Subtilisin E is an alkaline serine protease produced by Bacillus subtilis 168 (Ikemura et al., 1987). The primary gene product for the enzyme consists of pre-pro-subtilisin, having a unique 77-residue pro-sequence between the signal peptide (pre-sequence) and the 275-residue mature sequence. The pro-sequence has been shown to be essential to produce active subtilisin in vivo and is autoprocessed upon the completion of folding of prosubtilisin (Ikemura et al., 1988; Ohta et al., 1990, 1991; Zhu et al., 1989). The autoprocessing of the propeptide is blocked when any one of the three residues at the active center (Asp, His, and Ser) is substituted with other amino acid residues; Asp Asn (Zhu et al., 1989), His Ala (Shinde and Inouye, 1995), and Ser Ala (Li and Inouye, 1994). Interestingly, prothiolsubtilisin, in which Ser was replaced by Cys, could still autoprocess the propeptide despite the fact that thiolsubtilisin lost its protease activity against a synthetic substrate (Li and Inouye, 1994).

The requirement for the propeptide for the formation of active subtilisin has been demonstrated in vitro by the finding that the addition of propeptide is essential to renature subtilisin E denatured by 6 M guanidine HCl (Zhu et al., 1992; Ohta et al., 1991). A number of mutations within the propeptide have been isolated, which are defective in producing active subtilisin, indicating that specific amino acid residues and/or regions in the pro-sequence play important roles in the process of folding the mature subtilisin (Kobayashi and Inouye, 1992). Because of its intramolecular requirement for folding into active subtilisin, the propeptide has been termed an intramolecular chaperone (Inouye, 1991; Shinde and Inouye, 1993; Shinde et al., 1993). The existence of such intramolecular chaperones required for the formation of active enzymes has also been demonstrated for a number of proteases outside the subtilisin family, such as -lytic protease from Lysobacter enzymogenes (Silen et al., 1989; Silen and Agard, 1989) and vacuolar carboxypeptidase Y from Saccharomyces cerevisiae (Winther and Sorensen, 1991; Ramos et al., 1994).

Amino acid substitution mutations in the pro-sequence that were unable to produce active subtilisin in vivo have been isolated within almost the entire pro-sequence region (Kobayashi and Inouye, 1992). In the present paper, we selected six of the 25 mutations previously isolated, and overexpressed them using a T7 expression system in Escherichia coli. These mutant propeptides were purified to near homogeneity, and their abilities to refold denatured subtilisin in vitro were examined. We found that some mutant propeptides were able to refold denatured subtilisin quite efficiently, while others were very inefficient or incapable of renaturing unfolded subtilisin. The efficiencies of the mutant propeptides were found to correlate well to the Kvalues exhibited by these propeptides, indicating that the binding affinities of the mutant propeptides to mature subtilisin are directly related to their capacities to function as intramolecular chaperones.

EXPERIMENTAL PROCEDURES

Materials

Strains and Media

In Vivo Subtilisin Protease Assay

Construction of T7 Expression System

Six mutant propeptide genes were cloned into the pET11a vector as follows. Since all those mutations within the propeptide region were created on the pHI215T vector (Kobayashi and Inouye, 1992), an NdeI site was first created at the initiation codon of the gene by PCR using pHI215T containing desired mutations as a template. The two primers used for PCR were 5`-GCTCTAGACATATGGCCGGAAAAAGCAGTAC-3` and 5`-GGTCGGATCCTTAATATTCATG-3`. The former, which was used as the 5`-end primer, contained an NdeI site at its 5`-end (underlined). The latter was the 3`-end primer, which annealed to the junction region between the propeptide and the mature enzyme. Thus, the resulting PCR products contained the entire propeptide region. The PCR reaction was carried out with 20 ng of plasmid DNA, each primer at 400 nM, each dNTP at 0.2 mM, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl, and 0.5 unit of Taq polymerase. The final volume was 50 µl. Denaturation was carried out at 93 °C for 1 min, annealing at 50 °C for 2 min, and elongation at 72 °C for 1.5 min. This cycle was repeated 25 times (Saiki et al., 1988). Subsequently, the PCR product (about 260 base pairs in length) thus obtained was digested with the NdeI restriction enzyme. Since there is another NdeI site located approximately 30 base pairs upstream of the COOH terminus of the propeptide, a 200-base pair NdeI-NdeI fragment that contained most of the propeptide coding region from the NH terminus resulted from the digestion. The fragments were then cloned into pET11a-propeptide expression vector, from which the wild-type NdeI-NdeI propeptide region had been removed. All constructs were verified by DNA sequencing.

