Increased proteolytic resistance of ribonuclease A by protein engineering

Yvonne Markert, Jens Köditz, Johanna Mansfeld, Ulrich Arnold,1 and Renate Ulbrich-Hofmann,2

Department of Biochemistry/Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle, Germany


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 Conclusions
 References
 
Although highly stable toward unfolding, native ribonuclease A is known to be cleaved by unspecific proteases in the flexible loop region near Ala20. With the aim to create a protease-resistant ribonuclease A, Ala20 was substituted for Pro by site-directed mutagenesis. The resulting mutant enzyme was nearly identical to the wild-type enzyme in the near-UV and far-UV circular dichroism spectra, in its activity to 2',3'-cCMP and in its thermodynamic stability. However, the proteolytic resistance to proteinase K and subtilisin Carlsberg was extremely increased. Pseudo-first-order rate constants of proteolysis, determined by densitometric analysis of the bands of intact protein in SDS–PAGE, decreased by two orders of magnitude. In contrast, the rate constant of proteolysis with elastase was similar to that of the wild-type enzyme. These differences can be explained by the analysis of the fragments occurring in proteolysis with elastase. Ser21–Ser22 was identified as the main primary cleavage site in the degradation of the mutant enzyme by elastase. Obviously, this bond is not cleavable by proteinase K or subtilisin Carlsberg. The results demonstrate the high potential of a single mutation in protein stabilization to proteolytic degradation.

Keywords: elastase/proteinase K/ribonuclease A/site-directed mutagenesis/subtilisin Carlsberg


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 Conclusions
 References
 
The resistance to proteolytic attack is one of the most important criteria for the industrial application of enzymes. Proteolytic stability generally correlates with the thermal stability of proteins (McLendon and Radany, 1978Go; Daniel et al., 1982Go; Parsell and Sauer, 1989Go; Akasako et al., 1995Go). The reason for this coherence is that most proteins in their native conformation are resistant to proteases, whereas they are rapidly degraded after unfolding. Unspecific proteases, however, are also able to attack a protein in its native conformation if it contains loop regions that are accessible and flexible enough for a cleavage (Price and Johnson, 1990Go; Hubbard, 1998Go). Even small, highly compact and thermostable proteins may contain protease-sensitive regions. An example is the well known bovine pancreatic ribonuclease A (RNase A). Although it is characterized by a very compact global tertiary structure, which is stabilized by four disulfide bridges (Wlodawer et al., 1982Go), and a high thermodynamic stability (Pfeil, 1998Go), it is cleaved by subtilisin Carlsberg even at 25°C (Richards and Vithayathil, 1959Go). The nicked protein which is called ribonuclease S is still active under native conditions but it is separated into two fragments in SDS–PAGE. The fragments comprising the amino acid residues 1–20 (S-peptide) and 21–124 (S-protein) indicate that native RNase A is mainly cleaved between the residues Ala20 and Ser21 which are located in the well accessible loop between two helices (Figure 1Go). A similar fragmentation of RNase A was found by the likewise unspecific proteases proteinase K and elastase. While for proteinase K the Ala20–Ser21 peptide bond was also identified as the primary cleavage site (Rauber et al., 1978Go), elastase was reported to cleave the neighbored Ala19–Ala20 peptide bond (Klee, 1965Go).



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Fig. 1. Tertiary structure of RNase A. The model was taken from the Brookhaven protein data bank and drawn with MOLSCRIPT, version 1.4. {alpha}-Helices and ß-sheets are presented as ribbons.

 
Although protein engineering enables tailoring of many structural properties of proteins, the improvement of proteolytic resistance has rarely been the goal of directed or random mutagenesis. Frenken et al. increased the stability of Pseudomonas glumae lipase to subtilisin by introduction of charged amino acid or proline residues near the primary proteolytic cleavage site (Frenken et al., 1993Go). The best variant in which the two amino acids at that cleavage site were replaced by two arginine residues was five times more stable than the wild-type enzyme. The thermostability of the Asp49-mutant of subtilisin J was improved by deleting Tyr58 at the primary autoproteolytic cleavage site (Bae et al., 1995Go), whereas the removal of one of the autoproteolytic cleavage sites of neutral protease from Bacillus subtilis resulted in a new cleavage site and had no effect on the thermal stability of the enzyme (Van den Burg et al., 1998Go). Successful stabilization to autoproteolysis of rat trypsin was gained by two mutations (K61N, R117N) at main autolysis sites (Várallyay et al., 1998Go). Degradation of the Escherichia coli outer membrane protease OmpT could be abolished by introducing the mutations G216K and K217G in the primary autolysis site (Kramer et al., 2000Go).

