in the determination of the molecular mass, the values obtained from ultracentrifugation were taken as representative of the aggregation state of the polypeptides.

To explore the possibility that the M. tuberculosis cpn10 protein could adopt a heptameric form, a binding test was carried out with recombinant E. coli cpn60 (GroEL). Thus, it is well established that in order to exert its activity, the co-chaperone cpn10 protein must bind to cpn60 in the presence of Mg/ATP (see, for example, (28) ). Furthermore, electron microscopy studies have shown that both proteins share a 7-fold axis of symmetry when in the complexed form (see, for example, (29) ). Indeed, M. tuberculosis cpn10 bound to GroEL, and the complex thus obtained was a functional one in a refolding assay. These data suggest either that GroEL acts as a chaperone for the cpn10 protein by changing its aggregation state from 4 to 7 or that the smaller protein binds to GroEL in a tetrameric state. Alternatively, the transition between the two different aggregation states is due to the presence in the buffer of either ATP or Mg ions or both. This hypothesis was verified by additional SEC experiments in the presence of Mg/ATP. Magnesium ions alone were sufficient to change the aggregation state of cpn10 to 7 ( Fig. 2and Table 1). Ultracentrifugation studies conducted under the same conditions confirmed this conclusion (Table 1). A similar, although not quite so dramatic effect has been recently described for the cpn60 protein. Cross-linking of the native GroEL tetradecamer is accelerated by saturating amounts (10 mM) of Mg ions(30) .

Mg could be substituted with Mn and Ca ions in inducing the change to heptamers, while monovalent ions, such as K, were ineffective (Table 1). In the case of Zn ions, a heptameric species and small amounts of larger aggregates were obtained (Table 1). As to the shorter fragments, their aggregation states were not influenced by the addition of divalent ions, their retention times being the same in the presence or absence of Mg (data not shown).

To evaluate whether parameters other than the divalent cations could influence the aggregation of the protein, additional SEC and AUC studies were carried out. Protein concentration and type of buffer were examined. Also, solution pH was studied (by ultracentrifugation only) because the CD results (see below) indicated that both the protein and its C-terminal fragments undergo a conformational change at acidic pH values.

When the protein concentration was kept between 0.1 and 0.2 mg/ml, an aggregation state of 4 was found irrespective of the buffer used (i.e. 0.1 mM Tris with or without 10 mM KCl and PBS without Mg/Ca) (Table 1). The addition of Mg converted the protein to a heptamer in all of these solvents. When the concentration of the protein was increased to 1 mg/ml and the solvent was 0.1 M phosphate, the heptamer was the most abundant species in solution (95%) even in the absence of Mg. Interestingly, lowering the phosphate concentration to that of PBS (i.e. 10 mM) while maintaining the protein concentration at 1 mg/ml gave aggregation species that ranged from that of a heptamer to either a dimer or trimer (Fig. 4A and Table 1). Finally, at acidic pH and in 10 mM phosphate buffer, the protein was still a tetramer (Table 1).

Figure 4: Size-exclusion chromatography of M. tuberculosis cpn10 (1 mg/ml) in PBS, pH 7.4 (top trace), and human cpn10 (0.2 mg/ml) in 0.1 M Tris buffer, pH 7.4 (bottom trace), illustrating the presence of different oligomeric structures.

Similar studies conducted on recombinant E. coli cpn10 (also known as GroES) showed that the protein existed in solution only as a heptamer (Table 1). Human cpn10 had instead a behavior intermediate between that of GroES and that of M. tuberculosis cpn10 since it was heptameric in all cases tested, except in a 0.1 M Tris solution, pH 7.4, where it was a mixture of various aggregation states (Fig. 4B), and in the same solution plus 10 mM KCl, where it was mainly tetrameric (Table 1). Finally, peptides 26-99 and 51-99, which were not influenced by all the parameters discussed above (data not shown), became dimers at pH 3.4, while peptide 59-99 was still monomeric at a similar pH (Table 1).