Protein Expression and Purification

The purification of the propeptides was carried out as follows. The supernatant obtained above was first applied to a cation-ion exchange CM-Sepharose Fast Flow FPLC column, which was equilibrated with 50 mM sodium phosphate buffer (pH 5.0). The propeptide was eluted with a NaCl gradient (0-0.4 M). The propeptide peak was detected, and the pooled fractions were then dialyzed against an excess volume of 50 mM Tris-HCl (pH 8.5). The dialysate was applied to an anion-ion exchange Mono Q-Sepharose Fast Flow column. The protein peak eluted with a 0-0.4 M NaCl gradient was collected.

In Vitro Renaturation of Denatured Subtilisin by Propeptides

Determination of the Inhibition Constants

RESULTS

In Vivo Subtilisin Activity Assay

Figure 1: Full-length amino acid sequence of the propeptide of subtilisin E from the NH-terminal -77 position (left) to the COOH-terminal -1 position (right). The large arrow on the COOH terminus indicates the cleavage site between the propeptide and the mature sequence. From the COOH terminus, every 10th amino acid residue is marked by a dot above it. The six single amino acid substitution mutations used in this study are shown below the corresponding residues. The open triangle indicates the position of nonsense mutation, which gives the truncated N59-mer from the NH-terminal end of the propeptide. All 20 amino acid residues are represented by standard single-letter symbols.

Figure 2: In vivo subtilisin activity assay. Halo formation around the colonies was carried out on a casein agar plate as described under ``Experimental Procedures.'' The plate was incubated at 37 °C for 1 day and then at room temperature for 6 more days. 215 is a positive control with cells carrying plasmid pHI215, and 216 is a negative control with cells carrying plasmid pHI216. A-30T, P-15L, I-48T, K-36E, I-67V, and G-44D represent six amino acid substitution mutations in the propeptide region (Ala Thr, Pro Leu, Ile Thr, Lys Glu, Ile Val, and Gly Asp, respectively).

Expression and Purification of Mutant Propeptides

In Vitro Renaturation of Denatured Subtilisin by Mutant Propeptides

Figure 3: Assays of the protease activity of denatured subtilisin BPN` renatured with the addition of various propeptides. A, the denatured subtilisin BPN` was renatured with the wild-type propeptide as well as with the mutant propeptides as described under ``Experimental Procedures.'' Next an aliquot was taken to assay subtilisin activity with a synthetic substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The x axis indicates the incubation time in seconds and the y axis the absorbance at 410 nm. B, relative recovery of the subtilisin activity. From the data in panel A, the initial rate of the reaction was calculated and was compared to that obtained with the wild-type propeptide. Mutant abbreviations are defined in legend to Fig. 2; WT, wild type.

Binding of Mutant Propeptides to Subtilisins

Fig. 4shows the time courses of p-nitroaniline release from the substrate by subtilisin BPN` (Fig. 4A) and subtilisin Carlsberg (Fig. 4B) in the presence of variable concentrations of the wild-type propeptide from subtilisin E. The hyperbolic progress curves for release of p-nitroaniline by active subtilisin in the presence of propeptide of subtilisin E indicate that the propeptide is a slow and tight binding inhibitor for subtilisin BPN` and Carlsberg.