The aim of the present study has been to examine the effect of the mutation Ala20Pro in RNase A on the proteolytic resistance of the enzyme to proteinase K, subtilisin Carlsberg and elastase under native conditions. The results are compared with the thermodynamic stability of the wild-type and mutated RNase A.


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 Conclusions
 References
 
Proteins and chemicals

RNase A was purchased from Sigma, St. Louis, MO, USA and purified to homogeneity using a MONO S column (Pharmacia-Biotech, Uppsala, Sweden). Porcine elastase (Boehringer Mannheim, Penzburg, Germany), subtilisin Carlsberg (Sigma) and proteinase K (Sigma) were used without further purification.

Guanidine hydrochloride (GdnHCl), ultra pure, was from ICN Biomedicals, Irvine, CA, USA, dithiothreitol (DTT) from Sigma, glutathione reduced (GSH) and oxidized (GSSG) from Boehringer Mannheim, phenylmethanesulfonyl fluoride (PMSF) from Merck, Darmstadt, Germany. Oligonucleotides were from MWG Biotech, Ebersberg and AvaI from New England Biolabs, Frankfurt/Main, Germany. The components for growth media were from Difco Laboratories, Detroit, MI, USA. Escherichia coli strains XL-1 Blue and BL21 (DE3) were from Stratagene, Heidelberg, Germany. All other chemicals were of purest grade commercially available.

Molecular modeling

Molecular modeling of the mutations A19P, A20P and S21P in RNase A was performed using the program WHAT IF (Vriend, 1990Go).

Site-directed mutagenesis

The plasmid pBXR containing the RNase A gene (delCardayré et al., 1995Go) was obtained from Ronald T.Raines (WI, USA). The RNase A gene was modified by use of the QuikChangeTM site-directed mutagenesis kit (Stratagene). The codon GCC for Ala20 was replaced by the codon CCG for Pro. The oligonucleotides 5'-GACTCCAGCACTTCCGCCCCGAGCAGCTCCAACTACTG-3' (forward) and 5'-CAGTAGTTGGAGCTGCTCGGGGCGGAAGTGCTGGAGTC-3' (reverse) were used where the nucleotide substitutions are bold-faced. The underlined sequences mark the AvaI restriction site introduced to facilitate the selection of positive clones. The mutation was verified by DNA sequencing according to Sanger et al. (Sanger et al., 1977Go) using the SequiThermExcelTM LongReadTM DNA sequencing kit (Biozym, Oldendorf, Germany) and a Li-COR 4000 DNA-sequencer (MWG). The plasmid carrying the correct DNA sequence was transformed into the E.coli expression host strain BL21 (DE3).