CD Spectroscopy

Figure 5: CD spectra of M. tuberculosis cpn10 and fragments. A, M. tuberculosis cpn10 and C-terminal fragments (0.1 mg/ml) in 0.1 M phosphate buffer, pH 7.4. The spectrum of peptide 1-58 is shown in the inset. The spectra of the C-terminal fragments had different intensities (see ``Results,'' the other panels of this figure, and Fig. 7). Therefore, to enable their visual comparison, the spectra of peptides 59-99 and 26-99 and the full-length protein were multiplied by factors of 6, 12, and 300, respectively. B, CD curves of peptide 59-99 at different pH values. All fragments and the full-length protein were scarcely soluble in the approximate pH range 4-5.5. Spectra at these pH values were therefore not obtained. C, as in B, but for peptide 51-99. D, as in B and C, but for peptide 26-99. WL, wavelength.

Figure 7: CD spectra of M. tuberculosis (Mt) cpn10 (0.1 mg/ml) in 0.1 M phosphate buffer. A, the protein at different pH values; B, spectra recorded in the absence and presence of Mg (7 mM); C, temperature studies carried out between 0 and 30 °C; D, protein in a 0.1 M phosphate/MeOH (35:65) mixture. WL, wavelength.

Peptide 59-99

pH titration experiments led to a decrease of the contribution at 202 nm and an increase in the minimum at 215 nm (Fig. 5B). At pH 3.0, where the peptide had a stable structure (32) and was monomeric, there was only the band at 215 nm.

Peptide 51-99

Peptide 26-99

Peptide 1-58

Peptide 1-99

The spectrum of the M. tuberculosis protein (Fig. 5A) was qualitatively similar to those of E. coli cpn10, synthetic rat cpn10(14) , and recombinant human cpn10. In particular, the E. coli protein had a minimum at 202 nm and a shoulder at 197 nm, while human cpn10 (and rat cpn10; the two proteins differ by one residue) had minima at 203 and 197 nm (Fig. 6). Thus, the band at 202-203 nm and the shoulder at 197-198 nm are characteristic of this class of proteins and independent of their origin or aggregation state.

Figure 6: CD spectra of E. coli and human (Hu) cpn10 proteins (0.1 mg/ml) in 0.1 M phosphate buffer, pH 7.4. WL, wavelength.

The intensity of the 204 nm band was, however, larger in the case of mammalian and E. coli proteins. The aggregation state was only partly responsible for these changes since the spectrum of the M. tuberculosis protein in its heptameric state (i.e. in the presence of Mg; see below) was still less intense than those of the other two chaperonins. Possible explanations for this difference could be either the presence in solution of small concentrations of tetrameric M. tuberculosis cpn10 or a difference balance, in the three proteins, of the residues/regions contributing to the 204 nm band and the less intense 198 nm band.

Lowering the pH of M. tuberculosis cpn10-containing solutions induced a shift to 204 nm of the broad signal at 203 nm similar to that seen for the C-terminal fragments, while the shoulder at 217 nm became more pronounced (Fig. 7A). An isosbestic point at 210.5 nm, together with invariance of the spectral features between pH 4.5 and 2 (spectra in this pH range were essentially identical; data not shown), indicated the existence of an equilibrium between at least two species, one (or more) at pH 7.4 and a second conformation stable in the pH 4.5 to 2 interval.

Qualitatively, the addition of Mg, which aggregation studies had shown to induce a transition from tetramers to heptamers, led to the same changes (i.e. shift of the 203 nm band to 204 nm and increase in the intensity of the 217 nm shoulder) observed during pH titration (Fig. 7B). In particular, virtually no changes were observed when 1 or <1 eq (C 0.01 mM) of magnesium ions/protein subunit was added, while a continuous change in the shape of the spectrum was obtained upon adding 5 eq of Mg (0.05 mM) and up to a total magnesium concentration of 5 mM. These results suggested that there was no stoichiometric binding of the ion to the cpn10 protein.

Modulation of the intensities of the minima at 200 nm also occurred during temperature studies. Thus, at 0 °C, the spectrum had two almost equally intense bands, at 199 and 203 nm, respectively, while raising the temperature to 30 °C led to a sharpening of the latter, which moved to 204 nm. At this temperature, the 198 nm contribution was reduced considerably (Fig. 7C).

Finally, the addition of MeOH to aqueous solutions of M. tuberculosis cpn10 led to a CD spectrum resembling that of an all--structure (Fig. 7D)(31) . This suggested that the ability to form anti-parallel -strands shown by the C-terminal fragments was maintained in the full-length protein.

