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. 1991 Sep 1;88(17):7715–7718. doi: 10.1073/pnas.88.17.7715

Cold denaturation and 2H2O stabilization of a staphylococcal nuclease mutant.

L C Antonino 1, R A Kautz 1, T Nakano 1, R O Fox 1, A L Fink 1
PMCID: PMC52373  PMID: 1652762

Abstract

Cold denaturation is now recognized as a general property of proteins but has been observed only under destabilizing conditions, such as moderate denaturant concentration or low pH. By destabilizing the protein using site-directed mutagenesis, we have observed cold denaturation at pH 7.0 in the absence of denaturants in a mutant of staphylococcal nuclease, which we call NCA S28G for a hybrid protein between staphylococcal nuclease and concanavalin A in which there is the point mutation Ser-28----Gly. The temperature of maximum stability (tmax) as determined by circular dichroism (CD) was 18.1 degrees C, and the midpoints of the thermal unfolding transitions (tm) were 0.6 degrees C and 30.0 degrees C. These values may be compared with the tm of 52.5 degrees C for wild-type staphylococcal nuclease, for which no cold denaturation was observed under these conditions. When the stability of the mutant was examined in 2H2O by NMR, CD, or fluorescence, a substantial increase in the amount of folded protein at the tmax was noted as well as a decrease in tmax, reflecting increased stability.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Calderon R. O., Stolowich N. J., Gerlt J. A., Sturtevant J. M. Thermal denaturation of staphylococcal nuclease. Biochemistry. 1985 Oct 22;24(22):6044–6049. doi: 10.1021/bi00343a004. [DOI] [PubMed] [Google Scholar]
  2. Chen B. L., Schellman J. A. Low-temperature unfolding of a mutant of phage T4 lysozyme. 1. Equilibrium studies. Biochemistry. 1989 Jan 24;28(2):685–691. doi: 10.1021/bi00428a041. [DOI] [PubMed] [Google Scholar]
  3. Epstein H. F., Schechter A. N., Chen R. F., Anfinsen C. B. Folding of staphylococcal nuclease: kinetic studies of two processes in acid renaturation. J Mol Biol. 1971 Sep 28;60(3):499–508. doi: 10.1016/0022-2836(71)90184-7. [DOI] [PubMed] [Google Scholar]
  4. Evans P. A., Kautz R. A., Fox R. O., Dobson C. M. A magnetization-transfer nuclear magnetic resonance study of the folding of staphylococcal nuclease. Biochemistry. 1989 Jan 10;28(1):362–370. doi: 10.1021/bi00427a050. [DOI] [PubMed] [Google Scholar]
  5. Franks F., Hatley R. H., Friedman H. L. The thermodynamics of protein stability. Cold destabilization as a general phenomenon. Biophys Chem. 1988 Sep;31(3):307–315. doi: 10.1016/0301-4622(88)80037-1. [DOI] [PubMed] [Google Scholar]
  6. Fuchs S., Cuatrecasas P., Anfinsen C. B. An improved method for the purification of staphylococcal nuclease. J Biol Chem. 1967 Oct 25;242(20):4768–4770. [PubMed] [Google Scholar]
  7. Griko Y. V., Privalov P. L., Sturtevant J. M., Venyaminov SYu Cold denaturation of staphylococcal nuclease. Proc Natl Acad Sci U S A. 1988 May;85(10):3343–3347. doi: 10.1073/pnas.85.10.3343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hatley R. H., Franks F. The cold-induced denaturation of lactate dehydrogenase at sub-zero temperatures in the absence of perturbants. FEBS Lett. 1989 Oct 23;257(1):171–173. doi: 10.1016/0014-5793(89)81813-7. [DOI] [PubMed] [Google Scholar]
  9. Hynes T. R., Kautz R. A., Goodman M. A., Gill J. F., Fox R. O. Transfer of a beta-turn structure to a new protein context. Nature. 1989 May 4;339(6219):73–76. doi: 10.1038/339073a0. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Masson P., Laurentie M. Stability of butyrylcholinesterase: thermal inactivation in water and deuterium oxide. Biochim Biophys Acta. 1988 Nov 2;957(1):111–121. doi: 10.1016/0167-4838(88)90163-x. [DOI] [PubMed] [Google Scholar]
  12. Pace C. N., Laurents D. V. A new method for determining the heat capacity change for protein folding. Biochemistry. 1989 Mar 21;28(6):2520–2525. doi: 10.1021/bi00432a026. [DOI] [PubMed] [Google Scholar]
  13. Pace N. C., Tanford C. Thermodynamics of the unfolding of beta-lactoglobulin A in aqueous urea solutions between 5 and 55 degrees. Biochemistry. 1968 Jan;7(1):198–208. doi: 10.1021/bi00841a025. [DOI] [PubMed] [Google Scholar]
  14. Privalov P. L. Cold denaturation of proteins. Crit Rev Biochem Mol Biol. 1990;25(4):281–305. doi: 10.3109/10409239009090612. [DOI] [PubMed] [Google Scholar]
  15. Privalov P. L., Griko YuV, Venyaminov SYu, Kutyshenko V. P. Cold denaturation of myoglobin. J Mol Biol. 1986 Aug 5;190(3):487–498. doi: 10.1016/0022-2836(86)90017-3. [DOI] [PubMed] [Google Scholar]
  16. Privalov P. L., Tiktopulo E. I., Venyaminov SYu, Griko YuV, Makhatadze G. I., Khechinashvili N. N. Heat capacity and conformation of proteins in the denatured state. J Mol Biol. 1989 Feb 20;205(4):737–750. doi: 10.1016/0022-2836(89)90318-5. [DOI] [PubMed] [Google Scholar]
  17. Shortle D., Meeker A. K., Freire E. Stability mutants of staphylococcal nuclease: large compensating enthalpy-entropy changes for the reversible denaturation reaction. Biochemistry. 1988 Jun 28;27(13):4761–4768. doi: 10.1021/bi00413a027. [DOI] [PubMed] [Google Scholar]
  18. Woodfin B. M., Henderson R. F., Henderson T. R. Effects of D2O on the association-dissociation equilibrium in subunit proteins. J Biol Chem. 1970 Aug 10;245(15):3733–3737. [PubMed] [Google Scholar]
  19. de Gómez-Puyou M. T., Gómez-Puyou A., Cerbón J. Increased conformational stability of mitochondrial soluble ATPase (F1) by substitution of H2O for D2O. Arch Biochem Biophys. 1978 Apr 15;187(1):72–77. doi: 10.1016/0003-9861(78)90007-3. [DOI] [PubMed] [Google Scholar]

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