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. 1992 Sep;1(9):1162–1172. doi: 10.1002/pro.5560010910

Structure of a rapidly formed intermediate in ribonuclease T1 folding.

T Kiefhaber 1, F X Schmid 1, K Willaert 1, Y Engelborghs 1, A Chaffotte 1
PMCID: PMC2142177  PMID: 1304394

Abstract

Kinetic intermediates in protein folding are short-lived and therefore difficult to detect and to characterize. In the folding of polypeptide chains with incorrect isomers of Xaa-Pro peptide bonds the final rate-limiting transition to the native state is slow, since it is coupled to prolyl isomerization. Incorrect prolyl isomers thus act as effective traps for folding intermediates and allow their properties to be studied more easily. We employed this strategy to investigate the mechanism of slow folding of ribonuclease T1. In our experiments we use a mutant form of this protein with a single cis peptide bond at proline 39. During refolding, protein chains with an incorrect trans proline 39 can rapidly form extensive secondary structure. The CD signal in the amide region is regained within the dead-time of stopped-flow mixing (15 ms), indicating a fast formation of the single alpha-helix of ribonuclease T1. This step is correlated with partial formation of a hydrophobic core, because the fluorescence emission maximum of tryptophan 59 is shifted from 349 nm to 325 nm within less than a second. After about 20 s of refolding an intermediate is present that shows about 40% enzymatic activity compared to the completely refolded protein. In addition, the solvent accessibility of tryptophan 59 is drastically reduced in this intermediate and comparable to that of the native state as determined by acrylamide quenching of the tryptophan fluorescence. Activity and quenching measurements have long dead-times and therefore we do not know whether enzymatic activity and solvent accessibility also change in the time range of milliseconds. At this stage of folding at least part of the beta-sheet structure is already present, since it hosts the active site of the enzyme. The trans to cis isomerization of the tyrosine 38-proline 39 peptide bond in the intermediate and consequently the formation of native protein is very slow (tau = 6,500 s at pH 5.0 and 10 degrees C). It is accompanied by an additional increase in tryptophan fluorescence, by the development of the fine structure of the tryptophan emission spectrum, and by the regain of the full enzymatic activity. This indicates that the packing of the hydrophobic core, which involves both tryptophan 59 and proline 39, is optimized in this step. Apparently, refolding polypeptide chains with an incorrect prolyl isomer can very rapidly form partially folded intermediates with native-like properties.