DISCUSSION

The work that we describe here provides, for the first time, CD data on the secondary structure of M. tuberculosis cpn10, leads to a hypothesis for the tertiary structure, and demonstrates, surprisingly, that the main quaternary unit is a tetramer. The following is a detailed discussion of the main findings.

Aggregation

The heptameric structure of M. tuberculosis cpn10 acts as a molecular chaperone by binding to E. coli cpn60 and generating a complex functional in a refolding assay. This shows that both M. tuberculosis cpn10 and E. coli cpn10 associate in the same way, i.e. as heptamers with the cpn60 tetradecamer.

What is the significance of tetrameric M. tuberculosis cpn10? The first clue comes from the our observation that M. tuberculosis cpn10 is tetrameric in low protein and low divalent ionic solutions. This suggests that, in nature, where a wide variety of conditions are present, the tetrameric form may predominate. Indeed, this appears to be the case since M. tuberculosis lysate has only one species that binds to anti-cpn10 monoclonal antibody and has the molecular mass of a tetramer. (It is important to note that the monoclonal antibody used in this experiment binds to both tetrameric and heptameric forms of M. tuberculosis chaperonin, ()ruling out the existence of heptameric species in mycobacterial lysate.)

Is M. tuberculosis cpn10 biologically different from E. coli or mammalian cpn10? The most obvious difference is immunogenicity. For example, M. tuberculosis cpn10 is highly antigenic (3, 4, 5, 6, 7) while E. coli and mammalian cpn10 proteins are not(9) . Furthermore, recent data suggest that M. tuberculosis cpn10 can stimulate monocytes(10) , macrophages(11) , and synovial fibroblast-like cells. ()In contrast, human and E. coli cpn10 proteins are poor immunogens(9) . It is very difficult even to raise low affinity antibodies against human cpn10 by repeated injections into animals(9) . Thus, the different behavior toward aggregation shown by the cpn10 proteins described in this work and, in particular, the ability of the M. tuberculosis homologue to form stable tetrameric species may explain the different biological activities of cpn10 proteins.

The data on aggregation using the protein's fragments suggest where, in the sequence, the regions involved in subunit interactions are approximately located. Thus, the behavior of peptides 1-58 and 26-99 and the full-length protein clearly indicates that sequence 1-25 is pivotal to aggregation to tetramers/heptamers. Interestingly, the motif h+PLxD + hhhq, which spans residues 6-15, has been proposed as the cpn10 protein fingerprint (Fig. 1). Here, it is proposed that this sequence is one of the regions required for tetramer/heptamer formation. Another aggregation region may be in the C-terminal half of the protein, although the data are not sufficient for a more precise and unequivocal location.

Secondary Structure

Here, a -contribution seemed likely due to the structure of peptide 59-99, which, at low pH, was assigned to a -sheet, and the existence in the spectra of all other polypeptides of minima/shoulders at 215-220 nm. Furthermore, the spectrum of M. tuberculosis cpn10 in water/MeOH mixtures was that of proteins with a high -pleated sheet content. The shoulder between 225 and 230 nm seen in the spectrum of peptide 1-58 could also be interpreted as deriving from -sheets since proteins with this fold and scarce aromatic contribution exhibit a minimum in this region of the spectrum(31) . A contribution of the random coil type to the structure of the protein and fragments was also probable since the H- chemical shift of residues contained in sequence 17-32 of GroES (sequence 19-34 in M. tuberculosis cpn10) has been shown to be virtually identical to those reported for random coil peptides(36) .

The minimum at 203-204 was more difficult to interpret. Its assignment to an -helix seemed unlikely due to the lack of the intense band at 222 nm. On the other hand, the spectra of both polyproline II (37) and type I -turns (Woody's class C spectrum) (38) have minima in this wavelength range.

A more positive assignment derived from the observation that the main band of monomeric peptides 51-99 and 26-99 is at 198 nm while the minimum at 203-204 nm and the shoulder at 215-217 nm characterize the spectra of dimeric peptides 51-99 and 26-99 and tetrameric and heptameric cpn10 proteins. Thus, based on these observations, it was concluded that the 203-204 nm band correlates with aggregation, whereas the opposite applies to the minimum at 198 nm. Whether these changes in quaternary structure are accompanied by changes in secondary structure (for instance, random coil to polyproline II-like structure) of parts of the molecule or whether the 203-204 nm contribution derives directly from the interactions between subunits cannot be concluded on the basis of these data only.

