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. 1990 Jul;9(7):2315–2319. doi: 10.1002/j.1460-2075.1990.tb07403.x

The Escherichia coli heat shock proteins GroEL and GroES modulate the folding of the beta-lactamase precursor.

A A Laminet 1, T Ziegelhoffer 1, C Georgopoulos 1, A Plückthun 1
PMCID: PMC551958  PMID: 2192863

Abstract

One of the fundamental problems in biochemistry is the role of accessory proteins in the process of protein folding. The Escherichia coli heat shock protein complex GroEL/ES has been suggested to be a 'chaperonin' and be involved in both oligomer assembly as well as protein transport through the membrane. We show here that the folding of the purified precursor of beta-lactamase is inhibited by purified GroEL or the GroEL/ES complex with a stoichiometry of one particle per molecule of pre-beta-lactamase. Purified GroES alone has no effect on folding. After Mg2+ ATP addition folding resumes and the yield of active enzyme is higher than in the absence of GroEL or GroEL/ES. Unexpectedly, GroEL or GroEL/ES, when added to folded pre-beta-lactamase, lead to an apparent net 'unfolding', probably to a collapsed state of the protein, which can be reversed by the addition of Mg2+ ATP. The reversible and Mg2+ ATP-dependent association of GroEL/ES with non-native proteins might explain its postulated role in both protein transport and oligomer assembly.

