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. 1997 Dec;6(12):2617–2623. doi: 10.1002/pro.5560061213

Thermal unfolding of dodecameric glutamine synthetase: inhibition of aggregation by urea.

N J Nosworthy 1, A Ginsburg 1
PMCID: PMC2143615  PMID: 9416610

Abstract

Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 M(r)) at pH 7 and approximately 0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm approximately 42 degrees C; delta H approximately equal to 0.9 J/g) followed by a large irreversible transition (Tm approximately 81 degrees C; delta H approximately equal to 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55 degrees C) and inhibits aggregation accompanying unfolding at < or = 0.2 mg protein/mL. With increasing temperature (30-70 degrees C) or incubation times at 25 degrees C (5-35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo-first-order (t1/2 = 1,030 s at 20.0 degrees C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax approximately 64 degrees C; delta H = 17 +/- 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (delta H = 57 +/- 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4: 1544-1552), after correcting for the binding of urea to protein sites exposed during unfolding (-42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.

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Selected References

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  1. Aghajanian S. A., Martin S. R., Engel P. C. Urea-induced inactivation and denaturation of clostridial glutamate dehydrogenase: the absence of stable dimeric or trimeric intermediates. Biochem J. 1995 Nov 1;311(Pt 3):905–910. doi: 10.1042/bj3110905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baldwin R. L. The nature of protein folding pathways: the classical versus the new view. J Biomol NMR. 1995 Feb;5(2):103–109. doi: 10.1007/BF00208801. [DOI] [PubMed] [Google Scholar]
  3. Colombo G., Villafranca J. J. Amino acid sequence of Escherichia coli glutamine synthetase deduced from the DNA nucleotide sequence. J Biol Chem. 1986 Aug 15;261(23):10587–10591. [PubMed] [Google Scholar]
  4. Dobson C. M. Finding the right fold. Nat Struct Biol. 1995 Jul;2(7):513–517. doi: 10.1038/nsb0795-513. [DOI] [PubMed] [Google Scholar]
  5. Fisher M. T. The effect of groES on the groEL-dependent assembly of dodecameric glutamine synthetase in the presence of ATP and ADP. J Biol Chem. 1994 May 6;269(18):13629–13636. [PubMed] [Google Scholar]
  6. Freire E., van Osdol W. W., Mayorga O. L., Sanchez-Ruiz J. M. Calorimetrically determined dynamics of complex unfolding transitions in proteins. Annu Rev Biophys Biophys Chem. 1990;19:159–188. doi: 10.1146/annurev.bb.19.060190.001111. [DOI] [PubMed] [Google Scholar]
  7. Ginsburg A., Yeh J., Hennig S. B., Denton M. D. Some effects of adenylylation on the biosynthetic properties of the glutamine synthetase from Escherichia coli. Biochemistry. 1970 Feb 3;9(3):633–649. doi: 10.1021/bi00805a025. [DOI] [PubMed] [Google Scholar]
  8. Ginsburg A., Zolkiewski M. Differential scanning calorimetry study of reversible, partial unfolding transitions in dodecameric glutamine synthetase from Escherichia coli. Biochemistry. 1991 Oct 1;30(39):9421–9429. doi: 10.1021/bi00103a005. [DOI] [PubMed] [Google Scholar]
  9. Haschemeyer R. H., Wall J. S., Hainfeld J., Maurizi M. R. Scanning transmission electron microscopy of submolecular oligomers of stabilized glutamine synthetase from Escherichia coli. J Biol Chem. 1982 Jun 25;257(12):7252–7253. [PubMed] [Google Scholar]
  10. Hunt J. B., Ginsburg A. Mn2+ and substrate interactions with glutamine synthetase from Escherichia coli. J Biol Chem. 1980 Jan 25;255(2):590–594. [PubMed] [Google Scholar]
  11. Hunt J. B., Smyrniotis P. Z., Ginsburg A., Stadtman E. R. Metal ion requirement by glutamine synthetase of Escherichia coli in catalysis of gamma-glutamyl transfer. Arch Biochem Biophys. 1975 Jan;166(1):102–124. doi: 10.1016/0003-9861(75)90370-7. [DOI] [PubMed] [Google Scholar]
  12. Jaenicke R. Protein folding: local structures, domains, subunits, and assemblies. Biochemistry. 1991 Apr 2;30(13):3147–3161. doi: 10.1021/bi00227a001. [DOI] [PubMed] [Google Scholar]
