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. 1999 Apr 15;339(Pt 2):309–317.

Prediction and experimental testing of Bacillus acidocaldarius thioredoxin stability.

E Pedone 1, R Cannio 1, M Saviano 1, M Rossi 1, S Bartolucci 1
PMCID: PMC1220159  PMID: 10191261

Abstract

In order to investigate further the determinants of protein stability, four mutants of thioredoxin from Bacillus acidocaldarius were designed: K18G, R82E, K18G/R82E, and D102X, in which the last four amino acids were deleted. The mutants were constructed on the basis of molecular dynamic studies and the prediction of the structure of thioredoxin from B. acidocaldarius, performed by a comparative molecular modelling technique using Escherichia coli thioredoxin as the reference protein. The mutants obtained by PCR strategy were expressed in E. coli and then characterized. CD spectroscopy, spectrofluorimetry and thermodynamic comparative studies permitted comparison of the relative physicochemical behaviour of the four proteins with that of the wild-type protein. As predicted for the molecular dynamic analysis at 500 K in vacuo, the wild-type structure was more stable than that of the mutants; in fact the Tm of the four proteins showed a decrease of about 15 degrees C for the double and the truncated mutants, and a decrease of about 12 degrees C for the single mutants. A difference in the resistance of the proteins to denaturants such as guanidine HCl and urea was revealed; the wild-type protein always proved to be the most resistant. The results obtained show the importance of hydrogen bonds and ion pairs in determining protein stability and confirm that simulation methods are able to direct protein engineering in site-directed mutagenesis.