Figure 4: Progress curves for the onset of slow binding inhibition of subtilisin BPN` (panel A), subtilisin Carlsberg (panel B), and subtilisin E (panel C) by the propeptide of subtilisin E. At the indicated concentrations, the substrate was premixed with the propeptide before the enzyme was added. A, the final concentrations in the reaction mixture were 50 mM Tris-HCl (pH 7.0), KCl = 0.1 M, CaCl = 1.0 mM, [s-AAPF-pNA] = 0.3 mM, and the reaction was carried out at room temperature with the subtilisin BPN` at concentration of 2.0 10 mg/ml. B, the buffer condition was the same as above and the concentration of subtilisin Carlsberg was 3.0 10 mg/ml. C, the final concentrations in the reaction mixture were 50 mM Tris-HCl (pH 8.0), KCl = 0.1 M, CaCl = 1.0 mM, [s-AAPF-pNA] = 0.5 mM, at 25 °C and the concentration of subtilisin E was 0.002 mg/ml. D, inhibition of subtilisin BPN` by the Ile Thr mutant propeptide. The mutant propeptide behaves as a rapid equilibrium competitive inhibitor of subtilisin BPN`. Assay conditions were the same as in panel A, and the concentrations of the mutant propeptide are indicated in the figure.

The hyperbolic progress curves for release of p-nitroaniline from s-AAPF-pNA by active subtilisin in the presence of propeptide E indicated that propeptide E is a slow and tight binding inhibitor (Morrison and Walsh, 1988) of subtilisin BPN` and Carlsberg.

Competitive slow binding inhibition generally fits one of the two mechanisms described below (Cha, 1975; Morrison and Walsh, 1988).

Both mechanisms can be described by the same equation (Cha, 1975), where , , and are the enzymatic hydrolysis rate (at time t), initial rate, and steady state rate, respectively.

The apparent first order rate constant k, however, has different significance, for a single-step process, as shown by ,

and for a two-step process, as shown in .

The obtained from the on-set progress curves (Fig. 4) is independent of inhibitor concentration [I] for both subtilisin BPN` and Carlsberg (data not shown). That confirms that inhibition of subtilisins by pro-peptide E follows , similar to inhibition of cathepsin B by its propeptide (Fox et al., 1992), in which

In contrast, is a function of [I] if inhibition occurs in a two-step process.

At constant substrate concentration [S], integration of gives the following equation, where A and A are the absorbance of the hydrolysis product at time t and zero.

Hence, and k could be obtained by non-linear least square fitting of the onset (starting with enzyme) progress curves (A versus t). The progress curves starting with substrate also confirmed the slow binding mechanism, but they were not used here due to the depletion of pro-peptide as a result of proteolytic digestion during preincubation. Then, K is estimated by non-linear least square fitting of a set of versus [I] to the competitive inhibition equation shown below.

The propeptide is also a good substrate of subtilisins (data not shown). This observation makes the measurement of K much more complex. A meaningful K could only be obtained when the concentrations of propeptide and subtilisin were carefully balanced. In other words, the chosen propeptide concentration has to be high enough to slow down the hydrolysis and to maintain a relatively constant inhibitor concentration, and small enough to give a measurable steady state velocity.

The K value of the propeptide of subtilisin E was calculated to be 1.4 10M against subtilisin BPN` and 1.02 10M against subtilisin Carlsberg. Streptomyces subtilisin inhibitor, one of the strongest inhibitors known of subtilisin, was also found to behave as a slow binding inhibitor (K is 0.1 nM against subtilisin BPN` according to our protocol). However, the propeptide of subtilisin E is a very weak inhibitor for subtilisin E itself (Fig. 4C). This is probably due to the fact that the propeptide of subtilisin E is a better substrate of subtilisin E than of subtilisin BPN` and Carlsberg, and therefore it is digested promptly by active subtilisin E (Hu, 1994). Since no steady state kinetic behavior could be reached due to the digestion, no attempts were made to calculate an accurate K value for subtilisin E. For this reason, subtilisin BPN` was used for the present study.

