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. 1999 May;8(5):1116–1126. doi: 10.1110/ps.8.5.1116

A stable intermediate in the equilibrium unfolding of Escherichia coli citrate synthase.

A Ayed 1, H W Duckworth 1
PMCID: PMC2144337  PMID: 10338022

Abstract

Urea-induced unfolding of Escherichia coli citrate synthase occurs in two phases, as monitored by circular dichroism at 222 nm (measuring secondary structure) or by tryptophan fluorescence. In this paper we characterize the intermediate state, which retains about 40% of the ellipticity of the native state, and is stable between 2.5 M and 5.5 M urea, approximately. This intermediate binds significant amounts of the probe for hydrophobic surfaces, anilinonaphthalene sulfonate, but forms aggregates at least as high as an octamer, as shown by transverse urea gradient polyacrylamide electrophoresis. Thermal denaturation of E. coli citrate synthase also produces an intermediate at temperatures near 60 degrees C, which also retains about 40% of the native ellipticity and forms aggregates, as measured by electrospray-ionization/time-of-flight mass spectrometry. We have used a collection of "cavity-forming" mutant proteins, in which bulky buried hydrophobic residues are replaced by alanines, to explore the nature of the intermediate state further. A certain amount of these mutant proteins shows a destabilized intermediate, as measured by the urea concentration range in which the intermediate is observed. These mutants are found in parts of the citrate synthase sequence that, in a native state, form helices G, M, N, Q, R, and S. From this and other evidence, it is argued that the intermediate state is an aggregated state in which these six helices, or parts of them, remain folded, and that formation of this intermediate is also likely to be a key step in the folding of E. coli citrate synthase.