Changes in the intensities of the 198 and 203-204 nm minima were also observed during the temperature studies of the full-length protein. In particular, the band at 198 nm became more intense at 0 °C and vanished almost completely at 30 °C, where it was replaced by the 204 nm minimum. Since we had correlated similar changes in secondary structure with changes in the aggregation state and due to the fact that hydrophobic interactions are weaker at low temperature(39) , we conclude that the oligomeric structure of the protein is stabilized by hydrophobic interactions.

Conclusions

CD results were consistent with the existence of aggregation equilibria. The latter could be followed by monitoring the intensity of the minima at 198 and 203-204 nm. The CD data also permitted us to conclude that the protein adopts a mainly anti-parallel -fold consisting of two different regions, each containing an anti-parallel -sheet: the first region comprises residues 1-45, and the second comprises either peptide 55-99 or 59-99, with peptide 46-54 (46-58) forming a large loop connecting the two sheets. The reasons for these assumptions were (i) our data that indicate that peptide 59-99 has a spectroscopic and aggregation behavior different from that of the other fragments; (ii) the assignment of peptide 46-58 to a loop region containing the protein antibody-binding site(5, 27) ; (iii) the ability of the protein to adopt a mainly -fold, as shown by its CD spectrum in water/MeOH mixtures; and (iv) the recently published indication that the nearly complete crystal structure of GroES is made of identical subunits with a mainly -barrel fold(40) . -Barrels are generally formed by two -sheets that are joined together and packed against each other.

CD data were also used to conclude that the secondary structure composition of subunits is similar in both stabilized tetramers (i.e. the CD structure at acidic pH values) and heptamers and that contact between subunits is mainly through hydrophobic forces. Finally, the spectra of E. coli and human cpn10 molecules were also characterized by minima at 198 and 202-204 nm. Thus, the latter, which was found to correlate with aggregation, appears to be a general feature of cpn10 molecules.

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.

§

Present address: Max Planck Institute for Colloid and Interface Research, Colloid Chemistry Department, Kantstr. 55, D-14513 Telfow, FRG.

¶

To whom correspondence should be addressed. Tel.: 39-2-64433090; Fax: 39-2-66011579.

()

The abbreviations used are: cpn10, chaperonin 10; HPLC, high performance liquid chromatography; SEC, size-exclusion chromatography; PBS, phosphate-buffered saline; AUC, analytical ultracentrifugation.

()

G. Galli, P. Ghezzi, P. Mascagni, F. Marcucci, and M. Fratelli, submitted for publication.

()

H. L. Ball and P. Mascagni, submitted for publication.

()

G. Fossati, G. Legname, P. Lucietto, H. L. Ball, P. Giuliani, A. R. Coates, and P. Mascagni, manuscript in preparation.

()

F. Marelli, P. Mascagni, and A. R. Coates, unpublished observation.

()