Expression, renaturation and purification of A20P-RNase A

An overnight culture of the E.coli strain BL21(DE3) carrying the mutated pBXR vector was grown at 37°C and 250 r.p.m. (incubator shaker innova 4300, New Brunswick Scientific, Nurtingen, Germany), in terrific broth medium (TM, delCardayré et al., 1995Go) with 100 µg/ml ampicillin. An aliquot of 5 ml of this culture was used to inoculate 500 ml of TB (containing 400 µg/ml ampicillin). After the cells were grown at 37°C and 250 r.p.m. to an OD600 of 2, expression of the mutant enzyme was induced by addition of isopropyl-ß-D-thiogalactopyranoside to a final concentration of 1 mM. After 4 h of expression, the cells were harvested by centrifugation at 5 000 g and 4°C for 15 min. Cell lysis was performed in 20 mM Tris–HCl buffer, pH 8.0, containing 10 mM EDTA by treatment with lysozyme (1.5 mg/g cells) at 4°C for 1 h and homogenization with a Gaulin homogenizer. The inclusion bodies were isolated by centrifugation at 30 000 g and 4°C for 30 min and resolubilized in 20 mM Tris–HCl buffer, pH 8.0, containing 6 M GdnHCl, 100 mM DTT and 10 mM EDTA under stirring at room temperature for 2 h. After adjusting the pH to 4.0, the protein solution was dialyzed against 2x10 l of 20 mM acetic acid overnight at 4°C. Precipitates formed during dialysis were removed by centrifugation at 15 000 g and 4°C for 30 min. The protein solution obtained from 500 ml of culture was added dropwise to 500 ml of renaturation buffer with a final concentration of 0.1 M Tris–acetic acid, pH 8.5, 0.1 M NaCl, 3 mM GSH, 0.3 mM GSSG, 5 mM EDTA at room temperature and kept for 48 h. The renatured protein was purified using an S-Sepharose column (Pharmacia) which had been equilibrated with 20 mM sodium formiate, pH 4.0. The elution of the protein was carried out with a linear gradient from 50 mM Tris–HCl buffer, pH 7.8, to 50 mM Tris–HCl buffer, pH 8.0, containing 0.5 M NaCl. The combined peak fractions were applied to a SuperdexTM 75 column (Pharmacia Biotech) in 50 mM Tris–HCl buffer, pH 8.0, eluted and stored at 4°C.

Determination of the protein concentration

The protein concentration of RNase A and A20P-RNase A was determined by using the molar absorption coefficient of {varepsilon}278nm = 9800 M cm, (Sela and Anfinsen, 1957Go).

RNase activity

The activity of RNase A and A20P-RNase A was determined according to Crook et al. (Crook et al., 1960Go) in 50 mM Tris–HCl buffer, pH 8.0, containing 0.2 mM 2',3'-cCMP at 25°C. The changes in absorbance were followed in a 1 cm cuvette at 286 nm using a Hitachi (Tokyo, Japan) U2000 spectrophotometer. For calculation of activity, {Delta}{varepsilon} = 1450 M cm (delCardayré and Raines, 1995Go) was used.

Circular dichroism spectroscopy

Circular dichroism (CD) spectra of RNase A and A20P-RNase A were recorded on a CD spectrometer 62 ADS (AVIV, Lakewood, NJ, USA) at 10°C. The measurements were performed at a protein concentration of 2 mg/ml in 50 mM Tris–HCl buffer, pH 8.0, using a 1 cm cuvette in the near-UV region and in 10 mM Tris–HCl, pH 8.0, using a 0.01 cm cuvette in the far-UV region.

Transition curves in GdnHCl and thermodynamic stability

GdnHCl-induced transition curves of RNase A and A20P-RNase A were obtained by fluorescence spectroscopy using a fluorescence spectrophotometer F-4500 (Hitachi). The measurements were carried out at a protein concentration of 50 µg/ml RNase in 50 mM Tris–HCl buffer, pH 8.0, at 25°C. The excitation wavelength was 278 nm and the emission was detected at 302.8 nm for RNase A and at 304.2 nm for A20P-RNase A using a slit width of 5 nm for both excitation and emission.

[GdnHCL]1/2 and m values were determined from the spectroscopic data according to Pace et al. (Pace et al., 1989Go) using the linear relation:

(1)

Proteolysis

Proteolysis was carried out at 25°C with final concentrations of 0.2 mg/ml of RNase A or A20P-RNase A and 0.5–100 µg/ml of protease. The reaction was started by addition of 15 µl of protease in 50 mM Tris–HCl buffer, pH 8.0, containing 10 mM CaCl2, to 135 µl of RNase A or A20P-RNase A in 50 mM Tris–HCl buffer, pH 8.0. After defined time intervals, samples of 10 µl were withdrawn, immediately mixed with 10 µl of 100 mM PMSF to stop the reaction, dried under nitrogen and analyzed by SDS–PAGE.

SDS-PAGE and determination of the rate constants of proteolysis (kp)

Electrophoresis was carried out on a Midget electrophoresis unit (Hoefer, San Francisco, CA, USA) according to Schägger and von Jagow (Schägger and von Jagow, 1987Go) using 10 and 18% acrylamide for stacking and separating gels and working without spacer gel. The gels were stained with Coomassie Brillant Blue G250. After staining, the gels were evaluated at 595 nm using a densitometer CD 60 (Desaga, Heidelberg, Germany).

kp values were calculated from the decrease of the peak areas of the intact RNase A or A20P-RNase A bands in SDS–PAGE gels as a function of time of proteolysis, which followed pseudo-first-order kinetics.

