Department of Biochemistry/Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle, Germany
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: elastase/proteinase K/ribonuclease A/site-directed mutagenesis/subtilisin Carlsberg
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, 1990).
Site-directed mutagenesis
The plasmid pBXR containing the RNase A gene (delCardayré et al., 1995) 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., 1977
) 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., 1995) 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 TrisHCl 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 TrisHCl 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 Trisacetic 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 TrisHCl buffer, pH 7.8, to 50 mM TrisHCl 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 TrisHCl 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 278nm = 9800 M cm, (Sela and Anfinsen, 1957
).
RNase activity
The activity of RNase A and A20P-RNase A was determined according to Crook et al. (Crook et al., 1960) in 50 mM TrisHCl 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,
= 1450 M cm (delCardayré and Raines, 1995
) 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 TrisHCl buffer, pH 8.0, using a 1 cm cuvette in the near-UV region and in 10 mM TrisHCl, 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 TrisHCl 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., 1989) 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 0.5100 µg/ml of protease. The reaction was started by addition of 15 µl of protease in 50 mM TrisHCl buffer, pH 8.0, containing 10 mM CaCl2, to 135 µl of RNase A or A20P-RNase A in 50 mM TrisHCl 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 SDSPAGE.
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, 1987) 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 SDSPAGE 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., 1996) 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1993) 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., 1986
). Moreover, the introduction of a proline residue is expected to reduce the flexibility of the concerned structural region (Jaenicke, 2000
).
Starting from literature reports which described Ala20Ser21 as the main cleavage site in native RNase A for subtilisin Carlsberg as well as for proteinase K (Richards and Vithayathil, 1959; Rauber et al., 1978
) and Ala19Ala20 for elastase (Klee, 1965
), 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 SDSPAGE 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 2a) characterizing secondary structures and the CD spectra in the near-UV region (Figure 2b
) 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 I
).
|
|
|
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 SDSPAGE. In Figure 4, typical fragment patterns are shown as a function of time of proteolysis. Figure 4ac
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 Ser21Val124. While this fragment is the primary cleavage product of proteinase K (Rauber et al., 1978
) and subtilisin Carlsberg (Richards and Vithayathil, 1959
), it is formed from the primary cleavage product of elastase (Ala20Val124) by the fast subsequent release of Ala20 (Klee, 1965
). 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 e
), whereas the degradation by elastase was similar to that of RNase A (Figure 4f
).
|
|
|
The situation is completely different with elastase. After proteolysis, a large fragment could be accumulated even for A20P-RNase A (Figure 4f). 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: Lys1Ser21 and Ser22Val124 (Table III
), identifying Ser21Ser22 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, 1965
), who postulated that the main degradation route of RNase A by elastase proceeds via Ala19Ala20, we identified Ser21Ser22 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, 1973
).
|
![]() |
Conclusions |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
2 To whom correspondence should be addressed. E-mail: ulbrich-hofmann{at}biochemtech.uni-halle.de
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arnold,U. and Ulbrich-Hofmann,R. (1997) Biochemistry, 36, 21662172.[ISI][Medline]
Arnold,U. and Ulbrich-Hofmann,R. (2001) Eur. J. Biochem., 268, 9397.
Arnold,U., Rücknagel,K.P., Schierhorn,A. and Ulbrich-Hofmann,R. (1996) Eur. J. Biochem., 237, 862869.[Abstract]
Bae,K.H., Jang,J.S., Park,K.S., Lee,S.H. and Byun,S.M. (1995) Biochem. Biophys. Res. Commun., 207, 2024.[ISI][Medline]
Betzel,C., Singh,T.P., Visanji,M., Peters,K., Fittkau,S., Saenger,W. and Wilson,K.S. (1993) J. Biol. Chem., 268, 1585415858.
Brömme,D., Peters,K., Fink,S. and Fittkau,S. (1986) Arch. Biochem. Biophys., 244, 439446.[ISI][Medline]
Carter,P., Nilsson,B., Burnier,J.P., Burdick,D. and Wells,J.A. (1989) Proteins, 6, 240248.[ISI][Medline]
Crook,E.M., Mathias,A.P. and Rabin,B.R. (1960) Biochem. J., 74, 234238.
