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. 1988 May;85(9):2894–2898. doi: 10.1073/pnas.85.9.2894

pH-induced changes in Rhodospirillum rubrum cytochrome c2 and subsequent renaturation: an 15N NMR study.

L P Yu 1, G M Smith 1
PMCID: PMC280109  PMID: 2834719

Abstract

The 15N-enriched ferrocytochrome c2 from Rhodospirillum rubrum was studied by 15N NMR at different solvent pH values. The mobility and chemical shift of the N-terminal glutamic acid (335.4 ppm at pH 5.1) were found to depend on pH. It was least mobile between pH 8 and 9.0, which is explained in terms of pH-dependent conformational changes and formation of salt linkages and/or hydrogen bonds. The resonances of the lysine side chains are centered around 341.7 ppm at low pH and move upfield with pH by about 8.4 ppm with pKa values of 10.8. The exchange rates of the epsilon NH protons are lowest near their pKa values. The protein is very stable in the pH range between 4.9 and 10.0 but unfolds abruptly at pH 10.5-11. Denaturation was verified by the measurement of several parameters by NMR. The renaturation of the protein demonstrates that the folding begins with reformation of heme coordination and establishment of a hydrophobic core, followed by positioning of side chains and peptide backbones linking the nucleation centers. The repositioning processes had time scales of minutes to hours in contrast to the reported values of seconds in some studies.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. ANFINSEN C. B. The limited digestion of ribonuclease with pepsin. J Biol Chem. 1956 Jul;221(1):405–412. [PubMed] [Google Scholar]
  2. Anfinsen C. B. Principles that govern the folding of protein chains. Science. 1973 Jul 20;181(4096):223–230. doi: 10.1126/science.181.4096.223. [DOI] [PubMed] [Google Scholar]
  3. Bogusky M. J., Tsang P., Opella S. J. One- and two- dimensional 15N/1H NMR of filamentous phage coat proteins in solution. Biochem Biophys Res Commun. 1985 Mar 15;127(2):540–545. doi: 10.1016/s0006-291x(85)80193-5. [DOI] [PubMed] [Google Scholar]
  4. Corradin G., Harbury H. A. Reconstitution of horse heart cytochrome c: interaction of the components obtained upon cleavage of the peptide bond following methionine residue 65. Proc Natl Acad Sci U S A. 1971 Dec;68(12):3036–3039. doi: 10.1073/pnas.68.12.3036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dill K. A. Theory for the folding and stability of globular proteins. Biochemistry. 1985 Mar 12;24(6):1501–1509. doi: 10.1021/bi00327a032. [DOI] [PubMed] [Google Scholar]
  6. Dus K., Sletten K., Kamen M. D. Cytochrome c2 of Rhodospirillum rubrum. II. Complete amino acid sequence and phylogenetic relationships. J Biol Chem. 1968 Oct 25;243(20):5507–5518. [PubMed] [Google Scholar]
  7. Englander S. W., Wand A. J. Main-chain-directed strategy for the assignment of 1H NMR spectra of proteins. Biochemistry. 1987 Sep 22;26(19):5953–5958. doi: 10.1021/bi00393a001. [DOI] [PubMed] [Google Scholar]
  8. Epstein H. F., Schechter A. N., Cohen J. S. Folding of staphylococcal nuclease: magnetic resonance and fluorescence studies of individual residues. Proc Natl Acad Sci U S A. 1971 Sep;68(9):2042–2046. doi: 10.1073/pnas.68.9.2042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Griffey R. H., Redfield A. G. Proton-detected heteronuclear edited and correlated nuclear magnetic resonance and nuclear Overhauser effect in solution. Q Rev Biophys. 1987 Feb;19(1-2):51–82. doi: 10.1017/s0033583500004029. [DOI] [PubMed] [Google Scholar]
  10. Jardetzky O., Thielmann H., Arata Y., Markley J. L., Williams M. N. Tentative sequential model for the unfolding and refolding of staphylococcal nuclease at high pH. Cold Spring Harb Symp Quant Biol. 1972;36:257–261. doi: 10.1101/sqb.1972.036.01.034. [DOI] [PubMed] [Google Scholar]
  11. Kim P. S., Baldwin R. L. Specific intermediates in the folding reactions of small proteins and the mechanism of protein folding. Annu Rev Biochem. 1982;51:459–489. doi: 10.1146/annurev.bi.51.070182.002331. [DOI] [PubMed] [Google Scholar]
  12. Leszczynski J. F., Rose G. D. Loops in globular proteins: a novel category of secondary structure. Science. 1986 Nov 14;234(4778):849–855. doi: 10.1126/science.3775366. [DOI] [PubMed] [Google Scholar]