  13. Kawahara K., Tanford C. Viscosity and density of aqueous solutions of urea and guanidine hydrochloride. J Biol Chem. 1966 Jul 10;241(13):3228–3232. [PubMed] [Google Scholar]
  14. Makhatadze G. I., Privalov P. L. Protein interactions with urea and guanidinium chloride. A calorimetric study. J Mol Biol. 1992 Jul 20;226(2):491–505. doi: 10.1016/0022-2836(92)90963-k. [DOI] [PubMed] [Google Scholar]
  15. Maurizi M. R., Ginsburg A. Active-site ligand binding and subunit interactions in glutamine synthetase from Escherichia coli. Curr Top Cell Regul. 1985;26:191–206. doi: 10.1016/b978-0-12-152826-3.50022-x. [DOI] [PubMed] [Google Scholar]
  16. Maurizi M. R., Ginsburg A. Adenosine 5'-triphosphate analogues as structural probes for Escherichia coli glutamine synthetase. Biochemistry. 1986 Jan 14;25(1):131–140. doi: 10.1021/bi00349a020. [DOI] [PubMed] [Google Scholar]
  17. Pinkofsky H. B., Ginsburg A., Reardon I., Heinrikson R. L. Lysyl residue 47 is near the subunit ATP-binding site of glutamine synthetase from Escherichia coli. J Biol Chem. 1984 Aug 10;259(15):9616–9622. [PubMed] [Google Scholar]
  18. Privalov P. L. Intermediate states in protein folding. J Mol Biol. 1996 May 24;258(5):707–725. doi: 10.1006/jmbi.1996.0280. [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. Sackett D. L., Wolff J. Nile red as a polarity-sensitive fluorescent probe of hydrophobic protein surfaces. Anal Biochem. 1987 Dec;167(2):228–234. doi: 10.1016/0003-2697(87)90157-6. [DOI] [PubMed] [Google Scholar]
  21. Shapiro B. M., Ginsburg A. Effects of specific divalent cations on some physical and chemical properties of glutamine synthetase from Escherichia coli. Taut and relaxed enzyme forms. Biochemistry. 1968 Jun;7(6):2153–2167. doi: 10.1021/bi00846a018. [DOI] [PubMed] [Google Scholar]
  22. Shrake A., Fisher M. T., McFarland P. J., Ginsburg A. Partial unfolding of dodecameric glutamine synthetase from Escherichia coli: temperature-induced, reversible transitions of two domains. Biochemistry. 1989 Jul 25;28(15):6281–6294. doi: 10.1021/bi00441a021. [DOI] [PubMed] [Google Scholar]
  23. Sosnick T. R., Mayne L., Hiller R., Englander S. W. The barriers in protein folding. Nat Struct Biol. 1994 Mar;1(3):149–156. doi: 10.1038/nsb0394-149. [DOI] [PubMed] [Google Scholar]
  24. Tanford C. Protein denaturation. Adv Protein Chem. 1968;23:121–282. doi: 10.1016/s0065-3233(08)60401-5. [DOI] [PubMed] [Google Scholar]
  25. Villafranca J. J., Ash D. E., Wedler F. C. Manganese (II) and substrate interaction with unadenylylated glutamine synthetase (Escherichia coli w). II. Electron paramagnetic resonance and nuclear magnetic resonance studies of enzyme-bound manganese(II) with substrates and a potential transition-state analogue, methionine sulfoximine. Biochemistry. 1976 Feb 10;15(3):544–553. doi: 10.1021/bi00648a014. [DOI] [PubMed] [Google Scholar]
  26. Woolfolk C. A., Shapiro B., Stadtman E. R. Regulation of glutamine synthetase. I. Purification and properties of glutamine synthetase from Escherichia coli. Arch Biochem Biophys. 1966 Sep 26;116(1):177–192. doi: 10.1016/0003-9861(66)90026-9. [DOI] [PubMed] [Google Scholar]
  27. Woolfolk C. A., Stadtman E. R. Regulation of glutamine synthetase. IV. Reversible dissociation and inactivation of glutamine synthetase from Escherichia coli by the concerted action of EDTA and urea. Arch Biochem Biophys. 1967 Oct;122(1):174–189. doi: 10.1016/0003-9861(67)90137-3. [DOI] [PubMed] [Google Scholar]
  28. Yamashita M. M., Almassy R. J., Janson C. A., Cascio D., Eisenberg D. Refined atomic model of glutamine synthetase at 3.5 A resolution. J Biol Chem. 1989 Oct 25;264(30):17681–17690. doi: 10.2210/pdb2gls/pdb. [DOI] [PubMed] [Google Scholar]
  29. Zolkiewski M., Ginsburg A. Thermodynamic effects of active-site ligands on the reversible, partial unfolding of dodecameric glutamine synthetase from Escherichia coli: calorimetric studies. Biochemistry. 1992 Dec 8;31(48):11991–12000. doi: 10.1021/bi00163a006. [DOI] [PubMed] [Google Scholar]
  30. Zolkiewski M., Nosworthy N. J., Ginsburg A. Urea-induced dissociation and unfolding of dodecameric glutamine synthetase from Escherichia coli: calorimetric and spectral studies. Protein Sci. 1995 Aug;4(8):1544–1552. doi: 10.1002/pro.5560040812. [DOI] [PMC free article] [PubMed] [Google Scholar]

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