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A novel method to determine thermal transition curves of disulfide-containing proteins and their structured folding intermediates
BBRC Biochemical and Biophysical Research Communications 311 (2003) 514–517 www.elsevier.com/locate/ybbrc
A novel method to determine thermal transition curves of disulfide-containing proteins and their structured folding intermediates Guoqiang Xu, Mahesh Narayan, Ervin Welker, and Harold A. Scheraga* Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853-1301, USA Received 30 September 2003
Abstract The stability of a protein or of its folding intermediates is frequently characterized by its resistance to chemical and/or thermal denaturation. The folding/unfolding process is generally followed by spectroscopic methods such as absorbance, fluorescence, circular dichroism spectroscopy, etc. Here, we demonstrate a new method, by using HPLC, for determining the thermal unfolding transitions of disulfide-containing proteins and their structured folding intermediates. The thermal transitions of a model protein, ribonuclease A (RNase A), and a recently found unfolding intermediate of onconase (ONC), des [30–75], have been estimated by this method. Finally, the advantages of this method over traditional techniques are discussed by providing specific examples. Ó 2003 Elsevier Inc. All rights reserved. Keywords: RNase A; Onconase; Thermal transition curve; Melting point; Disulfide; Folding; Unfolding; Reduction-pulse
Knowledge of the stability of a protein or of its folding intermediates is of fundamental importance in biochemistry and is often obtained by estimating its resistance to chemical and/or thermal denaturation. The environment around a reporter group within the protein, such as a Tyr or Trp residue (or even groups of residues such as those forming an a-helix), is monitored by spectroscopic methods such as absorbance [1,2], fluorescence [3], and circular dichroism spectroscopy [1,2,4], as a function of a chemical denaturing agent such as GdnHCl1 or physical denaturing agents such as pH, temperature or pressure [5–8]. During each step of the procedure, an equilibration between the folded and unfolded forms of the protein is established, and the ratio between the native and unfolded state varies as the denaturation condition is altered. *
Corresponding author. Fax: 1-607-254-4700. E-mail address: [email protected] (H.A. Scheraga). 1 Abbreviations: RNase A, bovine pancreatic ribonuclease A; ONC, onconase; des [x–y], intermediate of ONC having all native disulfide bonds but lacking the (x–y) disulfide bond; AEMTS, 2-amino ethylmethylthiosulfonate; DTTred , reduced dithiothreitol; GdnHCl, guanidine hydrochloride; EDTA, ethylenediaminetetraacetic acid; Hepes, N -2-hydroxyethylpiperizine-N 0 -2-ethanesulfonic acid; HPLC, high performance liquid chromatography; RP, reduction-pulse. 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.10.039
The method introduced here and applied to determine the thermal transition of a four-disulfide-containing model protein, bovine pancreatic ribonuclease A (RNase A), makes use of such an equilibrium. A small amount of reducing agent is used to distinguish between the unfolded species (U) and the folded protein (N). This is possible because the disulfides of a conformationally unfolded protein can easily be reduced by the small amount of reducing agent within a very short time, whereas the folded protein retains its three-dimensional structure with intact and protected disulfides under the same conditions [9]. Then the thiols of the reduced species resulting from reduction of the unfolded fraction of the protein are blocked immediately with an excess amount of 2-amino ethylmethylthiosulfonate (AEMTS) and analyzed on a cation-exchange HPLC column which facilitates the separation of the AEMTS-blocked reduced species from the disulfide-intact protein. The thermal transition curve and the midpoint of the thermal transition (Tm ) are determined by assessing the percentages of the native and reduced proteins from the areas of their respective peaks as a function of increasing temperature. After testing the accuracy of this method using RNase A, whose conformational folding and unfolding have been determined previously using other
G. Xu et al. / Biochemical and Biophysical Research Communications 311 (2003) 514–517
techniques [1,2,4], the method is applied to a recently obtained stable unfolding intermediate (des [30–75]) of ONC (a homologue of RNase A) [10,11]. The thermal transition of this intermediate, missing the (30–75) disulfide bond (i.e., des [30–75]), is especially interesting because it can reflect the contribution of that particular disulfide bond to the stability of the whole protein. The method described here has advantages over traditional techniques, such as UV-spectroscopy and CD, when the sample concentration is very small. Furthermore, in situations in which a mixture of structured intermediates exists (which can be separated from one another by HPLC), this method can be applied to determine the mid-points of their respective thermal transitions simultaneously.
