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Clitocypin, a new cysteine proteinase inhibitor, is monomeric: impact on the mechanism of folding
BBRC Biochemical and Biophysical Research Communications 324 (2004) 576–578 www.elsevier.com/locate/ybbrc
Clitocypin, a new cysteine proteinase inhibitor, is monomeric: impact on the mechanism of foldingq Katja Galesˇaa, Richard M. Thomasb, Marjetka Kidricˇa, Roger H. Paina,* a
Department of Biochemistry and Molecular Biology, Jozˇef Stefan Institute, Jamova, 39, 1000 Ljubljana, Slovenia b Institut fu¨r Polymere, ETH-Zentrum, CH-8092 Zu¨rich, Switzerland Received 31 August 2004
Abstract The molecular mass of clitocypin, a new type of cysteine proteinase inhibitor from the mushroom Clitocybe nebularis, has been determined by analytical ultracentrifugation and gel exclusion chromatography. The result is in agreement with the formula mass of 16.8 kDa, demonstrating that the inhibitor is a monomer in aqueous solution. This enables the kinetics of unfolding and refolding to be interpreted in terms of folding in a kinetically two state, highly cooperative transition from the thermally unfolded state. Ó 2004 Published by Elsevier Inc. Keywords: Analytical ultracentrifugation; Clitocypin; Cysteine proteinase inhibitors; Folding; Molecular mass; Monomeric structure; Mushrooms
Clitocypin is a novel cysteine proteinase inhibitor isolated from the fruiting body of the mushroom Clitocybe nebularis [1], and is the first to be characterized from higher fungi. Its sequence of 150 amino acid residues, M = 16.9 kDa, contains no cysteine or methionine. No sequence elements typical of other cysteine proteinase inhibitors are present and it thus appears to be a member of a new class of protein inhibitors. Its circular dichroism (CD) spectrum has unusual features [2] that are similar to those of a group of small b-strand proteins [3–6]. Fourier transform infrared spectroscopy (FTIR) has shown it to possess a high content of b-structure [2]. It was originally reported to possess a homodimeric structure [1], which constrained interpretation of the kinetics of reversible unfolding in terms of an unusual model [2]. We report here a re-examination of the molecular weight in solution, with implications for interq Abbreviations: CD, circular dichroism; FTIR, Fourier transform infrared. * Corresponding author. Fax: +386 1 257 3594. E-mail address: [email protected] (R.H. Pain).
0006-291X/$ - see front matter Ó 2004 Published by Elsevier Inc. doi:10.1016/j.bbrc.2004.09.092
preting the previously reported kinetics of folding and unfolding.
Materials and methods Clitocypin. The isolation and purification of clitocypin from fruit bodies of C. nebularis has been reported [2]. The same sample was used in the present study. Gel exclusion chromatography. Molecular weight was determined ¨ kta Explorer using a Superdex HR 75 column (length 30 cm) and an A analytical system (both from Amersham Pharmacia Biotech). The buffer was 100 mM phosphate, 200 mM NaCl, pH 7.0. The column was calibrated with bovine serum albumin, ovalbumin, chymotrypsinogen A, myoglobin, cytochrome c, and aprotinin (Serva). Analytical ultracentrifugation. Conventional sedimentation equilibrium experiments were conducted using a Beckman XL-A analytical ultracentrifuge equipped with an AN60Ti rotor. Double sector charcoal-filled epon centrepieces were employed and fluorocarbon FC43 was used to provide a false bottom. A buffer of 0.05 M phosphate, pH 6.6, was employed throughout. Radial data were collected at a spacing of 0.001 cm and at least 10 scans were averaged for each data set. Achievement of equilibrium was confirmed by the superposition of data collected at suitable time intervals. The apparent partial specific volume (v20 = 0.726) was calculated from the amino acid composition [7], and solvent density (q20 = 1.0053) was measured with a Paar
K. Galesˇa et al. / Biochemical and Biophysical Research Communications 324 (2004) 576–578 DSA48 density and sound analyser. Equilibrium data were analysed with the program SEDPHAT [8,9].
Results and discussion Calibrated gel exclusion chromatography indicated a molecular mass for clitocypin of 16,400, strongly suggesting that the inhibitor is a monomer in dilute aqueous solution. In order to confirm this and to exclude the possibility of its association to a dimer at higher concentrations, we used analytical ultracentrifugation as an absolute method for determining molecular mass. Results obtained at 24 and 48 h for 0.04 mg cm 3 protein (Mrav = 16,250 ± 1300) were the same, within experimental error, as those acquired at the same times at 0.8 mg cm 3 (Mrav = 15,710 ± 190), confirming both attainment of equilibrium and lack of reversible association over a twentyfold concentration range. Global fitting of all four data sets gave Mr = 15,720 ± 180 (Fig. 1). These results clearly indicate that clitocypin is monomeric in solution over this concentration range.