  13. McIntosh L. P., Griffey R. H., Muchmore D. C., Nielson C. P., Redfield A. G., Dahlquist F. W. Proton NMR measurements of bacteriophage T4 lysozyme aided by 15N isotopic labeling: structural and dynamic studies of larger proteins. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1244–1248. doi: 10.1073/pnas.84.5.1244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Moore G. R., Robinson M. N., Williams G., Williams R. J. Solution structure of mitochondrial cytochrome c. II. 1H nuclear magnetic resonance of ferrocytochrome c. J Mol Biol. 1985 Jun 5;183(3):429–446. doi: 10.1016/0022-2836(85)90012-9. [DOI] [PubMed] [Google Scholar]
  15. Pettigrew G. W., Bartsch R. G., Meyer T. E., Kamen M. D. Redox potentials of the photosynthetic bacterial cytochromes c2 and the structural bases for variability. Biochim Biophys Acta. 1978 Sep 7;503(3):509–523. doi: 10.1016/0005-2728(78)90150-0. [DOI] [PubMed] [Google Scholar]
  16. Pettigrew G. W., Meyer T. E., Bartsch R. G., Kamen M. D. pH dependence of the oxidation-reduction potential of cytochrome c2. Biochim Biophys Acta. 1976 May 14;430(2):197–208. doi: 10.1016/0005-2728(76)90079-7. [DOI] [PubMed] [Google Scholar]
  17. Pettigrew G. W., Smith G. M. Novel N-terminal protein blocking group identified as dimethylproline. Nature. 1977 Feb 17;265(5595):661–662. doi: 10.1038/265661a0. [DOI] [PubMed] [Google Scholar]
  18. Pfeil W., Privalov P. L. Thermodynamic investigations of proteins. I. Standard functions for proteins with lysozyme as an example. Biophys Chem. 1976 Jan;4(1):23–32. doi: 10.1016/0301-4622(76)80003-8. [DOI] [PubMed] [Google Scholar]
  19. Privalov P. L. Stability of proteins: small globular proteins. Adv Protein Chem. 1979;33:167–241. doi: 10.1016/s0065-3233(08)60460-x. [DOI] [PubMed] [Google Scholar]
  20. Salemme F. R., Freer S. T., Xuong N. H., Alden R. A., Kraut J. The structure of oxidized cytochrome c 2 of Rhodospirillum rubrum. J Biol Chem. 1973 Jun 10;248(11):3910–3921. doi: 10.2210/pdb1c2c/pdb. [DOI] [PubMed] [Google Scholar]
  21. Smith G. M. Proton nuclear magnetic resonance studies of Rhodospirillum rubrum cytochrome. Biochemistry. 1979 Apr 17;18(8):1628–1634. doi: 10.1021/bi00575a039. [DOI] [PubMed] [Google Scholar]
  22. Smith G. M., Yu L. P., Domingues D. J. Directly observed 15N NMR spectra of uniformly enriched proteins. Biochemistry. 1987 Apr 21;26(8):2202–2207. doi: 10.1021/bi00382a020. [DOI] [PubMed] [Google Scholar]
  23. Swanson R., Trus B. L., Mandel N., Mandel G., Kallai O. B., Dickerson R. E. Tuna cytochrome c at 2.0 A resolution. I. Ferricytochrome structure analysis. J Biol Chem. 1977 Jan 25;252(2):759–775. [PubMed] [Google Scholar]
  24. Takano T., Trus B. L., Mandel N., Mandel G., Kallai O. B., Swanson R., Dickerson R. E. Tuna cytochrome c at 2.0 A resolution. II. Ferrocytochrome structure analysis. J Biol Chem. 1977 Jan 25;252(2):776–785. [PubMed] [Google Scholar]
  25. Vaucheret H., Signon L., Le Bras G., Garel J. R. Mechanism of renaturation of a large protein, aspartokinase-homoserine dehydrogenase. Biochemistry. 1987 May 19;26(10):2785–2790. doi: 10.1021/bi00384a020. [DOI] [PubMed] [Google Scholar]
  26. Wand A. J., Englander S. W. Two-dimensional 1H NMR studies of cytochrome c. Biochemistry. 1985 Sep 24;24(20):5290–5294. doi: 10.1021/bi00341a002. [DOI] [PubMed] [Google Scholar]
  27. Wetlaufer D. B. Folding of protein fragments. Adv Protein Chem. 1981;34:61–92. doi: 10.1016/s0065-3233(08)60518-5. [DOI] [PubMed] [Google Scholar]
  28. Williams G., Moore G. R., Porteous R., Robinson M. N., Soffe N., Williams R. J. Solution structure of mitochondrial cytochrome c. I. 1H nuclear magnetic resonance of ferricytochrome c. J Mol Biol. 1985 Jun 5;183(3):409–428. doi: 10.1016/0022-2836(85)90011-7. [DOI] [PubMed] [Google Scholar]
  29. Wooten J. B., Cohen J. S., Vig I., Schejter A. pH-induced conformational transitions of ferricytochrome c: a carbon-13 and deuterium nuclear magnetic resonance study. Biochemistry. 1981 Sep 15;20(19):5394–5402. doi: 10.1021/bi00522a007. [DOI] [PubMed] [Google Scholar]

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