Materials and methods Materials. AEMTS (>99% pure) was purchased from Anatrace and used without further purification. RNase A and DTTred were obtained from Sigma. RNase A was purified by the method described previously [12]. All other chemicals were of the highest grade commercially available. Native ONC was expressed in our laboratory as described previously [10]. Isolation of des [30–75] ONC. The des [30–75] intermediate of ONC was isolated by subjecting 1 mg native ONC to reducing conditions (pH 8, 100 mM DTTred , 15 °C) described previously [10]. The mixture was quenched by glacial acetic acid after 100 min. Removal of buffer salts, DTTred , and trace amounts of native ONC was accomplished by reversed-phase HPLC. Water and organic solvents were removed by lyophilization. Determination of thermal transition curves of RNase A and des [30– 75] ONC. RNase A (0.2 mg) and des [30–75] ONC (0.1 mg) were each incubated separately in 200 mL of a 10 mM Hepes buffer at pH 7 along with 0.5 mM EDTA. After a 10-min incubation at the desired temperature, a reduction-pulse (RP) using a small amount of DTTred (5 mM final concentration, pH 8) was applied for 2 min at the same temperature as that of the denaturing reaction. AEMTS was then added to the sample to block any free thiols, followed by HPLC separation (see Scheme 1). The percentages of the folded and unfolded proteins were calculated from the areas of the corresponding peaks in the chromatograms. The thermal transition curves and their midpoints were obtained from those data.
515
Fig. 1. Typical chromatogram of wild-type RNase A, which was incubated at 60 °C, and pH 7, for 10 min, and then subjected to a 2-min reduction-pulse, followed by blocking with AEMTS.
species shifts slightly to the unfolded state since the ensemble of molecules of the disulfide-intact conformationally folded protein shifts somewhat from N to U due to the rapid removal of U by reduction. Therefore, the concentration of the remaining native protein is slightly less than it should be at the same temperature in the equilibrium. Fig. 2 is a plot of the percentage of native RNase A as a function of increasing temperature. The curve is the fitted result using a sigmoidal function, from which both the midpoint of the thermal transition, Tm , as well as the standard deviation of the experimental data from the mathematical function, can easily be obtained. Since the equilibrium shifted slightly from N to U due to the reduction-pulse that was carried out at the same temperature as the denaturing temperature, the resulting midpoint of the thermal transition curve was slightly underestimated. The value obtained here for RNase A is 3–5 °C lower than in other types of experiments, such as differential scanning calorimetry (DSC) and spectroscopic measurements at similar pH values [1–4,13–16].
Results Fig. 1 shows the HPLC chromatogram for wild-type RNase A incubated at 60 °C and subjected to a reduction-pulse at that temperature. In the chromatogram, two species are evident, native (N) and fully reduced proteins (R). It should be noted that, in this method, after reduction of the conformationally unfolded protein, the equilibrium between the folded and unfolded
Scheme 1.
Fig. 2. Percentage of native RNase A at different temperatures at pH 7. The solid curve is the fit of a sigmoidal function to the experimental data.