G. Fossati, unpublished observation.

REFERENCES

1. Ellis, R. J., and Van der Vies, S. M. (1991) Annu. Rev. Biochem. 60, 321-347 [CrossRef][Medline] [Order article via Infotrieve]
2. Saibil, H., Dong, Z., Wood, S., and Auf Der Mauer, A. (1991) Nature 353, 25-26 [Medline] [Order article via Infotrieve]
3. Coates, A. R. M. (1995) in The Chaperonins (Ellis, R. J., ed) Academic Press Ltd., London, in press
4. Mehra, V., Bloom, B. R., Bajardi, A. C., Grisso, C. L., Sieling, P. A., Alland, D., Convit, J., Fan, X., Hunter, S. W., Brennan, P. J., Rea, T. H., and Modlin, R. L. (1992) J. Exp. Med. 175, 275-284 [Abstract/Free Full Text]
5. Barnes, P. F., Mehra, V., Rivoire, B., Fong, S. J., Brennan, P. J., Voegtline, M. S., Minden, P., Houghten, R. A., Bloom, B. R., and Modlin, R. L. (1992) J. Immunol. 148, 1835-1840 [Abstract]
6. De Bruyn, J., Bosmans, R., Turneer, M., Weckx, M., Nyabenda, J., Van Vooren, J. P., Falmagne, P., Wiker, H. G., and Harboe, M. (1987) Infect. Immun. 5, 245-252
7. Oftung, F., Mustafa, A. S., Husson, R., Young, R. A., and Godal, T. (1987) J. Immunol. 138, 927-931 [Abstract]
8. Orme, I. M. (1988) J. Immunol. 140, 3589-3593 [Abstract]
9. Cavanagh, A. C., and Morton, H. (1994) Eur. J. Biochem. 222, 551-560 [Medline] [Order article via Infotrieve]
10. Friedland, J. S., Shattock, R., Remick, D. G, and Griffin, G. E. (1993) Clin. Exp. Immunol. 91, 58-62 [Medline] [Order article via Infotrieve]
11. Petermans, W. E., Raats, L. J., Langermans, J. A., and van Furth, R. (1994) Scan. J. Immunol. 39, 613-617 [CrossRef][Medline] [Order article via Infotrieve]
12. Chou, P. Y., and Fasman, G. D. (1974) Biochemistry 13, 222-245 [CrossRef][Medline] [Order article via Infotrieve]
13. Fossati, G., Lucietto, P., Giuliani, P., Ball, H. L., Chan, A. W. E., Leoni, F., Gromo, G., Coates, A. R. M., and Mascagni, P. (1993) in Proceedings of the Second Japanese Symposium on Peptide Chemistry (Yanaihara, N., ed) pp. 235-238, ESCOM Science Publishers B. V., Leiden, The Netherlands
14. Mascagni, P., Chan, A. W. E., Coates, A. R. M., Fossati, G., Giuliani, P., and Lucietto, P. (1994) in Proceedings of the Thirteenth American Peptide Symposium (Hodges, R. S., and Smith, J. A., eds) pp. 763-765, ESCOM Science Publishers B. V., Leiden, The Netherlands
15. Ball, H. L., and Mascagni, P. (1992) Int. J. Pept. Protein Res. 40, 370-379 [Medline] [Order article via Infotrieve]
16. Ball, H. L., Bertolini, G., Levi, S., and Mascagni, P. (1994) J. Chromatogr. 686, 73-83 [CrossRef]
17. Ball, H. L., Giuliani, P., Lucietto, P., Fossati, G., and Mascagni, P. (1995) Biochem. Pept. Proteins Nucleic Acids 1, 39-44
18. Garnier, J., Osguthorpe, D. J., and Robson, B. (1978) J. Mol. Biol. 120, 97-120 [CrossRef][Medline] [Order article via Infotrieve]
19. Legname, G., Fossati, G., Gromo, G., Monzini, N., Marcucci, F., and Modena, D. (1995) FEBS Lett. 361, 211-214 [CrossRef][Medline] [Order article via Infotrieve]
20. Atkins, D., Al-Ghusein, H., Prehaud, C., and Coates, A. R. M. (1994) Gene (Amst.) 150, 145-148 [CrossRef][Medline] [Order article via Infotrieve]
21. Yphantis, D. A. (1964) Biochemistry 3, 297-317
22. Teller, D. C. (1973) Methods Enzymol. 27, 346-441 [Medline] [Order article via Infotrieve]
23. Harding, S. E., Horton, J. C., and Morgan P. J. (1992) in Analytical Ultracentrifugation in Biochemistry and Polymer Science (Harding, S. E., Rowe, A. J., and Horton, J. C., eds) Royal Society of Chemistry, Cambridge
24. Kratky, O., Leopold, H., and Stabinger, H. (1973) Methods Enzymol. 27, 98-110 [Medline] [Order article via Infotrieve]
25. Perkins, S. J. (1986) Eur. J. Biochem. 57, 169-173 [CrossRef][Medline] [Order article via Infotrieve]
26. Minden, P., Houghten, R. A., Spear, J. R., and Shinnick, T. M. (1986) Infect. Immun. 53, 560-564 [Abstract/Free Full Text]
27. Verbon, A., Hartskeerl, R. A., and Kolk, A. H. J. (1991) Clin. Exp. Immunol. 86, 6-12 [Medline] [Order article via Infotrieve]
28. Goloubinoff, P., Christeller, J. T., Gatenby, A. A., and Lorimer, G. H. (1989) Nature 342, 884-889 [CrossRef][Medline] [Order article via Infotrieve]
29. Saibil, H., and Wood, S. (1993) Curr. Biol. 3, 207-213
30. Azem, A., Diamant, S., and Goloubinoff, P. (1994) Biochemistry 33, 6671-6675 [CrossRef][Medline] [Order article via Infotrieve]
31. Perczel, A., Park, K., and Fasman, G. D. (1992) Proteins Struct. Funct. Genet. 13, 57-69 [CrossRef][Medline] [Order article via Infotrieve]
32. Chan, E., Fossati, G., Giuliani, P., Lucietto, P., Zaliani, A., Coates, A. R. M., and Mascagni, P. (1995) Biochem. Biophys. Res. Commun. 211, 14-20 [CrossRef][Medline] [Order article via Infotrieve]
33. Ellis, R. J. (1990) Science 250, 954-959 [Abstract/Free Full Text]
34. Richardson, J. S. (1981) Adv. Protein Chem. 34, 167-339, and references therein [Medline] [Order article via Infotrieve]
35. Manavalan, P., and Johnson, W. C., Jr. (1983) Nature 305, 831-832 [CrossRef]
36. Landry, S. J., Zeilstra-Ryalls, J., Fayet, O., Georgopoulos, C., and Gierasch, L. M. (1993) Nature 364, 255-258 [CrossRef][Medline] [Order article via Infotrieve]
37. Woody, R. W. (1992) Adv. Biophys. Chem. 2, 37-79
38. Woody, R. W. (1985) in The Peptides (Hruby, V. J., ed) Vol. 7, pp. 15-115, Academic Press, New York
39. Pratt, L. R., and Chandler, D. (1977) J. Chem. Phys. 67, 3683-3695 [CrossRef]
40. Saibil, H. R. (1994) Nature Struct. Biol. 1, 838-842 [CrossRef][Medline] [Order article via Infotrieve]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				G. Fossati, G. Izzo, E. Rizzi, E. Gancia, D. Modena, M. L. Moras, N. Niccolai, E. Giannozzi, O. Spiga, L. Bono, et al. Mycobacterium tuberculosis Chaperonin 10 Is Secreted in the Macrophage Phagosome: Is Secretion Due to Dissociation and Adoption of a Partially Helical Structure at the Membrane? J. Bacteriol., July 15, 2003; 185(14): 4256 - 4267. [Abstract] [Full Text] [PDF]