577
The deviation from the mass calculated from the amino acid composition (16,854) was probably due to small uncertainties in experimental parameters, such as a 1–2% error in the estimation of the apparent partial specific volume. In our previous paper [2] we showed that the central features of the reversible thermal transition to an essentially unfolded state are (i) cooperativity of folding of secondary and tertiary structures, as shown by monitoring tryptophan and peptide ellipticity by UV CD and tyrosine and b-structure by FTIR, (ii) slow kinetics of folding and unfolding, slower than those normally observed for dimerization, and (iii) unfolding and refolding rates that are independent of protein concentration. Taking into account the earlier reported dimeric state of native clitocypin [1], these characteristics led to a model in which folding to monomer had to be the rate limiting step, and the dimerization step spectroscopically silent for both aromatic and secondary structure elements. The experimental demonstration in this paper that clitocypin is a monomer now removes these constraints and the kinetics reveal a protein molecule whose appreciable stability is dependent solely on non-covalent, intra-chain interactions and which folds in a two state, highly cooperative, yet relatively slow manner. The basis of this slow unfolding and refolding remains to be elucidated.
Acknowledgments The financial support of the Ministry of Education, Science and Sport of the Republic of Slovenia is acknowledged. The authors thank Dr. Jozˇe Brzin for providing the purified sample of clitocypin.
References
Fig. 1. A typical sedimentation equilibrium profile for clitocypin at 0.8 mg cm 3 (48 lM) in 0.05 M phosphate, pH 6.6, buffer at 20 °C, after 44 h at 19,000 rpm. The lower panel shows data acquired at 291 nm (s) and a fit to the data (—) derived from the combination of four datasets obtained at concentrations of 0.8 and 0.04 mg cm 3, each after 24 and 44 h equilibration times. The upper panel shows the residuals to the fit.
[1] J. Brzin, B. Rogelj, T. Popovic´, B. Sˇtrukelj, A. Ritonja, Clitocypin, a new type of cysteine proteinase inhibitor from fruit bodies of mushroom Clitocybe nebularis, J. Biol. Chem. 275 (2000) 20104– 20109. [2] M. Kidricˇ, H. Fabian, J. Brzin, T. Popovicˇ, R.H. Pain, Folding, stability, and secondary structure of a new dimeric cysteine proteinase inhibitor, Biochem. Biophys. Res. Commun. 297 (2002) 962–967. [3] E.K. Koepf, H.M. Petrassi, M. Sudol, J.W. Kelly, WW: An isolated three-stranded antiparallel beta-sheet domain that unfolds and refolds reversibly: evidence for a structured hydrophobic cluster in urea and GdnHCl and a disordered thermal unfolded state, Protein Sci. 8 (1999) 841–853. [4] A.R. Viguera, J.L. Arrondo, A. Musacchio, M. Saraste, L. Serrano, Characterization of the interaction of natural proline-rich peptides with five different SH3 domains, Biochemistry 33 (1994) 10925–10933. [5] S. Knapp, P.T. Mattson, P. Christova, K.D. Berndt, A. Karshikoff, M. Vihinen, C.I. Smith, R. Ladenstein, Thermal unfolding of small proteins with SH3 domain folding pattern, Proteins: Struct. Funct. Genet. 31 (1998) 309–319.
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K. Galesˇa et al. / Biochemical and Biophysical Research Communications 324 (2004) 576–578
[6] K.L. Reid, H.M. Rodriguez, B.J. Hillier, L.M. Gregoret, Stability and folding properties of a model beta-sheet protein, Escherichia coli CspA, Protein Sci. 7 (1998) 470–479. [7] T.M. Laue, B.D. Shah, T.M. Ridgeway, S.L. Pelletier, Computeraided interpretation of analytical sedimentation data for proteins, in: S.E. Harding, A.J. Rowe, J.C. Horton (Eds.), Analytical Ultracentrifugation in Biochemistry and Polymer Science, Royal Society of Chemistry, Cambridge, UK, 1992.
[8] P. Schuck, Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling, Biophys. J. 78 (2000) 1606–1609. [9] J. Vistica, J. Dam, J. Balbo, E. Yikilmaz, R.A. Mariuzza, T.A. Rouault, P. Schuck, Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition, Anal. Biochem. 326 (2004) 234–256.