516
G. Xu et al. / Biochemical and Biophysical Research Communications 311 (2003) 514–517
This method was then applied to a recently found stable intermediate from the reductive unfolding of ONC [10,11]. The thermal transition curve of des [30– 75] ONC and the Tm of this structured intermediate are shown in Fig. 3 and Table 1, together with the Tm of RNase A from this experiment and from the literature. Although two free cysteines are present in this ONC intermediate, the reshuffling is very limited under conditions in which the folded structure is stable. Moreover, the experiments were carried out at pH 7, where the reshuffling rate is lower than that at higher pH. The small amount of EDTA used in the experiments reduces the rate of metal-catalyzed air oxidation of free cysteines. The prevalence of such possible oxidation is of special concern, especially in the measurements of the thermal transition of thiol-containing intermediates, as the equilibrium temperature is increased.
would have been replaced by alanines. The Tm obtained with this new method was underestimated since the equilibrium shifted from the conformationally folded and unfolded species slightly during the reducing reaction, as shown by the use of RNase A as a test case. Diminishing the concentration of reducing agent in the reduction-pulse and decreasing the reduction time had little effect on the determination of the melting point for RNase A indicating that the method does not depend critically on the exact conditions of the reduction procedure. It should be mentioned that this method will become more accurate for proteins with slow conformational folding and/or unfolding rates since the principle used here is to block the equilibrated sample in the conformationally unfolded state by using a reduction-pulse. Ideally, if the rate of the conformationally unfolding reaction is larger than the rate of reduction (but much shorter than the equilibration time), this method is very accurate. The method would not work with proteins with a relatively large surfaceexposed disulfide that is reducible or becomes reducible at a much lower temperature than the global unfolding of the protein. This method is different from that of disulfide scrambling used to determine the chemical denaturation curves for tick anticoagulant peptide, a-lactalbumin, and leech carboxypeptidase inhibitor [17–19]. That method explores a delicate balance between the concentrations of the protein disulfide and the applied reducing reagent. Controlling the air-oxidation can be difficult at higher temperature during the thermal transition and might result in the oxidation of the reducing agent, which would translate into an error in the value of Tm . Besides, our method is much more accurate when HPLC cannot separate the native protein from the scrambled species. One major advantage of our method is that the reduced and native proteins can frequently be separated using sodium dodecyl sulfate gel electrophoresis followed by immunochemical detection, which reduces the required amount of protein (to the nanogram range) [20,21]. Furthermore, our method can be applied to determine the thermal transitions of a mixture of structured intermediates that may arise during oxidation regeneration, as long as they can be separated from one another, for example, by HPLC. This method can be applied to obtain the Tm ’s of structured intermediates of different proteins without resorting to cysteine mutations.
Discussion
Acknowledgments
This is the first time that the melting point of a structured disulfide-containing folding/unfolding intermediate of a protein has been estimated directly without using its mutant homologues in which cysteines
We thank Laura Hathaway for help during the expression of ONC. This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health (Grant No. GM-24893). Support was also received from the National Foundation for Cancer Research.
Fig. 3. Percentage of des [30–75] ONC at different temperatures at pH 7. The solid curve is the fit of a sigmoidal function to the experimental data. Table 1 Midpoints of the thermal transition curves of RNase A and the des [30–75] intermediate of ONC at pH 7 obtained from the fitted curves in Figs. 2 and 3 Protein and intermediate
Tm (°C)
Des [30–75] ONC RNase A Literature values for RNase Aa
56.7 0.3 58.0 0.1 61.3, 61.8
The standard deviations were obtained by fitting the experimental data with a sigmoidal function. a The literature Tm ’s of RNase A came from [13,14], which were obtained from DSC measurements at pH 7.