In the same manner, all six single amino acid substitution mutant propeptides and the truncated N59-mer propeptide were examined for their inhibitory behavior toward subtilisin BPN`. The calculated K values are listed in Table 1. Three of the six single mutant propeptides, Ala Thr, Lys Glu, and Pro Leu, have comparable K values with the wild-type propeptide (1.4 10M): 1.15 10, 1.61 10, and 2.10 10M, respectively. The other two propeptides, Gly Asp and Ile Val, have approximately 7-8-fold higher K values than the wild-type propeptide: 11.0 10 and 10.0 10M for the Gly Asp and the Ile Val propeptides, respectively. The Ile Thr mutant gave the most dramatic result, with a K value (118 10M) that is 80 times higher than that for the wild-type propeptide. This mutant propeptide is no longer a slow binding inhibitor of subtilisin BPN` (Fig. 5D). The N59-mer showed no inhibition toward subtilisin BPN`, even at 10 µM concentration (data not shown).

Figure 5: Plot of folding efficiency of the mutant propeptides versus the reciprocals of its K. Each filled dot represents the mutant propeptide or wild-type propeptide (marked accordingly). Mutant abbreviations are defined in Fig. 2legend.

DISCUSSION

In the present study, we selected 6 mutations out of 25 amino acid substitution mutations previously isolated (Kobayashi and Inouye, 1992) for further study. These mutations were originally screened according to their inabilities to form a halo around colonies on an agar plate containing casein. E. coli cells producing active subtilisin form a halo on the plate. By incubating the plate for a much longer time, three mutants were found to be able to develop halos (see Fig. 2). The propeptides containing these mutations classified as class I mutations were capable of refolding denatured subtilisin at a level of 50-80% of the efficiency of the wild-type propeptide. The K values were found to be well correlated with the refolding abilities of the class I mutant propeptides; Ala Thr showed 80% refolding efficiency with K of 1.15 10M, Lys Glu 60% with K of 1.61 10M, and Pro Leu 54% with K of 2.10 10M. It is interesting to note that these three mutations located in the COOH-terminal half of the propeptide resulted in drastic amino acid substitutions; a change of charge from +1 to -1 (Lys Glu), a hydrophobic to a hydrophilic residue in a cluster of hydrophobic residues (Ala Thr), and proline to leucine at position -15. It appears that the propeptide is designed to maintain the chaperone activity, albeit at a lower level, even with drastic amino acid substitution mutations in the COOH-terminal region. However, the fact that the deletion of the COOH-terminal 18 residues (N59-mer) resulted in the complete loss of refolding ability of the propeptide indicates that the COOH-terminal region is still essential for the chaperone function. The second class of mutations (class II; Ile Val and Gly Asp) formed no halo around colonies even after longer incubation, although the propeptides with the class II mutations were able to refold denatured subtilisin at a level of 10-20% of the efficiency of the wild-type propeptide. In this class, the K values are approximately 7-8 times higher than the K value of the wild-type propeptide. The class III mutations, Ile Thr, had a K value that was 80 times higher than that of the wild-type propeptide, and was barely able to refold denatured subtilisin.

Interestingly, a plot of folding efficiency of the mutant propeptide versus the reciprocals of its K leads to a linear relationship (Fig. 5). This suggests that the abilities of the mutant propeptides to bind to the mature subtilisin are directly related to their abilities to renature denatured subtilisin. The propeptide's ability to function as a chaperone for subtilisin folding is therefore related as to how well the propeptide is able to bind the mature active subtilisin to inhibit its activity. Neither of the mutant propeptides nor the wild-type propeptide have significant amounts of secondary structure, as judged by their circular dichroism spectra (not shown; see also Shinde et al., 1993). However, when the wild-type propeptide binds to mature active subtilisin, it acquires a substantial amount of secondary structure (Shinde et al., 1993). It has been suggested that the propeptide as a single turnover catalyst (Hu et al., 1994) exerts its catalytic power on a late step in the refolding process, in which the interaction between the propeptide and the mature sequence approximates the interaction between the propeptide and the active enzyme, i.e. in the transition state of the propeptide catalyzed folding, much of the secondary and tertiary structure found in the native structure is already formed. Therefore, the propeptides with the mutations described in the present paper are likely to become defective in formation of secondary structure. During the subtilisin folding process, the interaction between the propeptide and denatured subtilisin is believed to lead to the formation of secondary structures in both components. Thus, mutations that inhibit the acquisition of secondary structure in the propeptide may also render it incapable of refolding the denatured subtilisin.

