Abstract
The results presented here indicate that there are two slowly exchanging conformational isomers in unfolded bovine pancreatic ribonuclease A (RNase A) in the vicinity of Lys-41. The conformational heterogeneity is not observed in the fully folded protein. Therefore, one of the isomers may correspond to one of the slow-folding forms of the protein observed when refolding is initiated. These results were obtained from a chemically modified form of the protein, CL(7–41) RNase A, that has a dinitrophenyl cross-link between the ɛ-amino groups of Lys-7 and Lys-41. Extensive physical studies have shown that the cross-link does not significantly perturb the structure or the folding pathways of the protein. Therefore, the results obtained from this modified form of the protein are relevant to intact RNase A. The one-dimensional (1D) NMR spectrum of heat-unfolded CL(7–41) RNase A reveals that the singlet resonance for the C3H ring proton of the dinitrophenyl cross-link has been split into two unequal peaks in a 3:1 ratio, indicating that there are two distinct environments for the dinitrophenyl group. Variations in temperature, and the addition of urea, do not affect the relative peak intensities. The two peaks collapse into one after the protein is refolded. The observed splitting must originate from a slow reversible isomerization (>100 msec) in a neighboring bond. The two most likely candidates are either thecis/trans isomerization of the Lys-41-Pro-42 peptide bond or hindered rotation about the disulfide bond between Cys-40 and Cys-95.
Similar content being viewed by others
References
Adler, M., and Scheraga, H. A. (1988).Biochem. 27, 2471–2480.
Adler, M., and Scheraga, H. A. (1990).Biochem. 29, 8211–8216.
Chazin, W. J., Kordel, J., Drakenberg, T., Thulin, E., Brodin, P., Grundstrom, T., and Forsen, S. (1989).Proc. Natl. Acad. Sci. USA 86, 2195–2198.
Cook, K. H., Schmid, F. X., and Baldwin, R. L. (1979).Proc. Natl. Acad. Sci. USA 76, 6157–6161.
Evans, P. A., Dobson, C. M., Kautz, R. A., Hatfull, G., and Fox, R. O. (1987).Nature 329, 266–268.
Evans, P. A., Kautz, R. A., Fox, R. O., and Dobson, C. M. (1989).Biochem. 28, 362–370.
Ferrige, A. G., and Lindon, J. C. (1978).J. Mag. Reson. 31, 337–340.
Fox, R. O., Evans, P. A., and Dobson, C. M. (1986).Nature 320, 192–194.
Fraser, R. R., Boussard, G., Saunders, J. K., Lambert, J. B., and Mixan, C. E. (1971).J. Am. Chem. Soc. 93, 3822–3823.
Garel, J.-R., and Baldwin, R. L. (1973).Proc. Natl. Acad. Sci. USA 70, 3347–3351.
Garel, J.-R., Nall, B. T., and Baldwin, R. L. (1976).Proc. Natl. Acad. Sci. USA 73, 1853–1857.
Hagerman, P. J., Schmid, F. X., and Baldwin, R. L. (1979).Biochem. 18, 293–297.
Kessler, H., and Rundel, W. (1968).Chem. Ber. 101, 3350–3357.
Kim, P. S., and Baldwin, R. L. (1984).Nature 307, 329–334.
Lin, L.-N., and Brandts, J. F. (1983a).Biochem. 22, 564–573.
Lin, L.-N., and Brandts, J. F. (1983b).Biochem. 22, 559–563.
Lin, L.-N., and Brandts, J. F. (1984).Biochem. 23, 5713–5723.
Lin, L.-N., and Brandts, J. F. (1987a).Biochem. 26, 1826–1830.
Lin, L.-N., and Brandts, J. F. (1987b).Biochem. 26, 3537–3543.
Lin, S. H., Konishi, Y., Denton, M. E., and Scheraga, H. A. (1984).Biochem. 23, 5504–5512.
Lin, S. H., Konishi, Y., Nall, B. T., and Scheraga, H. A. (1985).Biochem. 24, 2680–2686.
Markley, J. L. (1975).Biochem. 14, 3546–3554.
Mui, P. W., Konishi, Y., and Scheraga, H. A. (1985).Biochem. 24, 4481–4489.
Rico, M., Santoro, J., Bermejo, F. J., Herranz, J., Nieto, J. L., Gallego, E., and Jiménez, M. A. (1986).Biopolymers 25, 1031–1053.
Schmid, F. X. (1986).FEBS Lett. 198, 217–220.
Schmid, F. X., and Baldwin, R. L. (1978).Proc. Natl. Acad. Sci. USA 75, 4764–4768.
Schmid, F. X., and Blaschek, H. (1981).Eur. J. Biochem. 114, 111–117.
Schmid, F. X., Grafl, R., Wrba, A., and Beintema, J. J. (1986).Proc. Natl. Acad. Sci. USA 83, 872–876.
Swadesh, J. K., Montelione, G. T., Thannhauser, T. W., and Scheraga, H. A. (1984).Proc. Natl. Acad. Sci. USA 81, 4606–4610.
Thannhauser, T. W., and Scheraga, H. A. (1985).Biochem. 24, 7681–7688.
Tsong, T. Y., Baldwin, R. L., and Elson, E. L. (1972).Proc. Natl. Acad. Sci. USA 69, 1809–1812.
Weber, P. C., Sheriff, S., Ohlendorf, D. H., Finzel, B. C., and Salemme, F. R. (1985).Proc. Natl. Acad. Sci. USA 82, 8473–8477.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Adler, M., Scheraga, H.A. Identification of a new site of conformational heterogeneity in unfolded ribonuclease A. J Protein Chem 9, 583–588 (1990). https://doi.org/10.1007/BF01025011
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF01025011