G. Xu et al. / Biochemical and Biophysical Research Communications 311 (2003) 514–517
References [1] J. Hermans Jr., H.A. Scheraga, Structural studies of ribonuclease. V. Reversible change of configuration, J. Am. Chem. Soc. 83 (1961) 3283–3292. [2] U. Arnold, K.P. Ruecknagel, A. Schierhorn, R. Ulbrich-Hofmann, Thermal unfolding and proteolytic susceptibility of ribonuclease A, Eur. J. Biochem. 237 (1996) 862–869. [3] R.A. Sendak, D.M. Rothwarf, W.J. Wedemeyer, W.A. Houry, H.A. Scheraga, Kinetic and thermodynamic studies of the folding/ unfolding of a tryptophan-containing mutant of ribonuclease A, Biochemistry 35 (1996) 12978–12992. [4] S.D. Stelea, P. Pancoska, A.S. Benight, T.A. Keiderling, Thermal unfolding of ribonuclease A in phosphate at neutral pH: deviations from the two-state model, Protein Sci. 10 (2001) 970–978. [5] S. Lapanje, Physicochemical Aspects of Protein Denaturation, John Wiley & Sons, New York, 1978. [6] J. Zhang, X. Peng, A. Jonas, J. Jonas, NMR study of the cold, heat, and pressure unfolding of ribonuclease A, Biochemistry 34 (1995) 8631–8641. [7] W.A. Houry, H.A. Scheraga, Nature of the unfolded state of ribonuclease A: effect of cis–trans X–Pro peptide bond isomerization, Biochemistry 35 (1996) 11719–11733. [8] D. Juminaga, W.J. Wedemeyer, R. Gardu~ no-J uarez, M.A. McDonald, H.A. Scheraga, Tyrosyl interactions in the folding and unfolding of bovine pancreatic ribonuclease A: A study of tyrosine-to-phenylalanine mutants, Biochemistry 36 (1997) 10131– 10145. [9] E. Welker, M. Narayan, W.J. Wedemeyer, H.A. Scheraga, Structural determinants of oxidative folding in proteins, Proc. Natl. Acad. Sci. USA 98 (2001) 2312–2316. [10] M. Narayan, G. Xu, D.R. Ripoll, H. Zhai, K. Breuker, C. Wanjalla, H.J. Leung, A. Navon, E. Welker, F.W. McLafferty, H.A. Scheraga, Dissimilarity in the reductive unfolding pathways of two ribonuclease variants, J. Mol. Biol. (to be submitted).
517
[11] G. Xu, M. Narayan, E. Welker, H.A. Scheraga, Characterization of the fast-forming intermediate, des [30–75], in the reductive unfolding of onconase, Biochemistry (to be submitted). [12] D.M. Rothwarf, H.A. Scheraga, Regeneration of bovine pancreatic ribonuclease A. 1. Steady-state distribution, Biochemistry 32 (1993) 2671–2679. [13] T.Y. Tsong, R.P. Hearn, D.P. Wrathall, J.M. Sturtevant, Calorimetric study of thermally induced conformational transitions of ribonuclease A and certain of its derivatives, Biochemistry 9 (1970) 2666–2677. [14] C.N. Pace, G.R. Grimsley, S.T. Thomas, G.I. Makhatadze, Heat capacity change for ribonuclease A folding, Protein Sci. 8 (1999) 1500–1504. [15] T.A. Klink, R.T. Raines, Conformational stability is a determinant of ribonuclease A cytotoxicity, J. Biol. Chem. 275 (2000) 17463–17467. [16] T.A. Klink, K.J. Woycechowsky, K.M. Taylor, R.T. Raines, Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A, Eur. J. Biochem. 267 (2000) 566–572. [17] J.Y. Chang, Denatured states of tick anticoagulant peptide. Compositional analysis of unfolded scrambled isomers, J. Biol. Chem. 274 (1999) 123–128. [18] J.Y. Chang, L. Li, The structure of denatured a-lactalbumin elucidated by the technique of disulfide scrambling: fractionation of conformational isomers of a-lactalbumin, J. Biol. Chem. 276 (2001) 9705–9712. [19] S. Salamanca, V. Villegas, J. Vendrell, L. Li, F.X. Aviles, J.Y. Chang, The unfolding pathway of leech carboxypeptidase inhibitor, J. Biol. Chem. 277 (2002) 17538–17543. [20] L.M. Herrmann, B. Caughey, The importance of the disulfide bond in prion protein conversion, Neuroreport 9 (1998) 2457– 2461. [21] E. Welker, L.D. Raymond, H.A. Scheraga, B. Caughey, Intramolecular versus intermolecular disulfide bonds in prion proteins, J. Biol. Chem. 277 (2002) 33477–33481.