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Selected References

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  1. Adler A. J., Greenfield N. J., Fasman G. D. Circular dichroism and optical rotatory dispersion of proteins and polypeptides. Methods Enzymol. 1973;27:675–735. doi: 10.1016/s0076-6879(73)27030-1. [DOI] [PubMed] [Google Scholar]
  2. Bycroft M., Matouschek A., Kellis J. T., Jr, Serrano L., Fersht A. R. Detection and characterization of a folding intermediate in barnase by NMR. Nature. 1990 Aug 2;346(6283):488–490. doi: 10.1038/346488a0. [DOI] [PubMed] [Google Scholar]
  3. Chen L. X., Longworth J. W., Fleming G. R. Picosecond time-resolved fluorescence of ribonuclease T1. A pH and substrate analogue binding study. Biophys J. 1987 Jun;51(6):865–873. doi: 10.1016/S0006-3495(87)83414-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cook K. H., Schmid F. X., Baldwin R. L. Role of proline isomerization in folding of ribonuclease A at low temperatures. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6157–6161. doi: 10.1073/pnas.76.12.6157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Creighton T. E. Protein folding. Biochem J. 1990 Aug 15;270(1):1–16. doi: 10.1042/bj2700001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Grinvald A., Steinberg I. Z. The fluorescence decay of tryptophan residues in native and denatured proteins. Biochim Biophys Acta. 1976 Apr 14;427(2):663–678. doi: 10.1016/0005-2795(76)90210-5. [DOI] [PubMed] [Google Scholar]
  7. Gryczynski I., Eftink M., Lakowicz J. R. Conformation heterogeneity in proteins as an origin of heterogeneous fluorescence decays, illustrated by native and denatured ribonuclease T1. Biochim Biophys Acta. 1988 Jun 13;954(3):244–252. doi: 10.1016/0167-4838(88)90079-9. [DOI] [PubMed] [Google Scholar]
  8. Khan M. Y., Villanueva G., Newman S. A. On the origin of the positive band in the far-ultraviolet circular dichroic spectrum of fibronectin. J Biol Chem. 1989 Feb 5;264(4):2139–2142. [PubMed] [Google Scholar]
  9. Kiefhaber T., Grunert H. P., Hahn U., Schmid F. X. Folding of RNase T1 is decelerated by a specific tertiary contact in a folding intermediate. Proteins. 1992 Feb;12(2):171–179. doi: 10.1002/prot.340120210. [DOI] [PubMed] [Google Scholar]
  10. Kiefhaber T., Grunert H. P., Hahn U., Schmid F. X. Replacement of a cis proline simplifies the mechanism of ribonuclease T1 folding. Biochemistry. 1990 Jul 10;29(27):6475–6480. doi: 10.1021/bi00479a020. [DOI] [PubMed] [Google Scholar]
  11. Kiefhaber T., Quaas R., Hahn U., Schmid F. X. Folding of ribonuclease T1. 1. Existence of multiple unfolded states created by proline isomerization. Biochemistry. 1990 Mar 27;29(12):3053–3061. doi: 10.1021/bi00464a023. [DOI] [PubMed] [Google Scholar]
  12. Kiefhaber T., Quaas R., Hahn U., Schmid F. X. Folding of ribonuclease T1. 2. Kinetic models for the folding and unfolding reactions. Biochemistry. 1990 Mar 27;29(12):3061–3070. doi: 10.1021/bi00464a024. [DOI] [PubMed] [Google Scholar]
  13. Kiefhaber T., Schmid F. X. Kinetic coupling between protein folding and prolyl isomerization. II. Folding of ribonuclease A and ribonuclease T1. J Mol Biol. 1992 Mar 5;224(1):231–240. doi: 10.1016/0022-2836(92)90586-9. [DOI] [PubMed] [Google Scholar]
  14. Kim P. S., Baldwin R. L. Intermediates in the folding reactions of small proteins. Annu Rev Biochem. 1990;59:631–660. doi: 10.1146/annurev.bi.59.070190.003215. [DOI] [PubMed] [Google Scholar]
  15. Krebs H., Schmid F. X., Jaenicke R. Native-like folding intermediates of homologous ribonucleases. Biochemistry. 1985 Jul 16;24(15):3846–3852. doi: 10.1021/bi00336a005. [DOI] [PubMed] [Google Scholar]
  16. Kuwajima K., Garvey E. P., Finn B. E., Matthews C. R., Sugai S. Transient intermediates in the folding of dihydrofolate reductase as detected by far-ultraviolet circular dichroism spectroscopy. Biochemistry. 1991 Aug 6;30(31):7693–7703. doi: 10.1021/bi00245a005. [DOI] [PubMed] [Google Scholar]
  17. Manning M. C., Woody R. W. Theoretical study of the contribution of aromatic side chains to the circular dichroism of basic bovine pancreatic trypsin inhibitor. Biochemistry. 1989 Oct 17;28(21):8609–8613. doi: 10.1021/bi00447a051. [DOI] [PubMed] [Google Scholar]
  18. Pace C. N. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 1986;131:266–280. doi: 10.1016/0076-6879(86)31045-0. [DOI] [PubMed] [Google Scholar]
  19. Roder H., Elöve G. A., Englander S. W. Structural characterization of folding intermediates in cytochrome c by H-exchange labelling and proton NMR. Nature. 1988 Oct 20;335(6192):700–704. doi: 10.1038/335700a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Santoro M. M., Bolen D. W. Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry. 1988 Oct 18;27(21):8063–8068. doi: 10.1021/bi00421a014. [DOI] [PubMed] [Google Scholar]
  21. Schmid F. X. Mechanism of folding of ribonuclease A. Slow refolding is a sequential reaction via structural intermediates. Biochemistry. 1983 Sep 27;22(20):4690–4696. doi: 10.1021/bi00289a013. [DOI] [PubMed] [Google Scholar]
  22. Udgaonkar J. B., Baldwin R. L. Early folding intermediate of ribonuclease A. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8197–8201. doi: 10.1073/pnas.87.21.8197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Udgaonkar J. B., Baldwin R. L. NMR evidence for an early framework intermediate on the folding pathway of ribonuclease A. Nature. 1988 Oct 20;335(6192):694–699. doi: 10.1038/335694a0. [DOI] [PubMed] [Google Scholar]
  24. Weissman J. S., Kim P. S. Reexamination of the folding of BPTI: predominance of native intermediates. Science. 1991 Sep 20;253(5026):1386–1393. doi: 10.1126/science.1716783. [DOI] [PubMed] [Google Scholar]

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