Daniel,R.M., Cowan,D.A., Morgan,H.W. and Curran,M.P. (1982) Biochem. J., 207, 641644.[ISI][Medline]
delCardayré,S.B. and Raines,R.T. (1995) J. Mol. Biol., 252, 328336.[ISI][Medline]
delCardayré,S.B., Ribo,M., Yokel,E.M., Quirk,D.J., Rutter,W.J. and Raines,R.T. (1995) Protein Eng., 8, 261273.[Abstract]
Frenken,L.G.J., Egmond,M.R., Batenburg,A.M. and Verrips,C.T. (1993) Protein Eng., 6, 637642.[Abstract]
Hubbard,S.J. (1998) Biochim. Biophys. Acta, 1382, 191206.[ISI][Medline]
Jaenicke,R. (2000) J. Biotechnol., 79, 193203.[ISI][Medline]
Klee,W.A. (1965) J. Biol. Chem., 240, 29002906.
Kramer,R.A., Zandwijken,D., Egmond,M.R. and Dekker,N. (2000) Eur. J. Biochem., 267, 885893.
Lu,W., Apostol,I., Qasim,M.A., Warne,N., Wynn,R., Zhang,W.L., Anderson,S., Chiang,Y.W., Ogin,E., Rothberg,I., Ryan,K. and Laskowski,M.,Jr, (1997) J. Mol. Biol., 266, 441461.[ISI][Medline]
McLendon,G. and Radany,E. (1978) J. Biol. Chem., 253, 63356337.[Abstract]
Meldal,M., Svendsen,I., Breddam,K. and Auzanneau,F.I. (1994) Proc. Natl Acad. Sci. USA, 91, 33143318.[Abstract]
Méndez,T.J., Johnson,J.V. and Richardson,D.E. (2000) Anal. Biochem., 279, 114118.[ISI][Medline]
Morihara,K. and Oka,T. (1977) Arch. Biochem. Biophys., 178, 188194.[ISI][Medline]
Neumann,U. and Hofsteenge,J. (1994) Protein Sci., 3, 248256.
Pace,C.N., Shirley,B.A. and Thomson,J.A. (1989) In Creighton,T.E. (ed.), Protein StructureA Practical Approach. IRL Press, Oxford, pp. 311330.
Parsell,D.A. and Sauer,R.T. (1989) J. Biol. Chem., 264, 75907595.
Pfeil,W. (ed.) (1998) Protein Stability and Folding. A Collection of Thermodynamic Data. Springer Verlag, Berlin, pp. 278281.
Price,N.C. and Johnson,C.M. (1990) In Beynon,R.J. and Bond,J.S. (eds), Proteolytic Enzymes. A Practical Approach. IRL Press, Oxford, pp. 163180.
Rauber,N.R.K., Jany,K.D. and Pfleiderer,G. (1978) Z. Naturforsch. [C], 33, 660663.[Medline]
Richards,F.M. and Vithayathil,P.J. (1959) J. Biol. Chem., 234, 14591465.
Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Natl Acad. Sci. USA, 74, 54635467.[Abstract]
Saxena,A.K., Singh,T.P., Peters,K., Fittkau,S. and Betzel,C. (1996) Protein Sci., 5, 24532458.
Schägger,H. and von Jagow,G. (1987) Anal. Biochem., 166, 368379.[ISI][Medline]
Sela,M. and Anfinsen,C.B. (1957) Biochim. Biophys. Acta, 24, 229235.[ISI][Medline]
Thompson,R.C. and Blout,E.R. (1973) Biochemistry, 12, 5157.[ISI][Medline]
Van den Burg,B., Eijsink,V.G.H., Vriend,G., Veltman,O.R. and Venema,G. (1998) Biotechnol. Appl. Biochem., 27, 125132.[ISI][Medline]
Várallyay,É., Pál,G., Patthy,A., Szilágyi,L. and Gráf,L. (1998) Biochem. Biophys. Commun., 243, 5660.[ISI][Medline]
Vriend,G. (1990) J. Mol. Graph., 8, 5256.[ISI][Medline]
Wlodawer,A., Bott,R. and Sjölin,L. (1982) J. Biol. Chem., 257, 13251332.
Received March 28, 2001; revised July 13, 2001; accepted July 16, 2001.