				B. Chua-Intra, R. J. Wilkinson, and J. Ivanyi Selective T-Cell Recognition of the N-Terminal Peptide of GroES in Tuberculosis Infect. Immun., March 1, 2002; 70(3): 1645 - 1647. [Abstract] [Full Text] [PDF]

				B. Taneja and S. C. Mande Metal ions modulate the plastic nature of Mycobacterium tuberculosis chaperonin-10 Protein Eng. Des. Sel., June 1, 2001; 14(6): 391 - 395. [Abstract] [Full Text] [PDF]

				I. Rosenkrands, K. Weldingh, P. Ravn, L. Brandt, P. Hojrup, P. B. Rasmussen, A. R. Coates, M. Singh, P. Mascagni, and P. Andersen Differential T-Cell Recognition of Native and Recombinant Mycobacterium tuberculosis GroES Infect. Immun., November 1, 1999; 67(11): 5552 - 5558. [Abstract] [Full Text] [PDF]

				S. Meghji, P. A. White, S. P. Nair, K. Reddi, K. Heron, B. Henderson, A. Zaliani, G. Fossati, P. Mascagni, J. F. Hunt, et al. Mycobacterium tuberculosis Chaperonin 10 Stimulates Bone Resorption: A Potential Contributory Factor in Pott's Disease J. Exp. Med., October 20, 1997; 186(8): 1241 - 1246. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fossati, G. Articles by Mascagni, P. Search for Related Content PubMed PubMed Citation Articles by Fossati, G. Articles by Mascagni, P. Social Bookmarking                      What's this?