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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. 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]
  2. Bernstein H. D., Poritz M. A., Strub K., Hoben P. J., Brenner S., Walter P. Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle. Nature. 1989 Aug 10;340(6233):482–486. doi: 10.1038/340482a0. [DOI] [PubMed] [Google Scholar]
  3. Blake M. S., Johnston K. H., Russell-Jones G. J., Gotschlich E. C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem. 1984 Jan;136(1):175–179. doi: 10.1016/0003-2697(84)90320-8. [DOI] [PubMed] [Google Scholar]
  4. Bochkareva E. S., Lissin N. M., Girshovich A. S. Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein. Nature. 1988 Nov 17;336(6196):254–257. doi: 10.1038/336254a0. [DOI] [PubMed] [Google Scholar]
  5. Bulleid N. J., Freedman R. B. Defective co-translational formation of disulphide bonds in protein disulphide-isomerase-deficient microsomes. Nature. 1988 Oct 13;335(6191):649–651. doi: 10.1038/335649a0. [DOI] [PubMed] [Google Scholar]
  6. Bychkova V. E., Pain R. H., Ptitsyn O. B. The 'molten globule' state is involved in the translocation of proteins across membranes? FEBS Lett. 1988 Oct 10;238(2):231–234. doi: 10.1016/0014-5793(88)80485-x. [DOI] [PubMed] [Google Scholar]
  7. Chandrasekhar G. N., Tilly K., Woolford C., Hendrix R., Georgopoulos C. Purification and properties of the groES morphogenetic protein of Escherichia coli. J Biol Chem. 1986 Sep 15;261(26):12414–12419. [PubMed] [Google Scholar]
  8. Chirico W. J., Waters M. G., Blobel G. 70K heat shock related proteins stimulate protein translocation into microsomes. Nature. 1988 Apr 28;332(6167):805–810. doi: 10.1038/332805a0. [DOI] [PubMed] [Google Scholar]
  9. Collier D. N., Bankaitis V. A., Weiss J. B., Bassford P. J., Jr The antifolding activity of SecB promotes the export of the E. coli maltose-binding protein. Cell. 1988 Apr 22;53(2):273–283. doi: 10.1016/0092-8674(88)90389-3. [DOI] [PubMed] [Google Scholar]
  10. Cover W. H., Ryan J. P., Bassford P. J., Jr, Walsh K. A., Bollinger J., Randall L. L. Suppression of a signal sequence mutation by an amino acid substitution in the mature portion of the maltose-binding protein. J Bacteriol. 1987 May;169(5):1794–1800. doi: 10.1128/jb.169.5.1794-1800.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Creighton T. E. Experimental studies of protein folding and unfolding. Prog Biophys Mol Biol. 1978;33(3):231–297. doi: 10.1016/0079-6107(79)90030-0. [DOI] [PubMed] [Google Scholar]
  12. Crooke E., Brundage L., Rice M., Wickner W. ProOmpA spontaneously folds in a membrane assembly competent state which trigger factor stabilizes. EMBO J. 1988 Jun;7(6):1831–1835. doi: 10.1002/j.1460-2075.1988.tb03015.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Crooke E., Wickner W. Trigger factor: a soluble protein that folds pro-OmpA into a membrane-assembly-competent form. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5216–5220. doi: 10.1073/pnas.84.15.5216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Deshaies R. J., Koch B. D., Werner-Washburne M., Craig E. A., Schekman R. A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature. 1988 Apr 28;332(6167):800–805. doi: 10.1038/332800a0. [DOI] [PubMed] [Google Scholar]
  15. Eilers M., Schatz G. Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. Nature. 1986 Jul 17;322(6076):228–232. doi: 10.1038/322228a0. [DOI] [PubMed] [Google Scholar]
  16. Ellis J. Proteins as molecular chaperones. 1987 Jul 30-Aug 5Nature. 328(6129):378–379. doi: 10.1038/328378a0. [DOI] [PubMed] [Google Scholar]
  17. Ellis R. J., Hemmingsen S. M. Molecular chaperones: proteins essential for the biogenesis of some macromolecular structures. Trends Biochem Sci. 1989 Aug;14(8):339–342. doi: 10.1016/0968-0004(89)90168-0. [DOI] [PubMed] [Google Scholar]
  18. Ellis R. J., van der Vies S. M., Hemmingsen S. M. The molecular chaperone concept. Biochem Soc Symp. 1989;55:145–153. [PubMed] [Google Scholar]
  19. Fayet O., Ziegelhoffer T., Georgopoulos C. The groES and groEL heat shock gene products of Escherichia coli are essential for bacterial growth at all temperatures. J Bacteriol. 1989 Mar;171(3):1379–1385. doi: 10.1128/jb.171.3.1379-1385.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fling S. P., Gregerson D. S. Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer system without urea. Anal Biochem. 1986 May 15;155(1):83–88. doi: 10.1016/0003-2697(86)90228-9. [DOI] [PubMed] [Google Scholar]
  21. Flynn G. C., Chappell T. G., Rothman J. E. Peptide binding and release by proteins implicated as catalysts of protein assembly. Science. 1989 Jul 28;245(4916):385–390. doi: 10.1126/science.2756425. [DOI] [PubMed] [Google Scholar]
  22. Georgopoulos C., Ang D. The Escherichia coli groE chaperonins. Semin Cell Biol. 1990 Feb;1(1):19–25. [PubMed] [Google Scholar]
  23. 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]
