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. 1994 Nov;3(11):1953–1960. doi: 10.1002/pro.5560031107

Folding and aggregation of TEM beta-lactamase: analogies with the formation of inclusion bodies in Escherichia coli.

G Georgiou 1, P Valax 1, M Ostermeier 1, P M Horowitz 1
PMCID: PMC2142649  PMID: 7703842

Abstract

The enzyme TEM beta-lactamase has been used as a model for understanding the pathway leading to formation of inclusion bodies in Escherichia coli. The equilibrium denaturation of TEM beta-lactamase revealed that an intermediate that has lost enzymatic activity, native protein fluorescence, and UV absorption, but retains 60% of the native circular dichroism signal, becomes populated at intermediate (1.0-1.4 M) concentrations of guanidium chloride (GdmCl). This species exhibits a large increase in bis-1-anilino-8-naphthalene sulfonic acid fluorescence, indicating the presence of exposed hydrophobic surfaces. When TEM beta-lactamase was unfolded in different initial concentrations of GdmCl and refolded to the same final conditions by dialysis a distinct minimum in the yield of active protein was observed for initial concentrations of GdmCl in the 1.0-1.5 M range. It was shown that the lower reactivation yield was solely due to the formation of noncovalently linked aggregates. We propose that the aggregation of TEM beta-lactamase involves the association of a compact state having partially exposed hydrophobic surfaces. This hypothesis is consistent with our recent findings that TEM beta-lactamase inclusion bodies contains extensive secondary structure (Przybycien TM, Dunn JP, Valax P, Georgiou G, 1994, Protein Eng 7:131-136). Finally, we have also shown that protein aggregation was enhanced at higher temperatures and in the presence of 5 mM dithiothreitol and was inhibited by the addition of sucrose. These conditions exert a similar effect on the formation of inclusion bodies in vivo.