A novel method to determine thermal transition curves of disulfide-containing proteins and their structured folding intermediates Guoqiang Xu, Mahesh Narayan, Ervin Welker, and Harold A. Scheraga* Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853-1301, USA Received 30 September 2003
Abstract The stability of a protein or of its folding intermediates is frequently characterized by its resistance to chemical and/or thermal denaturation. The folding/unfolding process is generally followed by spectroscopic methods such as absorbance, fluorescence, circular dichroism spectroscopy, etc. Here, we demonstrate a new method, by using HPLC, for determining the thermal unfolding transitions of disulfide-containing proteins and their structured folding intermediates. The thermal transitions of a model protein, ribonuclease A (RNase A), and a recently found unfolding intermediate of onconase (ONC), des [30–75], have been estimated by this method. Finally, the advantages of this method over traditional techniques are discussed by providing specific examples. Ó 2003 Elsevier Inc. All rights reserved. Keywords: RNase A; Onconase; Thermal transition curve; Melting point; Disulfide; Folding; Unfolding; Reduction-pulse
Knowledge of the stability of a protein or of its folding intermediates is of fundamental importance in biochemistry and is often obtained by estimating its resistance to chemical and/or thermal denaturation. The environment around a reporter group within the protein, such as a Tyr or Trp residue (or even groups of residues such as those forming an a-helix), is monitored by spectroscopic methods such as absorbance [1,2], fluorescence [3], and circular dichroism spectroscopy [1,2,4], as a function of a chemical denaturing agent such as GdnHCl1 or physical denaturing agents such as pH, temperature or pressure [5–8]. During each step of the procedure, an equilibration between the folded and unfolded forms of the protein is established, and the ratio between the native and unfolded state varies as the denaturation condition is altered. *
Corresponding author. Fax: 1-607-254-4700. E-mail address: [email protected] (H.A. Scheraga). 1 Abbreviations: RNase A, bovine pancreatic ribonuclease A; ONC, onconase; des [x–y], intermediate of ONC having all native disulfide bonds but lacking the (x–y) disulfide bond; AEMTS, 2-amino ethylmethylthiosulfonate; DTTred , reduced dithiothreitol; GdnHCl, guanidine hydrochloride; EDTA, ethylenediaminetetraacetic acid; Hepes, N -2-hydroxyethylpiperizine-N 0 -2-ethanesulfonic acid; HPLC, high performance liquid chromatography; RP, reduction-pulse. 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.10.039
The method introduced here and applied to determine the thermal transition of a four-disulfide-containing model protein, bovine pancreatic ribonuclease A (RNase A), makes use of such an equilibrium. A small amount of reducing agent is used to distinguish between the unfolded species (U) and the folded protein (N). This is possible because the disulfides of a conformationally unfolded protein can easily be reduced by the small amount of reducing agent within a very short time, whereas the folded protein retains its three-dimensional structure with intact and protected disulfides under the same conditions [9]. Then the thiols of the reduced species resulting from reduction of the unfolded fraction of the protein are blocked immediately with an excess amount of 2-amino ethylmethylthiosulfonate (AEMTS) and analyzed on a cation-exchange HPLC column which facilitates the separation of the AEMTS-blocked reduced species from the disulfide-intact protein. The thermal transition curve and the midpoint of the thermal transition (Tm ) are determined by assessing the percentages of the native and reduced proteins from the areas of their respective peaks as a function of increasing temperature. After testing the accuracy of this method using RNase A, whose conformational folding and unfolding have been determined previously using other
G. Xu et al. / Biochemical and Biophysical Research Communications 311 (2003) 514–517
techniques [1,2,4], the method is applied to a recently obtained stable unfolding intermediate (des [30–75]) of ONC (a homologue of RNase A) [10,11]. The thermal transition of this intermediate, missing the (30–75) disulfide bond (i.e., des [30–75]), is especially interesting because it can reflect the contribution of that particular disulfide bond to the stability of the whole protein. The method described here has advantages over traditional techniques, such as UV-spectroscopy and CD, when the sample concentration is very small. Furthermore, in situations in which a mixture of structured intermediates exists (which can be separated from one another by HPLC), this method can be applied to determine the mid-points of their respective thermal transitions simultaneously.