Our finding that the propeptides are also substrates for the active enzymes has direct relevance to the notion of a ``single turnover catalyst'' (Hu, 1994). By definition, as a catalyst, the propeptide would catalyze both the refolding and unfolding reactions. Proteolysis of the propeptide renders it ineffective as a catalyst (or indeed as an inhibitor) and traps the mature protein in its refolded state.

Slow binding inhibition behavior had been observed in the interaction of another serine protease -lytic protease with its propeptide (Baker et al., 1992) and in the interaction of the cystein protease cathepsin B with its propeptide (Fox et al., 1992). Therefore, the kinetic behavior reported in the present manuscript appears to be quite general for the inhibition of the active proteases by their propeptides.

It is possible, although it would clearly be a speculation, that the slow binding inhibition reflects that the final conformation of the propeptide that is bound to the active conformation of the mature enzyme is different from the conformation of the propeptide in the initial encounter complex. Such a conformational change of the bound propeptide would in fact be consistent with the observed kinetics.

It has been recently reported that denatured subtilisin could be refolded in the absence of the propeptide providing that subtilisin is attached to a solid matrix (Hayashi et al., 1993). In that experiment, active subtilisin was first immobilized on CNBr-activated agarose gel and then denatured by 6 M guanidine HCl. Under these conditions, certain sites on the surface of the native subtilisin molecule may preferentially interact with the solid surface, and this interaction might restrict the unfolding process of subtilisin. Furthermore, even if subtilisins were completely denatured in this experiment, the protection of denatured subtilisin at certain sites by the matrix may highly restrict random movement of the molecule being folded, and this may be sufficient to avoid undesired interactions between the secondary structures during the renaturation process. Such an unproductive alternative folding pathway might govern the folding of denatured subtilisin in solution in the absence of the propeptide and perhaps lead to aggregation.

Identification of the step at which the propeptide must exert its influence on the folding pathway of mature subtilisin is a major outstanding issue in this folding mechanism. In the absence of any structural information about the propeptide, the mutational study described in this paper provides a guide to some specific interactions between the propeptide and the mature subtilisin, helping us to understand the molecular mechanism of propeptide-mediated protein folding.

FOOTNOTES

*

Work at Rutgers was supported by the Charles and Johanna Busch Biomedical Grant. This work was also supported in part by a grant from Ajinomoto Co., Ltd. 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.

()

The numbering of the propeptide starts from Tyr and extends to Met, i.e. from the carboxyl terminus toward the amino terminus. The mutant N59-mer extends from Leu to Met.

()

The abbreviations used are: PCR, polymerase chain reaction; IPTG, isopropyl--D-thiolgalactopyranoside; s-AAPF-pNA, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide.

