Abstract
The effects of the strong stabilizing anion, phosphate, on the oxidative folding of bovine pancreatic ribonuclease A were examined. Phosphate was found to catalyze several steps involved in the oxidative folding process at pH 8.0 and 25°C, resulting in an increase in the rate of pre-equilibration of unstructured species on the folding pathway. In the presence of 400 mM phosphate, the overall increase in the rate of regeneration of native protein was caused primarily by the increased formation and stabilization of tertiary structure in the nativelike intermediates, des-[40-95] and des-[65-72], involved in the rate-determining step. Based on the regeneration of native protein and the stability of Cys→ Ala substituted mutant analogs of the des-species, (C40A, C95A) and (C65A, C72A), it is suggested that the primary role of phosphate is to catalyze the overall regeneration of native protein through nonspecific electrostatic and hydrogen-bonding effects on the protein and solvent.
Similar content being viewed by others
REFERENCES
Ahmad, F. and Bigelow, C. C. (1986). J. Prot. Chem. 5, 355-367.
Baldwin, R. L. (1996). Biophys. J. 71, 2056-2063.
Bruice, T. W. and Kenyon, G. L. (1982). J. Protein Chem. 1, 47-58.
Collins, K. D. and Washabaugh, M. W. (1985). Q. Rev. Biophys. 18, 323-422.
Creighton, T. E. (1984). Methods Enzymol. 107, 305-329.
Freedman, R. B., Hirst, T. R., and Tuite, M. F. (1994). Trends Biochem. Sci. 19, 331-336.
Gilbert, H. F. (1997). J. Biol. Chem. 272, 29399-29402.
Ginsburg, A. and Carroll, W. R. (1965). Biochemistry 4, 2159-2174.
Hantgan, R. R., Hammes, G. G., and Scheraga, H. A. (1974). Biochemistry 13, 3421-3431.
Hermans, J., Jr. and Scheraga, H. A. (1961). J. Am. Chem. Soc. 83, 3283-3292.
Iwaoka, M., Juminaga, D., and Scheraga, H. A. (1998). Biochemistry 37, 4490-4501.
Iwaoka, M., Wedemeyer, W. J., and Scheraga, H. A. (1999). Biochemistry 38, 2805-2815.
Jelesarov, I., Dürr, E., Thomas, R. M., and Bosshard, H. R. (1998). Biochemistry 37, 7539-7550.
Konishi, Y., Ooi, T., and Scheraga, H. A. (1982). Biochemistry 21, 4734-4740.
Laity, J. H., Lester, C. C., Shimotakahara, S., Zimmerman, D. E., Montelione, G. T., and Scheraga, H. A. (1997). Biochemistry 36, 12683-12699.
Low, L. K., Shin H.-C., Narayan, M., Wedemeyer, W. J., and Scheraga, H. A. (2000). FEBS Lett. 472, 67-72.
Lyles, M. M. and Gilbert, H. F. (1991). Biochemistry 30, 613-619.
Narayan, M., Welker, E., Wedemeyer, W. J., and Scheraga. H. A. (2000). Acc. Chem. Res. 33, 805-812.
Prajapati, S., Bhakuni, V., Babu, K. R., and Jain, S. K. (1998). Eur. J. Biochem. 255, 178-184.
Rothwarf, D. M. and Scheraga, H. A. (1993a). Biochemistry 32, 2671-2679.
Rothwarf, D. M. and Scheraga, H. A. (1993b). Biochemistry 32, 2680-2689.
Rothwarf, D. M., Li, Y.-J., and Scheraga, H. A. (1998a). Biochemistry 37, 3760-3766.
Rothwarf, D. M., Li, Y.-J., and Scheraga, H. A. (1998b). Biochemistry 37, 3767-3776.
Schaffer, S. W., Ahmed, A. K., and Wetlaufer, D. B. (1975). J. Biol. Chem. 250, 8483-8486.
Shimotakahara, S., Rios, C. B., Laity, J. H., Zimmerman, D. E., Scheraga, H. A., and Montelione, G. T. (1997). Biochemistry 36, 6915-6929.
Shin H.-C. and Scheraga, H. A. (1999). FEBS Lett. 456, 143-145.
Shin H.-C. and Scheraga, H. A. (2000). J. Mol. Biol. 300, 995-1003.
von Hippel, P. H., and Wong, K.-Y. (1965). J. Biol. Chem. 240, 3909-3923.
Weissman, J. S. and Kim, P. S. (1991). Science 253, 1386-1393.
Welker, E., Narayan, M., Volles, M. J., and Scheraga, H. A. (1999). FEBS Lett. 460, 477-479.
Welker, E., Narayan, M., Wedemeyer, W. J., and Scheraga, H. A. (2001). Proc. Natl. Acad. Sci., U.S.A., 98, 2312-2316.
Xu, X. and Scheraga, H. A. (1998). Biochemistry 37, 7561-7571.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Low, L.K., Shin, HC. & Scheraga, H.A. Oxidative Folding of Bovine Pancreatic Ribonuclease A: Insight into the Overall Catalysis of the Refolding Pathway by Phosphate. J Protein Chem 21, 19–27 (2002). https://doi.org/10.1023/A:1014174930972
Published:
Issue Date:
DOI: https://doi.org/10.1023/A:1014174930972