Materials and methods Materials. AEMTS (>99% pure) was purchased from Anatrace and used without further purification. RNase A and DTTred were obtained from Sigma. RNase A was purified by the method described previously [12]. All other chemicals were of the highest grade commercially available. Native ONC was expressed in our laboratory as described previously [10]. Isolation of des [30–75] ONC. The des [30–75] intermediate of ONC was isolated by subjecting 1 mg native ONC to reducing conditions (pH 8, 100 mM DTTred , 15 °C) described previously [10]. The mixture was quenched by glacial acetic acid after 100 min. Removal of buffer salts, DTTred , and trace amounts of native ONC was accomplished by reversed-phase HPLC. Water and organic solvents were removed by lyophilization. Determination of thermal transition curves of RNase A and des [30– 75] ONC. RNase A (0.2 mg) and des [30–75] ONC (0.1 mg) were each incubated separately in 200 mL of a 10 mM Hepes buffer at pH 7 along with 0.5 mM EDTA. After a 10-min incubation at the desired temperature, a reduction-pulse (RP) using a small amount of DTTred (5 mM final concentration, pH 8) was applied for 2 min at the same temperature as that of the denaturing reaction. AEMTS was then added to the sample to block any free thiols, followed by HPLC separation (see Scheme 1). The percentages of the folded and unfolded proteins were calculated from the areas of the corresponding peaks in the chromatograms. The thermal transition curves and their midpoints were obtained from those data.
515
Fig. 1. Typical chromatogram of wild-type RNase A, which was incubated at 60 °C, and pH 7, for 10 min, and then subjected to a 2-min reduction-pulse, followed by blocking with AEMTS.
species shifts slightly to the unfolded state since the ensemble of molecules of the disulfide-intact conformationally folded protein shifts somewhat from N to U due to the rapid removal of U by reduction. Therefore, the concentration of the remaining native protein is slightly less than it should be at the same temperature in the equilibrium. Fig. 2 is a plot of the percentage of native RNase A as a function of increasing temperature. The curve is the fitted result using a sigmoidal function, from which both the midpoint of the thermal transition, Tm , as well as the standard deviation of the experimental data from the mathematical function, can easily be obtained. Since the equilibrium shifted slightly from N to U due to the reduction-pulse that was carried out at the same temperature as the denaturing temperature, the resulting midpoint of the thermal transition curve was slightly underestimated. The value obtained here for RNase A is 3–5 °C lower than in other types of experiments, such as differential scanning calorimetry (DSC) and spectroscopic measurements at similar pH values [1–4,13–16].
Results Fig. 1 shows the HPLC chromatogram for wild-type RNase A incubated at 60 °C and subjected to a reduction-pulse at that temperature. In the chromatogram, two species are evident, native (N) and fully reduced proteins (R). It should be noted that, in this method, after reduction of the conformationally unfolded protein, the equilibrium between the folded and unfolded
Scheme 1.
Fig. 2. Percentage of native RNase A at different temperatures at pH 7. The solid curve is the fit of a sigmoidal function to the experimental data.