ACKNOWLEDGEMENTS

REFERENCES

1. Baker, D., Sohl, J. L., and Agard, D. A. (1992a) Nature 356,263-265 [CrossRef][Medline] [Order article via Infotrieve]
2. Baker, D., Sohl, J. L., and Agard, D. A. (1992b) Proteins Struct. Funct. Genet. 12,339-344 [CrossRef][Medline] [Order article via Infotrieve]
3. Cha, S. (1975) Biochem. Pharmacol. 24,2177-2185 [CrossRef][Medline] [Order article via Infotrieve]
4. Fox, T., de Miquel, E., Mort, J. S., and Storer, A. C. (1992) Biochemistry 31,12571-12576 [CrossRef][Medline] [Order article via Infotrieve]
5. Hayashi, T., Matsubara, M., Kurimoto, E., Nohara, D., and Sakai, T. (1993) Chem. Pharm. Bull. 41,2063-2065
6. Hu, Z. (1994) Role of the Propeptide in the Refolding of Subtilisin , Ph.D. thesis, Rutgers University
7. Hu, Z., Zhu, X., Jordan, F., and Inouye, M. (1994) Biochemistry 33,562-569 [CrossRef][Medline] [Order article via Infotrieve]
8. Ikemura, H., and Inouye, M. (1988) J. Biol. Chem. 263,12959-12963 [Abstract/Free Full Text]
9. Ikemura, H., Takagi, H., and Inouye, M. (1987) J. Biol. Chem. 262,7859-7864 [Abstract/Free Full Text]
10. Inouye, M. (1991) Enzymes (Basel) 45,314-321 [Medline] [Order article via Infotrieve]
11. Kobayashi, T., and Inouye, M. (1992) J. Mol. Biol. 226,931-933 [CrossRef][Medline] [Order article via Infotrieve]
12. Li, Y., and Inouye, M. (1994) J. Biol. Chem. 269,4169-4174 [Abstract/Free Full Text]
13. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
14. Morrison, J. C., and Walsh, C. T. (1988) Adv. Enzymol. 61,201-301
15. Nakamura, K., Masui, Y., and Inouye, M. (1982) J. Mol. Appl. Genet. 1,289-299 [Medline] [Order article via Infotrieve]
16. Ohta, Y., and Inouye, M. (1990) Mol. Microbiol. 4,295-304 [CrossRef][Medline] [Order article via Infotrieve]
17. Ohta, Y., Hojo, H., Aimoto, S., Kobayashi, T., Zhu, X., Jordan, F., and Inouye, M. (1991) Mol. Microbiol . 5,1507-1510 [CrossRef][Medline] [Order article via Infotrieve]
18. Ramos, C., Winther, J. R., and Kielland-Brandt, M. C. (1994) J. Biol. Chem. 269,7006-7012 [Abstract/Free Full Text]
19. Saiki, R. K., Gelfand, D. H. M., Dtoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Science 239,487-491 [Abstract/Free Full Text]
20. Silen, J. L., and Agard, D. A. (1989) Nature 341,462-464 [CrossRef][Medline] [Order article via Infotrieve]
21. Silen, J. L., Frank, D., Fujishige, A., Bone, R., and Agard, D. A. (1989) J. Bacteriol. 171,1320-1325 [Abstract/Free Full Text]
22. Shinde, U., and Inouye, M. (1993) Trends Biochem. Sci. 18,442-446 [CrossRef][Medline] [Order article via Infotrieve]
23. Shinde, U., and Inouye, M. (1995) J. Mol. Biol. 247,390-395 [CrossRef][Medline] [Order article via Infotrieve]
24. Shinde, U., Li, Y., Chatterjee, C., and Inouye, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,6924-6928 [Abstract/Free Full Text]
25. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubenderff, J. W. (1990) Methods Enzymol. 185,60-89 [Medline] [Order article via Infotrieve]
26. Winther, J. R., and Sorensen, P. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,9330-9334 [Abstract/Free Full Text]
27. Zhu, X., Ohta, Y., Jordan, F., and Inouye, M. (1989) Nature 339,483-484 [CrossRef][Medline] [Order article via Infotrieve]
28. Zhu, X., Ohta, Y., Cash, P., Tous, G., Stein, S., Inouye, M., and Jordan, F. (1992) Biochem. Life Sci. Adv. 11,35-47

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				A. A. Golabek, N. Dolzhanskaya, M. Walus, K. E. Wisniewski, and E. Kida Prosegment of Tripeptidyl Peptidase I Is a Potent, Slow-binding Inhibitor of Its Cognate Enzyme J. Biol. Chem., June 13, 2008; 283(24): 16497 - 16504. [Abstract] [Full Text] [PDF]