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Selected References

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  1. Blum P., Velligan M., Lin N., Matin A. DnaK-mediated alterations in human growth hormone protein inclusion bodies. Biotechnology (N Y) 1992 Mar;10(3):301–304. doi: 10.1038/nbt0392-301. [DOI] [PubMed] [Google Scholar]
  2. Bowden G. A., Georgiou G. Folding and aggregation of beta-lactamase in the periplasmic space of Escherichia coli. J Biol Chem. 1990 Oct 5;265(28):16760–16766. [PubMed] [Google Scholar]
  3. Bowden G. A., Paredes A. M., Georgiou G. Structure and morphology of protein inclusion bodies in Escherichia coli. Biotechnology (N Y) 1991 Aug;9(8):725–730. doi: 10.1038/nbt0891-725. [DOI] [PubMed] [Google Scholar]
  4. Calciano L. J., Escobar W. A., Millhauser G. L., Miick S. M., Rubaloff J., Todd A. P., Fink A. L. Side-chain mobility of the beta-lactamase A state probed by electron spin resonance spectroscopy. Biochemistry. 1993 Jun 1;32(21):5644–5649. doi: 10.1021/bi00072a021. [DOI] [PubMed] [Google Scholar]
  5. Chalmers J. J., Kim E., Telford J. N., Wong E. Y., Tacon W. C., Shuler M. L., Wilson D. B. Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor. Appl Environ Microbiol. 1990 Jan;56(1):104–111. doi: 10.1128/aem.56.1.104-111.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chrunyk B. A., Evans J., Lillquist J., Young P., Wetzel R. Inclusion body formation and protein stability in sequence variants of interleukin-1 beta. J Biol Chem. 1993 Aug 25;268(24):18053–18061. [PubMed] [Google Scholar]
  7. DeFelippis M. R., Alter L. A., Pekar A. H., Havel H. A., Brems D. N. Evidence for a self-associating equilibrium intermediate during folding of human growth hormone. Biochemistry. 1993 Feb 16;32(6):1555–1562. doi: 10.1021/bi00057a021. [DOI] [PubMed] [Google Scholar]
  8. Georgiou G., Telford J. N., Shuler M. L., Wilson D. B. Localization of inclusion bodies in Escherichia coli overproducing beta-lactamase or alkaline phosphatase. Appl Environ Microbiol. 1986 Nov;52(5):1157–1161. doi: 10.1128/aem.52.5.1157-1161.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Horowitz P. M., Butler M. Interactive intermediates are formed during the urea unfolding of rhodanese. J Biol Chem. 1993 Feb 5;268(4):2500–2504. [PubMed] [Google Scholar]
  10. Jelsch C., Mourey L., Masson J. M., Samama J. P. Crystal structure of Escherichia coli TEM1 beta-lactamase at 1.8 A resolution. Proteins. 1993 Aug;16(4):364–383. doi: 10.1002/prot.340160406. [DOI] [PubMed] [Google Scholar]
  11. Mitraki A., Betton J. M., Desmadril M., Yon J. M. Quasi-irreversibility in the unfolding-refolding transition of phosphoglycerate kinase induced by guanidine hydrochloride. Eur J Biochem. 1987 Feb 16;163(1):29–34. doi: 10.1111/j.1432-1033.1987.tb10732.x. [DOI] [PubMed] [Google Scholar]
  12. Mitraki A., Danner M., King J., Seckler R. Temperature-sensitive mutations and second-site suppressor substitutions affect folding of the P22 tailspike protein in vitro. J Biol Chem. 1993 Sep 25;268(27):20071–20075. [PubMed] [Google Scholar]
  13. Mitraki A., King J. Amino acid substitutions influencing intracellular protein folding pathways. FEBS Lett. 1992 Jul 27;307(1):20–25. doi: 10.1016/0014-5793(92)80894-m. [DOI] [PubMed] [Google Scholar]
  14. Oberg K., Chrunyk B. A., Wetzel R., Fink A. L. Nativelike secondary structure in interleukin-1 beta inclusion bodies by attenuated total reflectance FTIR. Biochemistry. 1994 Mar 8;33(9):2628–2634. doi: 10.1021/bi00175a035. [DOI] [PubMed] [Google Scholar]
  15. Przybycien T. M., Dunn J. P., Valax P., Georgiou G. Secondary structure characterization of beta-lactamase inclusion bodies. Protein Eng. 1994 Jan;7(1):131–136. doi: 10.1093/protein/7.1.131. [DOI] [PubMed] [Google Scholar]
  16. Ptitsyn O. B., Pain R. H., Semisotnov G. V., Zerovnik E., Razgulyaev O. I. Evidence for a molten globule state as a general intermediate in protein folding. FEBS Lett. 1990 Mar 12;262(1):20–24. doi: 10.1016/0014-5793(90)80143-7. [DOI] [PubMed] [Google Scholar]
  17. Rinas U., Tsai L. B., Lyons D., Fox G. M., Stearns G., Fieschko J., Fenton D., Bailey J. E. Cysteine to serine substitutions in basic fibroblast growth factor: effect on inclusion body formation and proteolytic susceptibility during in vitro refolding. Biotechnology (N Y) 1992 Apr;10(4):435–440. doi: 10.1038/nbt0492-435. [DOI] [PubMed] [Google Scholar]
  18. Robson B., Pain R. H. The mechanism of folding of globular proteins. Equilibria and kinetics of conformational transitions of penicillinase from Staphylococcus aureus involving a state of intermediate conformation. Biochem J. 1976 May 1;155(2):331–344. doi: 10.1042/bj1550331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Schultz S. C., Dalbadie-McFarland G., Neitzel J. J., Richards J. H. Stability of wild-type and mutant RTEM-1 beta-lactamases: effect of the disulfide bond. Proteins. 1987;2(4):290–297. doi: 10.1002/prot.340020405. [DOI] [PubMed] [Google Scholar]
  20. Sigal I. S., DeGrado W. F., Thomas B. J., Petteway S. R., Jr Purification and properties of thiol beta-lactamase. A mutant of pBR322 beta-lactamase in which the active site serine has been replaced with cysteine. J Biol Chem. 1984 Apr 25;259(8):5327–5332. [PubMed] [Google Scholar]
  21. Strynadka N. C., Adachi H., Jensen S. E., Johns K., Sielecki A., Betzel C., Sutoh K., James M. N. Molecular structure of the acyl-enzyme intermediate in beta-lactam hydrolysis at 1.7 A resolution. Nature. 1992 Oct 22;359(6397):700–705. doi: 10.1038/359700a0. [DOI] [PubMed] [Google Scholar]
  22. Tokatlidis K., Dhurjati P., Millet J., Béguin P., Aubert J. P. High activity of inclusion bodies formed in Escherichia coli overproducing Clostridium thermocellum endoglucanase D. FEBS Lett. 1991 Apr 22;282(1):205–208. doi: 10.1016/0014-5793(91)80478-l. [DOI] [PubMed] [Google Scholar]
  23. Valax P., Georgiou G. Molecular characterization of beta-lactamase inclusion bodies produced in Escherichia coli. 1. Composition. Biotechnol Prog. 1993 Sep-Oct;9(5):539–547. doi: 10.1021/bp00023a014. [DOI] [PubMed] [Google Scholar]
  24. Wetzel R. Mutations and off-pathway aggregation of proteins. Trends Biotechnol. 1994 May;12(5):193–198. doi: 10.1016/0167-7799(94)90082-5. [DOI] [PubMed] [Google Scholar]

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