516
G. Xu et al. / Biochemical and Biophysical Research Communications 311 (2003) 514–517
This method was then applied to a recently found stable intermediate from the reductive unfolding of ONC [10,11]. The thermal transition curve of des [30– 75] ONC and the Tm of this structured intermediate are shown in Fig. 3 and Table 1, together with the Tm of RNase A from this experiment and from the literature. Although two free cysteines are present in this ONC intermediate, the reshuffling is very limited under conditions in which the folded structure is stable. Moreover, the experiments were carried out at pH 7, where the reshuffling rate is lower than that at higher pH. The small amount of EDTA used in the experiments reduces the rate of metal-catalyzed air oxidation of free cysteines. The prevalence of such possible oxidation is of special concern, especially in the measurements of the thermal transition of thiol-containing intermediates, as the equilibrium temperature is increased.
would have been replaced by alanines. The Tm obtained with this new method was underestimated since the equilibrium shifted from the conformationally folded and unfolded species slightly during the reducing reaction, as shown by the use of RNase A as a test case. Diminishing the concentration of reducing agent in the reduction-pulse and decreasing the reduction time had little effect on the determination of the melting point for RNase A indicating that the method does not depend critically on the exact conditions of the reduction procedure. It should be mentioned that this method will become more accurate for proteins with slow conformational folding and/or unfolding rates since the principle used here is to block the equilibrated sample in the conformationally unfolded state by using a reduction-pulse. Ideally, if the rate of the conformationally unfolding reaction is larger than the rate of reduction (but much shorter than the equilibration time), this method is very accurate. The method would not work with proteins with a relatively large surfaceexposed disulfide that is reducible or becomes reducible at a much lower temperature than the global unfolding of the protein. This method is different from that of disulfide scrambling used to determine the chemical denaturation curves for tick anticoagulant peptide, a-lactalbumin, and leech carboxypeptidase inhibitor [17–19]. That method explores a delicate balance between the concentrations of the protein disulfide and the applied reducing reagent. Controlling the air-oxidation can be difficult at higher temperature during the thermal transition and might result in the oxidation of the reducing agent, which would translate into an error in the value of Tm . Besides, our method is much more accurate when HPLC cannot separate the native protein from the scrambled species. One major advantage of our method is that the reduced and native proteins can frequently be separated using sodium dodecyl sulfate gel electrophoresis followed by immunochemical detection, which reduces the required amount of protein (to the nanogram range) [20,21]. Furthermore, our method can be applied to determine the thermal transitions of a mixture of structured intermediates that may arise during oxidation regeneration, as long as they can be separated from one another, for example, by HPLC. This method can be applied to obtain the Tm ’s of structured intermediates of different proteins without resorting to cysteine mutations.
Discussion
Acknowledgments
This is the first time that the melting point of a structured disulfide-containing folding/unfolding intermediate of a protein has been estimated directly without using its mutant homologues in which cysteines
We thank Laura Hathaway for help during the expression of ONC. This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health (Grant No. GM-24893). Support was also received from the National Foundation for Cancer Research.
Fig. 3. Percentage of des [30–75] ONC at different temperatures at pH 7. The solid curve is the fit of a sigmoidal function to the experimental data. Table 1 Midpoints of the thermal transition curves of RNase A and the des [30–75] intermediate of ONC at pH 7 obtained from the fitted curves in Figs. 2 and 3 Protein and intermediate
Tm (°C)
Des [30–75] ONC RNase A Literature values for RNase Aa
56.7 0.3 58.0 0.1 61.3, 61.8
The standard deviations were obtained by fitting the experimental data with a sigmoidal function. a The literature Tm ’s of RNase A came from [13,14], which were obtained from DSC measurements at pH 7.