Analysis of the proteolytic fragments

RNase A and A20P-RNase A were treated with elastase for 30 min as described above.

The resulting RNase species were denatured by addition of GdnHCl to a final concentration of 5 M, and the fragments were separated on an inert HPLC system (Merck-Hitachi, Tokyo, Japan) with an octyl reversed-phase column (Vydac, Hesperia, CA, USA) using a solvent gradient from 0 to 40% acetonitrile produced by solvent A (degassed water containing 0.070% trifluoroacetic acid) and solvent B (degassed acetonitrile containing 0.063% trifluoroacetic acid). The absorbance was followed at 214 nm and fractions were collected manually. MALDI mass spectrometry and protein sequencing were performed as described by Arnold et al. (Arnold et al., 1996Go) on a reflectron-type time-of-flight mass spectrometer ReflexTM (Bruker-Franzen, Bremen, Germany) and a protein sequencer 476A (Applied Biosystems, Foster City, CA, USA).


    Results and discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 Conclusions
 References
 
Mutant design and production

If the proteolytic degradation of the target protein starts from its native conformation, two strategies are conceivable for increasing the proteolytic resistance. First, the primary structure of the site of proteolytic attack may be changed by introducing amino acids that are disadvantageous for the action of the considered protease or, second, the conformational flexibility of the attacked structural region can be restricted. A combination of both variants is the proline concept (Frenken et al., 1993Go) which was preferred in these studies. Proline is known to be an unfavorable amino acid in the P1 as well as in the P1'-position for most proteases (Brömme et al., 1986Go). Moreover, the introduction of a proline residue is expected to reduce the flexibility of the concerned structural region (Jaenicke, 2000Go).

Starting from literature reports which described Ala20–Ser21 as the main cleavage site in native RNase A for subtilisin Carlsberg as well as for proteinase K (Richards and Vithayathil, 1959Go; Rauber et al., 1978Go) and Ala19–Ala20 for elastase (Klee, 1965Go), the substitution of Pro for Ala19, Ala20 and Ala21 was considered. The exchange Ala20Pro seemed to be the most promising mutation, since the native Ala20 residue occupies the P1 position of subtilisin Carlsberg or proteinase K and the P1'-position of elastase. Proline residues in these positions are expected to hinder proteolytic cleavage. Molecular modeling was used to check that no severe structural changes in RNase A should occur.

The mutation Ala20Pro was introduced into the DNA sequence encoding for RNase A by site-directed mutagenesis. The mutated gene was expressed in E.coli where the enzyme (called A20P-RNase A in this paper) accumulated as inclusion bodies. The resolubilized inclusion bodies contained a large amount of undesired cell proteins which, however, precipitated during the dialysis in the renaturation procedure and could therefore easily be removed. After refolding and purification, the protein (28 mg/l culture medium) was homogeneous in SDS–PAGE and identical with commercial, purified RNase A in its electrophoretic behavior.

Activity and conformational characterization of the mutant enzyme

As the CD spectra in the far-UV region (Figure 2aGo) characterizing secondary structures and the CD spectra in the near-UV region (Figure 2bGo) characterizing the tertiary structure indicate, the mutation A20P has no significant influence on the conformation of the enzyme. Correspondingly, also the activities of RNase A and A20P-RNase A are identical within the margin of error (Table IGo).



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Fig. 2. CD spectra of RNase A (—) and A20P-RNase A (·····) in 50 mM Tris–HCl buffer, pH 8.0, at 25°C in the far-UV (a) and in the near-UV regions (b).

 

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Table I. Activity and stability parameters of A20P-RNase A and wild-type RNase A at 25°C
 
Fluorescence spectra taken in the presence of the denaturant GdnHCl were used to characterize the unfolding of A20P-RNase A in comparison to that of wild-type RNase A. Figure 3Go presents the transition curves constructed from the fluorescence emission signals at 25°C. They demonstrate that the thermodynamic stabilities of the two enzymes are similar. Table IGo shows the corresponding co-operativity indices m (Equation 1Go) and the GdnHCl concentration at the transition point [GdnHCl]1/2.