Clitocypin, a new cysteine proteinase inhibitor, is monomeric: impact on the mechanism of foldingq Katja Galesˇaa, Richard M. Thomasb, Marjetka Kidricˇa, Roger H. Paina,* a
Department of Biochemistry and Molecular Biology, Jozˇef Stefan Institute, Jamova, 39, 1000 Ljubljana, Slovenia b Institut fu¨r Polymere, ETH-Zentrum, CH-8092 Zu¨rich, Switzerland Received 31 August 2004
Abstract The molecular mass of clitocypin, a new type of cysteine proteinase inhibitor from the mushroom Clitocybe nebularis, has been determined by analytical ultracentrifugation and gel exclusion chromatography. The result is in agreement with the formula mass of 16.8 kDa, demonstrating that the inhibitor is a monomer in aqueous solution. This enables the kinetics of unfolding and refolding to be interpreted in terms of folding in a kinetically two state, highly cooperative transition from the thermally unfolded state. Ó 2004 Published by Elsevier Inc. Keywords: Analytical ultracentrifugation; Clitocypin; Cysteine proteinase inhibitors; Folding; Molecular mass; Monomeric structure; Mushrooms
Clitocypin is a novel cysteine proteinase inhibitor isolated from the fruiting body of the mushroom Clitocybe nebularis [1], and is the first to be characterized from higher fungi. Its sequence of 150 amino acid residues, M = 16.9 kDa, contains no cysteine or methionine. No sequence elements typical of other cysteine proteinase inhibitors are present and it thus appears to be a member of a new class of protein inhibitors. Its circular dichroism (CD) spectrum has unusual features [2] that are similar to those of a group of small b-strand proteins [3–6]. Fourier transform infrared spectroscopy (FTIR) has shown it to possess a high content of b-structure [2]. It was originally reported to possess a homodimeric structure [1], which constrained interpretation of the kinetics of reversible unfolding in terms of an unusual model [2]. We report here a re-examination of the molecular weight in solution, with implications for interq Abbreviations: CD, circular dichroism; FTIR, Fourier transform infrared. * Corresponding author. Fax: +386 1 257 3594. E-mail address: [email protected] (R.H. Pain).
0006-291X/$ - see front matter Ó 2004 Published by Elsevier Inc. doi:10.1016/j.bbrc.2004.09.092
preting the previously reported kinetics of folding and unfolding.
Materials and methods Clitocypin. The isolation and purification of clitocypin from fruit bodies of C. nebularis has been reported [2]. The same sample was used in the present study. Gel exclusion chromatography. Molecular weight was determined ¨ kta Explorer using a Superdex HR 75 column (length 30 cm) and an A analytical system (both from Amersham Pharmacia Biotech). The buffer was 100 mM phosphate, 200 mM NaCl, pH 7.0. The column was calibrated with bovine serum albumin, ovalbumin, chymotrypsinogen A, myoglobin, cytochrome c, and aprotinin (Serva). Analytical ultracentrifugation. Conventional sedimentation equilibrium experiments were conducted using a Beckman XL-A analytical ultracentrifuge equipped with an AN60Ti rotor. Double sector charcoal-filled epon centrepieces were employed and fluorocarbon FC43 was used to provide a false bottom. A buffer of 0.05 M phosphate, pH 6.6, was employed throughout. Radial data were collected at a spacing of 0.001 cm and at least 10 scans were averaged for each data set. Achievement of equilibrium was confirmed by the superposition of data collected at suitable time intervals. The apparent partial specific volume (v20 = 0.726) was calculated from the amino acid composition [7], and solvent density (q20 = 1.0053) was measured with a Paar
K. Galesˇa et al. / Biochemical and Biophysical Research Communications 324 (2004) 576–578 DSA48 density and sound analyser. Equilibrium data were analysed with the program SEDPHAT [8,9].
Results and discussion Calibrated gel exclusion chromatography indicated a molecular mass for clitocypin of 16,400, strongly suggesting that the inhibitor is a monomer in dilute aqueous solution. In order to confirm this and to exclude the possibility of its association to a dimer at higher concentrations, we used analytical ultracentrifugation as an absolute method for determining molecular mass. Results obtained at 24 and 48 h for 0.04 mg cm 3 protein (Mrav = 16,250 ± 1300) were the same, within experimental error, as those acquired at the same times at 0.8 mg cm 3 (Mrav = 15,710 ± 190), confirming both attainment of equilibrium and lack of reversible association over a twentyfold concentration range. Global fitting of all four data sets gave Mr = 15,720 ± 180 (Fig. 1). These results clearly indicate that clitocypin is monomeric in solution over this concentration range.