G. Xu et al. / Biochemical and Biophysical Research Communications 311 (2003) 514–517
References [1] J. Hermans Jr., H.A. Scheraga, Structural studies of ribonuclease. V. Reversible change of configuration, J. Am. Chem. Soc. 83 (1961) 3283–3292. [2] U. Arnold, K.P. Ruecknagel, A. Schierhorn, R. Ulbrich-Hofmann, Thermal unfolding and proteolytic susceptibility of ribonuclease A, Eur. J. Biochem. 237 (1996) 862–869. [3] R.A. Sendak, D.M. Rothwarf, W.J. Wedemeyer, W.A. Houry, H.A. Scheraga, Kinetic and thermodynamic studies of the folding/ unfolding of a tryptophan-containing mutant of ribonuclease A, Biochemistry 35 (1996) 12978–12992. [4] S.D. Stelea, P. Pancoska, A.S. Benight, T.A. Keiderling, Thermal unfolding of ribonuclease A in phosphate at neutral pH: deviations from the two-state model, Protein Sci. 10 (2001) 970–978. [5] S. Lapanje, Physicochemical Aspects of Protein Denaturation, John Wiley & Sons, New York, 1978. [6] J. Zhang, X. Peng, A. Jonas, J. Jonas, NMR study of the cold, heat, and pressure unfolding of ribonuclease A, Biochemistry 34 (1995) 8631–8641. [7] W.A. Houry, H.A. Scheraga, Nature of the unfolded state of ribonuclease A: effect of cis–trans X–Pro peptide bond isomerization, Biochemistry 35 (1996) 11719–11733. [8] D. Juminaga, W.J. Wedemeyer, R. Gardu~ no-J uarez, M.A. McDonald, H.A. Scheraga, Tyrosyl interactions in the folding and unfolding of bovine pancreatic ribonuclease A: A study of tyrosine-to-phenylalanine mutants, Biochemistry 36 (1997) 10131– 10145. [9] E. Welker, M. Narayan, W.J. Wedemeyer, H.A. Scheraga, Structural determinants of oxidative folding in proteins, Proc. Natl. Acad. Sci. USA 98 (2001) 2312–2316. [10] M. Narayan, G. Xu, D.R. Ripoll, H. Zhai, K. Breuker, C. Wanjalla, H.J. Leung, A. Navon, E. Welker, F.W. McLafferty, H.A. Scheraga, Dissimilarity in the reductive unfolding pathways of two ribonuclease variants, J. Mol. Biol. (to be submitted).
517
[11] G. Xu, M. Narayan, E. Welker, H.A. Scheraga, Characterization of the fast-forming intermediate, des [30–75], in the reductive unfolding of onconase, Biochemistry (to be submitted). [12] D.M. Rothwarf, H.A. Scheraga, Regeneration of bovine pancreatic ribonuclease A. 1. Steady-state distribution, Biochemistry 32 (1993) 2671–2679. [13] T.Y. Tsong, R.P. Hearn, D.P. Wrathall, J.M. Sturtevant, Calorimetric study of thermally induced conformational transitions of ribonuclease A and certain of its derivatives, Biochemistry 9 (1970) 2666–2677. [14] C.N. Pace, G.R. Grimsley, S.T. Thomas, G.I. Makhatadze, Heat capacity change for ribonuclease A folding, Protein Sci. 8 (1999) 1500–1504. [15] T.A. Klink, R.T. Raines, Conformational stability is a determinant of ribonuclease A cytotoxicity, J. Biol. Chem. 275 (2000) 17463–17467. [16] T.A. Klink, K.J. Woycechowsky, K.M. Taylor, R.T. Raines, Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A, Eur. J. Biochem. 267 (2000) 566–572. [17] J.Y. Chang, Denatured states of tick anticoagulant peptide. Compositional analysis of unfolded scrambled isomers, J. Biol. Chem. 274 (1999) 123–128. [18] J.Y. Chang, L. Li, The structure of denatured a-lactalbumin elucidated by the technique of disulfide scrambling: fractionation of conformational isomers of a-lactalbumin, J. Biol. Chem. 276 (2001) 9705–9712. [19] S. Salamanca, V. Villegas, J. Vendrell, L. Li, F.X. Aviles, J.Y. Chang, The unfolding pathway of leech carboxypeptidase inhibitor, J. Biol. Chem. 277 (2002) 17538–17543. [20] L.M. Herrmann, B. Caughey, The importance of the disulfide bond in prion protein conversion, Neuroreport 9 (1998) 2457– 2461. [21] E. Welker, L.D. Raymond, H.A. Scheraga, B. Caughey, Intramolecular versus intermolecular disulfide bonds in prion proteins, J. Biol. Chem. 277 (2002) 33477–33481.