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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. Anderson D. H., Donald L. J., Jacob M. V., Duckworth H. W. A mutant of Escherichia coli citrate synthase that affects the allosteric equilibrium. Biochem Cell Biol. 1991 Apr;69(4):232–238. doi: 10.1139/o91-035. [DOI] [PubMed] [Google Scholar]
  3. Anderson D. H., Duckworth H. W. In vitro mutagenesis of Escherichia coli citrate synthase to clarify the locations of ligand binding sites. J Biol Chem. 1988 Feb 15;263(5):2163–2169. [PubMed] [Google Scholar]
  4. Arcus V. L., Vuilleumier S., Freund S. M., Bycroft M., Fersht A. R. Toward solving the folding pathway of barnase: the complete backbone 13C, 15N, and 1H NMR assignments of its pH-denatured state. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9412–9416. doi: 10.1073/pnas.91.20.9412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ayed A., Krutchinsky A. N., Ens W., Standing K. G., Duckworth H. W. Quantitative evaluation of protein-protein and ligand-protein equilibria of a large allosteric enzyme by electrospray ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom. 1998;12(7):339–344. doi: 10.1002/(SICI)1097-0231(19980415)12:7<339::AID-RCM163>3.0.CO;2-6. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Buchner J., Schmidt M., Fuchs M., Jaenicke R., Rudolph R., Schmid F. X., Kiefhaber T. GroE facilitates refolding of citrate synthase by suppressing aggregation. Biochemistry. 1991 Feb 12;30(6):1586–1591. doi: 10.1021/bi00220a020. [DOI] [PubMed] [Google Scholar]
  8. Daugherty D. L., Rozema D., Hanson P. E., Gellman S. H. Artificial chaperone-assisted refolding of citrate synthase. J Biol Chem. 1998 Dec 18;273(51):33961–33971. doi: 10.1074/jbc.273.51.33961. [DOI] [PubMed] [Google Scholar]
  9. Donald L. J., Crane B. R., Anderson D. H., Duckworth H. W. The role of cysteine 206 in allosteric inhibition of Escherichia coli citrate synthase. Studies by chemical modification, site-directed mutagenesis, and 19F NMR. J Biol Chem. 1991 Nov 5;266(31):20709–20713. [PubMed] [Google Scholar]
  10. Duckworth H. W., Anderson D. H., Bell A. W., Donald L. J., Chu A. L., Brayer G. D. Structural basis for regulation in gram-negative bacterial citrate synthases. Biochem Soc Symp. 1987;54:83–92. [PubMed] [Google Scholar]
  11. Ehrnsperger M., Gräber S., Gaestel M., Buchner J. Binding of non-native protein to Hsp25 during heat shock creates a reservoir of folding intermediates for reactivation. EMBO J. 1997 Jan 15;16(2):221–229. doi: 10.1093/emboj/16.2.221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eriksson A. E., Baase W. A., Zhang X. J., Heinz D. W., Blaber M., Baldwin E. P., Matthews B. W. Response of a protein structure to cavity-creating mutations and its relation to the hydrophobic effect. Science. 1992 Jan 10;255(5041):178–183. doi: 10.1126/science.1553543. [DOI] [PubMed] [Google Scholar]
  13. Horwich A. L., Low K. B., Fenton W. A., Hirshfield I. N., Furtak K. Folding in vivo of bacterial cytoplasmic proteins: role of GroEL. Cell. 1993 Sep 10;74(5):909–917. doi: 10.1016/0092-8674(93)90470-b. [DOI] [PubMed] [Google Scholar]
  14. Jackson S. E. How do small single-domain proteins fold? Fold Des. 1998;3(4):R81–R91. doi: 10.1016/S1359-0278(98)00033-9. [DOI] [PubMed] [Google Scholar]
  15. Kelly S. M., Price N. C. Reactivation of denatured citrate synthase. Int J Biochem. 1992 Apr;24(4):627–630. doi: 10.1016/0020-711x(92)90338-2. [DOI] [PubMed] [Google Scholar]
  16. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  17. Kuwajima K. The molten globule state of alpha-lactalbumin. FASEB J. 1996 Jan;10(1):102–109. doi: 10.1096/fasebj.10.1.8566530. [DOI] [PubMed] [Google Scholar]
  18. Liao D. I., Karpusas M., Remington S. J. Crystal structure of an open conformation of citrate synthase from chicken heart at 2.8-A resolution. Biochemistry. 1991 Jun 18;30(24):6031–6036. doi: 10.1021/bi00238a029. [DOI] [PubMed] [Google Scholar]
  19. Logan T. M., Thériault Y., Fesik S. W. Structural characterization of the FK506 binding protein unfolded in urea and guanidine hydrochloride. J Mol Biol. 1994 Feb 18;236(2):637–648. doi: 10.1006/jmbi.1994.1173. [DOI] [PubMed] [Google Scholar]
  20. Matthews B. W. Structural and genetic analysis of the folding and function of T4 lysozyme. FASEB J. 1996 Jan;10(1):35–41. doi: 10.1096/fasebj.10.1.8566545. [DOI] [PubMed] [Google Scholar]
  21. Molgat G. F., Donald L. J., Duckworth H. W. Chimeric allosteric citrate synthases: construction and properties of citrate synthases containing domains from two different enzymes. Arch Biochem Biophys. 1992 Oct;298(1):238–246. doi: 10.1016/0003-9861(92)90118-g. [DOI] [PubMed] [Google Scholar]
  22. Neri D., Billeter M., Wider G., Wüthrich K. NMR determination of residual structure in a urea-denatured protein, the 434-repressor. Science. 1992 Sep 11;257(5076):1559–1563. doi: 10.1126/science.1523410. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Pereira D. S., Donald L. J., Hosfield D. J., Duckworth H. W. Active site mutants of Escherichia coli citrate synthase. Effects of mutations on catalytic and allosteric properties. J Biol Chem. 1994 Jan 7;269(1):412–417. [PubMed] [Google Scholar]
  25. Ptitsyn O. B. How the molten globule became. Trends Biochem Sci. 1995 Sep;20(9):376–379. doi: 10.1016/s0968-0004(00)89081-7. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Russell R. J., Ferguson J. M., Hough D. W., Danson M. J., Taylor G. L. The crystal structure of citrate synthase from the hyperthermophilic archaeon pyrococcus furiosus at 1.9 A resolution,. Biochemistry. 1997 Aug 19;36(33):9983–9994. doi: 10.1021/bi9705321. [DOI] [PubMed] [Google Scholar]
  28. Russell R. J., Gerike U., Danson M. J., Hough D. W., Taylor G. L. Structural adaptations of the cold-active citrate synthase from an Antarctic bacterium. Structure. 1998 Mar 15;6(3):351–361. doi: 10.1016/s0969-2126(98)00037-9. [DOI] [PubMed] [Google Scholar]
  29. Russell R. J., Hough D. W., Danson M. J., Taylor G. L. The crystal structure of citrate synthase from the thermophilic archaeon, Thermoplasma acidophilum. Structure. 1994 Dec 15;2(12):1157–1167. doi: 10.1016/s0969-2126(94)00118-9. [DOI] [PubMed] [Google Scholar]
  30. Semisotnov G. V., Rodionova N. A., Razgulyaev O. I., Uversky V. N., Gripas' A. F., Gilmanshin R. I. Study of the "molten globule" intermediate state in protein folding by a hydrophobic fluorescent probe. Biopolymers. 1991 Jan;31(1):119–128. doi: 10.1002/bip.360310111. [DOI] [PubMed] [Google Scholar]
  31. Talgoy M. M., Bell A. W., Duckworth H. W. The reactions of Escherichia coli citrate synthase with the sulfhydryl reagents 5,5'-dithiobis-(2-nitrobenzoic acid) and 4,4'-dithiodipyridine. Can J Biochem. 1979 Jun;57(6):822–833. doi: 10.1139/o79-102. [DOI] [PubMed] [Google Scholar]
  32. Tong E. K., Duckworth H. W. The quaternary structure of citrate synthase from Escherichia coli K12. Biochemistry. 1975 Jan 28;14(2):235–241. doi: 10.1021/bi00673a007. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Zhi W., Landry S. J., Gierasch L. M., Srere P. A. Renaturation of citrate synthase: influence of denaturant and folding assistants. Protein Sci. 1992 Apr;1(4):522–529. doi: 10.1002/pro.5560010407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]

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