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Selected References

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

  1. Bartolucci S., Guagliardi A., Pedone E., De Pascale D., Cannio R., Camardella L., Rossi M., Nicastro G., de Chiara C., Facci P. Thioredoxin from Bacillus acidocaldarius: characterization, high-level expression in Escherichia coli and molecular modelling. Biochem J. 1997 Nov 15;328(Pt 1):277–285. doi: 10.1042/bj3280277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Becktel W. J., Schellman J. A. Protein stability curves. Biopolymers. 1987 Nov;26(11):1859–1877. doi: 10.1002/bip.360261104. [DOI] [PubMed] [Google Scholar]
  3. Brent R., Ptashne M. Mechanism of action of the lexA gene product. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4204–4208. doi: 10.1073/pnas.78.7.4204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chang C. T., Wu C. S., Yang J. T. Circular dichroic analysis of protein conformation: inclusion of the beta-turns. Anal Biochem. 1978 Nov;91(1):13–31. doi: 10.1016/0003-2697(78)90812-6. [DOI] [PubMed] [Google Scholar]
  5. Eklund H., Gleason F. K., Holmgren A. Structural and functional relations among thioredoxins of different species. Proteins. 1991;11(1):13–28. doi: 10.1002/prot.340110103. [DOI] [PubMed] [Google Scholar]
  6. Hellinga H. W., Wynn R., Richards F. M. The hydrophobic core of Escherichia coli thioredoxin shows a high tolerance to nonconservative single amino acid substitutions. Biochemistry. 1992 Nov 17;31(45):11203–11209. doi: 10.1021/bi00160a034. [DOI] [PubMed] [Google Scholar]
  7. Holmgren A. Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide. J Biol Chem. 1979 Oct 10;254(19):9627–9632. [PubMed] [Google Scholar]
  8. Holmgren A. Tryptophan fluorescence study of conformational transitions of the oxidized and reduced form of thioredoxin. J Biol Chem. 1972 Apr 10;247(7):1992–1998. [PubMed] [Google Scholar]
  9. Jaenicke R. Stability and folding of ultrastable proteins: eye lens crystallins and enzymes from thermophiles. FASEB J. 1996 Jan;10(1):84–92. doi: 10.1096/fasebj.10.1.8566552. [DOI] [PubMed] [Google Scholar]
  10. Jeng M. F., Campbell A. P., Begley T., Holmgren A., Case D. A., Wright P. E., Dyson H. J. High-resolution solution structures of oxidized and reduced Escherichia coli thioredoxin. Structure. 1994 Sep 15;2(9):853–868. doi: 10.1016/s0969-2126(94)00086-7. [DOI] [PubMed] [Google Scholar]
  11. Johnson W. C., Jr Protein secondary structure and circular dichroism: a practical guide. Proteins. 1990;7(3):205–214. doi: 10.1002/prot.340070302. [DOI] [PubMed] [Google Scholar]
  12. Johnson W. C., Jr Secondary structure of proteins through circular dichroism spectroscopy. Annu Rev Biophys Biophys Chem. 1988;17:145–166. doi: 10.1146/annurev.bb.17.060188.001045. [DOI] [PubMed] [Google Scholar]
  13. Katti S. K., LeMaster D. M., Eklund H. Crystal structure of thioredoxin from Escherichia coli at 1.68 A resolution. J Mol Biol. 1990 Mar 5;212(1):167–184. doi: 10.1016/0022-2836(90)90313-B. [DOI] [PubMed] [Google Scholar]
  14. Kawamura S., Kakuta Y., Tanaka I., Hikichi K., Kuhara S., Yamasaki N., Kimura M. Glycine-15 in the bend between two alpha-helices can explain the thermostability of DNA binding protein HU from Bacillus stearothermophilus. Biochemistry. 1996 Jan 30;35(4):1195–1200. doi: 10.1021/bi951581l. [DOI] [PubMed] [Google Scholar]
  15. Kotik M., Zuber H. Mutations that significantly change the stability, flexibility and quaternary structure of the l-lactate dehydrogenase from Bacillus megaterium. Eur J Biochem. 1993 Jan 15;211(1-2):267–280. doi: 10.1111/j.1432-1033.1993.tb19895.x. [DOI] [PubMed] [Google Scholar]
  16. Ladbury J. E., Wynn R., Hellinga H. W., Sturtevant J. M. Stability of oxidized Escherichia coli thioredoxin and its dependence on protonation of the aspartic acid residue in the 26 position. Biochemistry. 1993 Jul 27;32(29):7526–7530. doi: 10.1021/bi00080a026. [DOI] [PubMed] [Google Scholar]
  17. Ladbury J. E., Wynn R., Thomson J. A., Sturtevant J. M. Substitution of charged residues into the hydrophobic core of Escherichia coli thioredoxin results in a change in heat capacity of the native protein. Biochemistry. 1995 Feb 21;34(7):2148–2152. doi: 10.1021/bi00007a007. [DOI] [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Menéndez-Arias L., Argos P. Engineering protein thermal stability. Sequence statistics point to residue substitutions in alpha-helices. J Mol Biol. 1989 Mar 20;206(2):397–406. doi: 10.1016/0022-2836(89)90488-9. [DOI] [PubMed] [Google Scholar]
  20. Pappenberger G., Schurig H., Jaenicke R. Disruption of an ionic network leads to accelerated thermal denaturation of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima. J Mol Biol. 1997 Dec 12;274(4):676–683. doi: 10.1006/jmbi.1997.1421. [DOI] [PubMed] [Google Scholar]
  21. Pedone E. M., Bartolucci S., Rossi M., Saviano M. Computational analysis of the thermal stability in thioredoxins: a molecular dynamics approach. J Biomol Struct Dyn. 1998 Oct;16(2):437–446. doi: 10.1080/07391102.1998.10508259. [DOI] [PubMed] [Google Scholar]
  22. Rabilloud T., Vuillard L., Gilly C., Lawrence J. J. Silver-staining of proteins in polyacrylamide gels: a general overview. Cell Mol Biol (Noisy-le-grand) 1994 Feb;40(1):57–75. [PubMed] [Google Scholar]
  23. 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]
  24. Saviano M., Aida M., Corongiu G. Molecular dynamics simulation in vacuo and in solution of cyclolinopeptide A: a conformational study. Biopolymers. 1991 Jul;31(8):1017–1024. doi: 10.1002/bip.360310811. [DOI] [PubMed] [Google Scholar]
  25. Serrano L., Horovitz A., Avron B., Bycroft M., Fersht A. R. Estimating the contribution of engineered surface electrostatic interactions to protein stability by using double-mutant cycles. Biochemistry. 1990 Oct 9;29(40):9343–9352. doi: 10.1021/bi00492a006. [DOI] [PubMed] [Google Scholar]
  26. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  27. Takagi H., Takahashi T., Momose H., Inouye M., Maeda Y., Matsuzawa H., Ohta T. Enhancement of the thermostability of subtilisin E by introduction of a disulfide bond engineered on the basis of structural comparison with a thermophilic serine protease. J Biol Chem. 1990 Apr 25;265(12):6874–6878. [PubMed] [Google Scholar]
  28. Wallon G., Kryger G., Lovett S. T., Oshima T., Ringe D., Petsko G. A. Crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus. J Mol Biol. 1997 Mar 14;266(5):1016–1031. doi: 10.1006/jmbi.1996.0797. [DOI] [PubMed] [Google Scholar]
  29. de Prat Gay G., Johnson C. M., Fersht A. R. Contribution of a proline residue and a salt bridge to the stability of a type I reverse turn in chymotrypsin inhibitor-2. Protein Eng. 1994 Jan;7(1):103–108. doi: 10.1093/protein/7.1.103. [DOI] [PubMed] [Google Scholar]

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