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. 1992 Apr;1(4):522–529. doi: 10.1002/pro.5560010407

Renaturation of citrate synthase: influence of denaturant and folding assistants.

W Zhi 1, S J Landry 1, L M Gierasch 1, P A Srere 1
PMCID: PMC2142213  PMID: 1363914

Abstract

Citrate synthase (CS), which has been denatured in either guanidine hydrochloride (GdnHCl) or urea can be assisted in its renaturation in a variety of ways. The addition of each of the assistants--bovine serum albumin (BSA), oxaloacetate (OAA), and glycerol--promotes renaturation. In combination, the effect of these substances is additive with respect to the yield of folded CS. The report of Buchner et al. (Buchner, J., Schmidt, M., Fuchs, M., Jaenicke, R., Rudolph, R., Schmid, F.X., & Kiefhaber, T., 1991, Biochemistry 30, 1586-1591) that refolding of CS is facilitated by the GroE system (an Escherichia coli chaperonin [cpn] that is composed of GroEL [cpn60] and GroES [cpn10]) has been confirmed. However, we observed substantially higher yield of reactivated CS, 82%, and almost no reactivation in the absence of GroES, < 5%, whereas Buchner et al. reported 28% and 16%, respectively. In addition, we find that GroE-assisted refolding is more efficient for CS denatured in GdnHCl than for CS denatured in urea. This result is discussed in light of the known difference in the denatured states generated in GdnHCl and urea. Because GroEL inhibits the BSA/glycerol/OAA-assisted refolding, this system will be useful in future studies on the mechanism of GroE-facilitated refolding.

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Selected References

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  1. Baker K. P., Schatz G. Mitochondrial proteins essential for viability mediate protein import into yeast mitochondria. Nature. 1991 Jan 17;349(6306):205–208. doi: 10.1038/349205a0. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Goloubinoff P., Christeller J. T., Gatenby A. A., Lorimer G. H. Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfoleded state depends on two chaperonin proteins and Mg-ATP. Nature. 1989 Dec 21;342(6252):884–889. doi: 10.1038/342884a0. [DOI] [PubMed] [Google Scholar]
  4. Greenblatt G. A., Sarkissian I. V. Some catalytic and structural aspects of reactivation of denatured citrate synthase. Life Sci II. 1972 Apr 8;11(7):375–380. doi: 10.1016/0024-3205(72)90077-x. [DOI] [PubMed] [Google Scholar]
  5. Johnson J. K., Srivastava D. K. Interaction of ligands with pig heart citrate synthase: conformational changes and catalysis. Arch Biochem Biophys. 1991 Jun;287(2):250–256. doi: 10.1016/0003-9861(91)90414-e. [DOI] [PubMed] [Google Scholar]
  6. Landry S. J., Gierasch L. M. The chaperonin GroEL binds a polypeptide in an alpha-helical conformation. Biochemistry. 1991 Jul 30;30(30):7359–7362. doi: 10.1021/bi00244a001. [DOI] [PubMed] [Google Scholar]
  7. McEvily A. J., Harrison J. H. Subunit equilibria of porcine heart citrate synthase. Effects of enzyme concentration, pH, and substrates. J Biol Chem. 1986 Feb 25;261(6):2593–2598. [PubMed] [Google Scholar]
  8. Mendoza J. A., Rogers E., Lorimer G. H., Horowitz P. M. Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese. J Biol Chem. 1991 Jul 15;266(20):13044–13049. [PubMed] [Google Scholar]
  9. Mendoza J. A., Rogers E., Lorimer G. H., Horowitz P. M. Unassisted refolding of urea unfolded rhodanese. J Biol Chem. 1991 Jul 25;266(21):13587–13591. [PubMed] [Google Scholar]
  10. Ostermann J., Horwich A. L., Neupert W., Hartl F. U. Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis. Nature. 1989 Sep 14;341(6238):125–130. doi: 10.1038/341125a0. [DOI] [PubMed] [Google Scholar]
  11. Pace C. N., Laurents D. V., Thomson J. A. pH dependence of the urea and guanidine hydrochloride denaturation of ribonuclease A and ribonuclease T1. Biochemistry. 1990 Mar 13;29(10):2564–2572. doi: 10.1021/bi00462a019. [DOI] [PubMed] [Google Scholar]
  12. Remington S., Wiegand G., Huber R. Crystallographic refinement and atomic models of two different forms of citrate synthase at 2.7 and 1.7 A resolution. J Mol Biol. 1982 Jun 15;158(1):111–152. doi: 10.1016/0022-2836(82)90452-1. [DOI] [PubMed] [Google Scholar]
  13. SRERE P. A., KOSICKI G. W. The purification of citrate-condensing enzyme. J Biol Chem. 1961 Oct;236:2557–2559. [PubMed] [Google Scholar]
  14. SRERE P. A. SPECTRAL EVIDENCE FOR COMPLEX FORMATION BETWEEN OXALOACETATE AND CITRATE-CONDENSING ENZYME. Biochim Biophys Acta. 1965 Apr 26;99:197–200. doi: 10.1016/s0926-6593(65)80028-5. [DOI] [PubMed] [Google Scholar]
  15. Singh M., Brooks G. C., Srere P. A. Subunit structure and chemical characteristics of pig heart citrate synthase. J Biol Chem. 1970 Sep 25;245(18):4636–4640. [PubMed] [Google Scholar]
  16. Srere P. A. Citrate-condensing enzyme-oxalacetate binary complex. Studies on its physical and chemical properties. J Biol Chem. 1966 May 10;241(9):2157–2165. [PubMed] [Google Scholar]
  17. Tandon S., Horowitz P. M. Detergent-assisted refolding of guanidinium chloride-denatured rhodanese. The effects of the concentration and type of detergent. J Biol Chem. 1987 Apr 5;262(10):4486–4491. [PubMed] [Google Scholar]
  18. West S. M., Kelly S. M., Price N. C. The unfolding and attempted refolding of citrate synthase from pig heart. Biochim Biophys Acta. 1990 Mar 1;1037(3):332–336. doi: 10.1016/0167-4838(90)90034-d. [DOI] [PubMed] [Google Scholar]
  19. Wu J. Y., Yang J. T. Physicochemical characterization of citrate synthase and its subunits. J Biol Chem. 1970 Jan 10;245(1):212–218. [PubMed] [Google Scholar]
  20. Zhi W., Srere P. A., Evans C. T. Conformational stability of pig citrate synthase and some active-site mutants. Biochemistry. 1991 Sep 24;30(38):9281–9286. doi: 10.1021/bi00102a021. [DOI] [PubMed] [Google Scholar]

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