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Fig. 3. GdnHCl-induced conformational transition of RNase A (filled circle) and A20P-RNase A (open circle) in 50 mM Tris–HCl buffer, pH 8.0, at 25°C.

 
Therefore, as far as detected by spectroscopic and activity measurements, the native state as well as the unfolding of RNase A are not seriously affected by the exchange of the amino acid in the flexible protease-sensitive loop.

Proteolytic stability

The proteolytic susceptibility of RNase A and A20P-RNase A to the unspecific proteases proteinase K, subtilisin Carlsberg and elastase was analyzed by SDS–PAGE. In Figure 4Go, typical fragment patterns are shown as a function of time of proteolysis. Figure 4a–cGo shows the expected behavior of RNase A. The protein band of the intact enzyme decreases with time, while, complementarily, the band of a large fragment appears. According to literature, this protein band was assigned to the fragment Ser21–Val124. While this fragment is the primary cleavage product of proteinase K (Rauber et al., 1978Go) and subtilisin Carlsberg (Richards and Vithayathil, 1959Go), it is formed from the primary cleavage product of elastase (Ala20–Val124) by the fast subsequent release of Ala20 (Klee, 1965Go). The mutant enzyme A20P-RNase A showed a completely different behavior. To proteinase K and subtilisin Carlsberg, A20P-RNase A was extremely stable (Figure 4d and eGo), whereas the degradation by elastase was similar to that of RNase A (Figure 4fGo).



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Fig. 4. SDS–PAGE of the proteolytic degradation of RNase A (ac) and A20P-RNase A (df) in 50 mM Tris–HCl buffer, pH 8.0, containing 1 mM CaCl2 at 25°C. RNase A was incubated with (a) proteinase K (1:1, w/w) for 15, 30, 45 s and 1, 2, 3 min (from left to right), (b) subtilisin Carlsberg (2:1, w/w) for 15, 30 s and 1, 2, 5, 8 min, (c) elastase (10:1, w/w) for 30 s and 2, 10, 15, 30, 45 min. A20P-RNase A was incubated with (d) proteinase K (1:1, w/w) for 30 s, 15, 30 min and 2, 4, 7 h (from left to right), (e) subtilisin Carlsberg (2:1, w/w) for 30 s, 30, 90 min and 3, 6, 9 h, (f) elastase (10:1, w/w) for 30 s and 2, 10, 15, 30, 45 min.

 
From the intensity of the Coomassie-stained bands of the non-degraded proteins, pseudo-first-order constants of proteolysis (kp) were determined (Table IIGo). The difference in the kp values for RNase A and A20P-RNase A amounts to more than two orders of magnitude for the proteolysis by proteinase K or subtilisin Carlsberg, whereas kp is in the same range for both enzymes with elastase under these conditions.


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Table II. Rate constants of proteolysis at 25°C
 
For proteolysis with proteinase K, kp values were also determined as a function of the protease concentration (8–200 µg/ml, corresponding to a ratio of RNase A or A20P-RNase A to protease of 1–40:1, w/w). The resulting linear relationship between kp and the protease concentration for RNase A as well as for A20P-RNase A (Figure 5Go) suggests that the degradation of both enzymes starts from the native enzyme conformation. In case that A20P-RNase A, where the cleavage of the Ala20–Ser21 peptide bond is impeded, would be degraded from the unfolded molecule species, a hyperbolic function is expected (Arnold and Ulbrich-Hofmann, 1997Go). In addition, kp should be in the range of the unfolding constant (kU), which is not the case. For RNase A, kU at 25°C can be estimated to be 5x10-8/s (Arnold and Ulbrich-Hofmann, 2001Go), whereas kp is approximately three orders of magnitude higher.



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Fig. 5. Rate constants of proteolysis for proteinase K acting on RNase A (filled circle, left axis) and A20P-RNase A (inverted filled triangle, right axis) as a function of the protease concentration. Proteolysis was carried out with 200 µg/ml RNase A or A20P-RNase A in 50 mM Tris–HCl buffer, pH 8.0, containing 1 mM CaCl2 at 25°C.