577
The deviation from the mass calculated from the amino acid composition (16,854) was probably due to small uncertainties in experimental parameters, such as a 1–2% error in the estimation of the apparent partial specific volume. In our previous paper [2] we showed that the central features of the reversible thermal transition to an essentially unfolded state are (i) cooperativity of folding of secondary and tertiary structures, as shown by monitoring tryptophan and peptide ellipticity by UV CD and tyrosine and b-structure by FTIR, (ii) slow kinetics of folding and unfolding, slower than those normally observed for dimerization, and (iii) unfolding and refolding rates that are independent of protein concentration. Taking into account the earlier reported dimeric state of native clitocypin [1], these characteristics led to a model in which folding to monomer had to be the rate limiting step, and the dimerization step spectroscopically silent for both aromatic and secondary structure elements. The experimental demonstration in this paper that clitocypin is a monomer now removes these constraints and the kinetics reveal a protein molecule whose appreciable stability is dependent solely on non-covalent, intra-chain interactions and which folds in a two state, highly cooperative, yet relatively slow manner. The basis of this slow unfolding and refolding remains to be elucidated.
Acknowledgments The financial support of the Ministry of Education, Science and Sport of the Republic of Slovenia is acknowledged. The authors thank Dr. Jozˇe Brzin for providing the purified sample of clitocypin.
References
Fig. 1. A typical sedimentation equilibrium profile for clitocypin at 0.8 mg cm 3 (48 lM) in 0.05 M phosphate, pH 6.6, buffer at 20 °C, after 44 h at 19,000 rpm. The lower panel shows data acquired at 291 nm (s) and a fit to the data (—) derived from the combination of four datasets obtained at concentrations of 0.8 and 0.04 mg cm 3, each after 24 and 44 h equilibration times. The upper panel shows the residuals to the fit.
[1] J. Brzin, B. Rogelj, T. Popovic´, B. Sˇtrukelj, A. Ritonja, Clitocypin, a new type of cysteine proteinase inhibitor from fruit bodies of mushroom Clitocybe nebularis, J. Biol. Chem. 275 (2000) 20104– 20109. [2] M. Kidricˇ, H. Fabian, J. Brzin, T. Popovicˇ, R.H. Pain, Folding, stability, and secondary structure of a new dimeric cysteine proteinase inhibitor, Biochem. Biophys. Res. Commun. 297 (2002) 962–967. [3] E.K. Koepf, H.M. Petrassi, M. Sudol, J.W. Kelly, WW: An isolated three-stranded antiparallel beta-sheet domain that unfolds and refolds reversibly: evidence for a structured hydrophobic cluster in urea and GdnHCl and a disordered thermal unfolded state, Protein Sci. 8 (1999) 841–853. [4] A.R. Viguera, J.L. Arrondo, A. Musacchio, M. Saraste, L. Serrano, Characterization of the interaction of natural proline-rich peptides with five different SH3 domains, Biochemistry 33 (1994) 10925–10933. [5] S. Knapp, P.T. Mattson, P. Christova, K.D. Berndt, A. Karshikoff, M. Vihinen, C.I. Smith, R. Ladenstein, Thermal unfolding of small proteins with SH3 domain folding pattern, Proteins: Struct. Funct. Genet. 31 (1998) 309–319.
578
K. Galesˇa et al. / Biochemical and Biophysical Research Communications 324 (2004) 576–578
[6] K.L. Reid, H.M. Rodriguez, B.J. Hillier, L.M. Gregoret, Stability and folding properties of a model beta-sheet protein, Escherichia coli CspA, Protein Sci. 7 (1998) 470–479. [7] T.M. Laue, B.D. Shah, T.M. Ridgeway, S.L. Pelletier, Computeraided interpretation of analytical sedimentation data for proteins, in: S.E. Harding, A.J. Rowe, J.C. Horton (Eds.), Analytical Ultracentrifugation in Biochemistry and Polymer Science, Royal Society of Chemistry, Cambridge, UK, 1992.
[8] P. Schuck, Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling, Biophys. J. 78 (2000) 1606–1609. [9] J. Vistica, J. Dam, J. Balbo, E. Yikilmaz, R.A. Mariuzza, T.A. Rouault, P. Schuck, Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition, Anal. Biochem. 326 (2004) 234–256.