  24. Goloubinoff P., Gatenby A. A., Lorimer G. H. GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli. Nature. 1989 Jan 5;337(6202):44–47. doi: 10.1038/337044a0. [DOI] [PubMed] [Google Scholar]
  25. Hemmingsen S. M., Woolford C., van der Vies S. M., Tilly K., Dennis D. T., Georgopoulos C. P., Hendrix R. W., Ellis R. J. Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature. 1988 May 26;333(6171):330–334. doi: 10.1038/333330a0. [DOI] [PubMed] [Google Scholar]
  26. Hendrix R. W. Purification and properties of groE, a host protein involved in bacteriophage assembly. J Mol Biol. 1979 Apr 15;129(3):375–392. doi: 10.1016/0022-2836(79)90502-3. [DOI] [PubMed] [Google Scholar]
  27. Hohn T., Hohn B., Engel A., Wurtz M., Smith P. R. Isolation and characterization of the host protein groE involved in bacteriophage lambda assembly. J Mol Biol. 1979 Apr 15;129(3):359–373. doi: 10.1016/0022-2836(79)90501-1. [DOI] [PubMed] [Google Scholar]
  28. Jaenicke R. Folding and association of proteins. Prog Biophys Mol Biol. 1987;49(2-3):117–237. doi: 10.1016/0079-6107(87)90011-3. [DOI] [PubMed] [Google Scholar]
  29. Kumamoto C. A., Gannon P. M. Effects of Escherichia coli secB mutations on pre-maltose binding protein conformation and export kinetics. J Biol Chem. 1988 Aug 15;263(23):11554–11558. [PubMed] [Google Scholar]
  30. Kurzchalia T. V., Wiedmann M., Girshovich A. S., Bochkareva E. S., Bielka H., Rapoport T. A. The signal sequence of nascent preprolactin interacts with the 54K polypeptide of the signal recognition particle. Nature. 1986 Apr 17;320(6063):634–636. doi: 10.1038/320634a0. [DOI] [PubMed] [Google Scholar]
  31. Kusukawa N., Yura T., Ueguchi C., Akiyama Y., Ito K. Effects of mutations in heat-shock genes groES and groEL on protein export in Escherichia coli. EMBO J. 1989 Nov;8(11):3517–3521. doi: 10.1002/j.1460-2075.1989.tb08517.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kuwajima K. The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure. Proteins. 1989;6(2):87–103. doi: 10.1002/prot.340060202. [DOI] [PubMed] [Google Scholar]
  33. Laminet A. A., Plückthun A. The precursor of beta-lactamase: purification, properties and folding kinetics. EMBO J. 1989 May;8(5):1469–1477. doi: 10.1002/j.1460-2075.1989.tb03530.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lang K., Schmid F. X., Fischer G. Catalysis of protein folding by prolyl isomerase. Nature. 1987 Sep 17;329(6136):268–270. doi: 10.1038/329268a0. [DOI] [PubMed] [Google Scholar]
  35. Lecker S., Lill R., Ziegelhoffer T., Georgopoulos C., Bassford P. J., Jr, Kumamoto C. A., Wickner W. Three pure chaperone proteins of Escherichia coli--SecB, trigger factor and GroEL--form soluble complexes with precursor proteins in vitro. EMBO J. 1989 Sep;8(9):2703–2709. doi: 10.1002/j.1460-2075.1989.tb08411.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McMullin T. W., Hallberg R. L. A highly evolutionarily conserved mitochondrial protein is structurally related to the protein encoded by the Escherichia coli groEL gene. Mol Cell Biol. 1988 Jan;8(1):371–380. doi: 10.1128/mcb.8.1.371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. O'Callaghan C. H., Morris A., Kirby S. M., Shingler A. H. Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substrate. Antimicrob Agents Chemother. 1972 Apr;1(4):283–288. doi: 10.1128/aac.1.4.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Pelham H. R. Speculations on the functions of the major heat shock and glucose-regulated proteins. Cell. 1986 Sep 26;46(7):959–961. doi: 10.1016/0092-8674(86)90693-8. [DOI] [PubMed] [Google Scholar]
  40. Randall L. L., Hardy S. J. Correlation of competence for export with lack of tertiary structure of the mature species: a study in vivo of maltose-binding protein in E. coli. Cell. 1986 Sep 12;46(6):921–928. doi: 10.1016/0092-8674(86)90074-7. [DOI] [PubMed] [Google Scholar]
  41. Rothman J. E. Polypeptide chain binding proteins: catalysts of protein folding and related processes in cells. Cell. 1989 Nov 17;59(4):591–601. doi: 10.1016/0092-8674(89)90005-6. [DOI] [PubMed] [Google Scholar]
  42. Römisch K., Webb J., Herz J., Prehn S., Frank R., Vingron M., Dobberstein B. Homology of 54K protein of signal-recognition particle, docking protein and two E. coli proteins with putative GTP-binding domains. Nature. 1989 Aug 10;340(6233):478–482. doi: 10.1038/340478a0. [DOI] [PubMed] [Google Scholar]
  43. Walter P., Gilmore R., Blobel G. Protein translocation across the endoplasmic reticulum. Cell. 1984 Aug;38(1):5–8. doi: 10.1016/0092-8674(84)90520-8. [DOI] [PubMed] [Google Scholar]
  44. Zimmermann R., Sagstetter M., Lewis M. J., Pelham H. R. Seventy-kilodalton heat shock proteins and an additional component from reticulocyte lysate stimulate import of M13 procoat protein into microsomes. EMBO J. 1988 Sep;7(9):2875–2880. doi: 10.1002/j.1460-2075.1988.tb03144.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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