 
Unfortunately, it was not possible to identify fragments of the primary cleavage of A20P-RNase A in the degradation with proteinase K or subtilisin Carlsberg. The primary proteolytic event was too slow in comparison to the consecutive degradation of the arising fragments. Therefore, it cannot be decided whether the cleavage of A20P-RNase A proceeds via the same peptide bond (20–21) as in RNase A or via one of the neighboring peptide bonds (e.g. 18–19, 19–20, 21–22 or 22–23) which all are potential cleavage sites for proteinase K and subtilisin Carlsberg. In fact, it has been shown that 10–15% of RNase A is cleaved at the peptide bond Ser21–Ser22 (Neumann and Hofsteenge, 1994Go), and the peptide bonds Ser16–Thr17, Ser18–Ala19 and Ala19–Ala20 participate for 2.1, 7.0 and 3.0%, respectively (Méndez et al., 2000Go). While, in the case of cleavage at the peptide bond 20–21, the introduced proline residue occupies the P1-position of the protease substrate, it would be in position P2', P1', P2 or P3 if 18–19, 19–20, 21–22 or 22–23 are the primarily cleaved peptide bonds. From kinetic studies on subtilisin Carlsberg (Morihara and Oka, 1977Go; Meldal et al., 1994Go; Lu et al., 1997Go) and proteinase K (Morihara and Oka, 1977Go; Brömme et al., 1986Go; Carter et al., 1989Go; Betzel et al., 1993Go; Saxena et al., 1996Go) with peptide substrates it can be concluded that proline in all these positions is unfavorable, which might explain the strongly decreased rates of proteolysis with subtilisin Carlsberg and proteinase K.

The situation is completely different with elastase. After proteolysis, a large fragment could be accumulated even for A20P-RNase A (Figure 4fGo). Separation of the proteolytic fragments by RP-HPLC and their analysis by MALDI-MS and N-terminal sequencing allowed the determination of two complementary fragments: Lys1–Ser21 and Ser22–Val124 (Table IIIGo), identifying Ser21–Ser22 as the main primary cleavage site in A20P-RNase A. Stimulated by this surprising result, we examined the primary cleavage for wild-type RNase A in the same way (data not shown). In completion of the results by Klee (Klee, 1965Go), who postulated that the main degradation route of RNase A by elastase proceeds via Ala19–Ala20, we identified Ser21–Ser22 as an additional primary cleavage site for elastase in RNase A. This result explains why the degradation of the enzyme by elastase is not hindered by the mutation Ala20Pro. In this case the Pro residue is at the P2-position of the substrate, which has no negative influence on the substrate affinity to elastase as concluded from studies on peptide substrates (Thompson and Blout, 1973Go).


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Table III. Data of primary fragments of A20P-RNase A after cleavage with elastase
 

    Conclusions
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 Conclusions
 References
 
The strong stabilization of RNase A toward the unspecific proteases proteinase K and subtilisin Carlsberg as a consequence of the single mutation Ala20Pro demonstrates the large potential of rational strategies of protein engineering for the improvement of protein properties. While the catalytic and conformational properties were not changed by the mutation, the rate of proteolytic degradation was decelerated by two orders of magnitude. The dramatic stabilization effect as observed toward proteinase K and subtilisin Carlsberg is not found in the proteolysis by elastase, which is caused by the preference of another primary cleavage site (Ser21–Ser22 instead of Ala20–Ser21).


    Notes
 
1 Present address: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA Back

2 To whom correspondence should be addressed. E-mail: ulbrich-hofmann{at}biochemtech.uni-halle.de Back


    Acknowledgments
 
The authors wish to thank Professor Dr Ronald T.Raines, Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA, for the gift of the pBXR-plasmid and Professor Dr Gerrit Vriend, Center for Molecular and Biomolecular Informatics, University of Nijmegen, The Netherlands, for molecular modeling. Dr Karl-Peter Rücknagel and Dr Angelika Schierhorn, Max-Planck-Forschungsstelle `Enzymologie der Proteinfaltung', Halle/S., are gratefully acknowledged for N-terminal protein sequencing and MALDI-MS measurements.


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 Abstract
 Introduction
 Material and methods
 Results and discussion
 Conclusions
 References
 
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Received March 28, 2001; revised July 13, 2001; accepted July 16, 2001.