				S.-i. Tanaka, K. Saito, H. Chon, H. Matsumura, Y. Koga, K. Takano, and S. Kanaya Crystal Structure of Unautoprocessed Precursor of Subtilisin from a Hyperthermophilic Archaeon: EVIDENCE FOR Ca2+-INDUCED FOLDING J. Biol. Chem., March 16, 2007; 282(11): 8246 - 8255. [Abstract] [Full Text] [PDF]

				M.A. Pulido, Y. Koga, K. Takano, and S. Kanaya Directed evolution of Tk-subtilisin from a hyperthermophilic archaeon: identification of a single amino acid substitution responsible for low-temperature adaptation Protein Eng. Des. Sel., March 9, 2007; (2007) gzm006v1. [Abstract] [Full Text] [PDF]

				M. Pulido, K. Saito, S.-I. Tanaka, Y. Koga, M. Morikawa, K. Takano, and S. Kanaya Ca2+-Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis: the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding. Appl. Envir. Microbiol., June 1, 2006; 72(6): 4154 - 4162. [Abstract] [Full Text] [PDF]

				L. Jean, F. Hackett, S. R. Martin, and M. J. Blackman Functional Characterization of the Propeptide of Plasmodium falciparum Subtilisin-like Protease-1 J. Biol. Chem., August 1, 2003; 278(31): 28572 - 28579. [Abstract] [Full Text] [PDF]

				Y. Yabuta, E. Subbian, C. Oiry, and U. Shinde Folding Pathway Mediated by an Intramolecular Chaperone. A FUNCTIONAL PEPTIDE CHAPERONE DESIGNED USING SEQUENCE DATABASES J. Biol. Chem., April 18, 2003; 278(17): 15246 - 15251. [Abstract] [Full Text] [PDF]

				I. Massimi, E. Park, K. Rice, W. Muller-Esterl, D. Sauder, and M. J. McGavin Identification of a Novel Maturation Mechanism and Restricted Substrate Specificity for the SspB Cysteine Protease of Staphylococcus aureus J. Biol. Chem., October 25, 2002; 277(44): 41770 - 41777. [Abstract] [Full Text] [PDF]

				P. S. Sijwali, B. R. Shenai, and P. J. Rosenthal Folding of the Plasmodium falciparum Cysteine Protease Falcipain-2 Is Mediated by a Chaperone-like Peptide and Not the Prodomain J. Biol. Chem., April 19, 2002; 277(17): 14910 - 14915. [Abstract] [Full Text] [PDF]

				E. D. Anderson, S. S. Molloy, F. Jean, H. Fei, S. Shimamura, and G. Thomas The Ordered and Compartment-specific Autoproteolytic Removal of the Furin Intramolecular Chaperone Is Required for Enzyme Activation J. Biol. Chem., April 5, 2002; 277(15): 12879 - 12890. [Abstract] [Full Text] [PDF]

				G. Heusipp, G. M. Young, and V. L. Miller HreP, an In Vivo-Expressed Protease of Yersinia enterocolitica, Is a New Member of the Family of Subtilisin/Kexin-Like Proteases J. Bacteriol., June 15, 2001; 183(12): 3556 - 3563. [Abstract] [Full Text]

				X. Fu, M. Inouye, and U. Shinde Folding Pathway Mediated by an Intramolecular Chaperone. THE INHIBITORY AND CHAPERONE FUNCTIONS OF THE SUBTILISIN PROPEPTIDE ARE NOT OBLIGATORILY LINKED J. Biol. Chem., May 26, 2000; 275(22): 16871 - 16878. [Abstract] [Full Text] [PDF]

U. Shinde, X. Fu, and M. Inouye A Pathway for Conformational Diversity in Proteins Mediated by Intramolecular Chaperones J. Biol. Chem., May 28, 1999;