Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1995 Dec;4(12):2510–2516. doi: 10.1002/pro.5560041207

Conservative substitutions in the hydrophobic core of Rhodobacter sphaeroides thioredoxin produce distinct functional effects.

K Assemat 1, P M Alzari 1, J Clément-Métral 1
PMCID: PMC2143044  PMID: 8580841

Abstract

The internal residue Phe 25 in Rhodobacter sphaeroides thioredoxin was changed to five amino acids (Ala, Val, Leu, Ile, Tyr) by site-directed mutagenesis, and the mutant proteins were characterized in vitro and in vivo using the mutant trxA genes in an Escherichia coli TrxA- background. The substitution F25A severely impaired the functional properties of the enzyme. Strains expressing all other mutations can grow on methionine sulfoxide with growth efficiencies of 45-60% that of the wild type at 37 degrees, and essentially identical at 42 degrees. At both temperatures, however, strains harboring the substitutions F25V and F25Y had lower growth rates and formed smaller colonies. In another in vivo assay, only the wild type and the F25I substitution allowed growth of phage T3/7 at 37 degrees, demonstrating that subtle modifications of the protein interior at position 25 Ile/Leu or Phe/Tyr) can produce significant biological effects. All F25 mutants were good substrates for E. coli thioredoxin reductase. Although turnover rates and apparent Km values were significantly lower for all mutants compared to the wild type, catalytic efficiency of thioredoxin reductase was similar for all substrates. Determination of the free energy of unfolding showed that the aliphatic substitutions (Val, Leu, Ile) significantly destabilized the protein, whereas the F25Y substitution did not affect protein stability. Thus, thermodynamic stability of R. sphaeroides thioredoxin variants is not correlated with the distinct functional effects observed both in vivo and in vitro.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

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

  1. Clement-Metral J. D., Holmgren A., Cambillau C., Jörnvall H., Eklund H., Thomas D., Lederer F. Amino acid sequence determination and three-dimensional modelling of thioredoxin from the photosynthetic bacterium Rhodobacter sphaeroides Y. Eur J Biochem. 1988 Mar 1;172(2):413–419. doi: 10.1111/j.1432-1033.1988.tb13902.x. [DOI] [PubMed] [Google Scholar]
  2. Dao-pin S., Anderson D. E., Baase W. A., Dahlquist F. W., Matthews B. W. Structural and thermodynamic consequences of burying a charged residue within the hydrophobic core of T4 lysozyme. Biochemistry. 1991 Dec 10;30(49):11521–11529. doi: 10.1021/bi00113a006. [DOI] [PubMed] [Google Scholar]
  3. Eklund H., Cambillau C., Sjöberg B. M., Holmgren A., Jörnvall H., Hög J. O., Brändén C. I. Conformational and functional similarities between glutaredoxin and thioredoxins. EMBO J. 1984 Jul;3(7):1443–1449. doi: 10.1002/j.1460-2075.1984.tb01994.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Gleason F. K. Mutation of conserved residues in Escherichia coli thioredoxin: effects on stability and function. Protein Sci. 1992 May;1(5):609–616. doi: 10.1002/pro.5560010507. [DOI] [PMC free article] [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. 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]
  8. Huber H. E., Russel M., Model P., Richardson C. C. Interaction of mutant thioredoxins of Escherichia coli with the gene 5 protein of phage T7. The redox capacity of thioredoxin is not required for stimulation of DNA polymerase activity. J Biol Chem. 1986 Nov 15;261(32):15006–15012. [PubMed] [Google Scholar]
  9. 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]
  10. Kelley R. F., Richards F. M. Replacement of proline-76 with alanine eliminates the slowest kinetic phase in thioredoxin folding. Biochemistry. 1987 Oct 20;26(21):6765–6774. doi: 10.1021/bi00395a028. [DOI] [PubMed] [Google Scholar]
  11. Kelley R. F., Stellwagen E. Conformational transitions of thioredoxin in guanidine hydrochloride. Biochemistry. 1984 Oct 23;23(22):5095–5102. doi: 10.1021/bi00317a003. [DOI] [PubMed] [Google Scholar]
  12. Kellis J. T., Jr, Nyberg K., Sali D., Fersht A. R. Contribution of hydrophobic interactions to protein stability. Nature. 1988 Jun 23;333(6175):784–786. doi: 10.1038/333784a0. [DOI] [PubMed] [Google Scholar]
  13. Krause G., Holmgren A. Substitution of the conserved tryptophan 31 in Escherichia coli thioredoxin by site-directed mutagenesis and structure-function analysis. J Biol Chem. 1991 Mar 5;266(7):4056–4066. [PubMed] [Google Scholar]
  14. Lim W. A., Farruggio D. C., Sauer R. T. Structural and energetic consequences of disruptive mutations in a protein core. Biochemistry. 1992 May 5;31(17):4324–4333. doi: 10.1021/bi00132a025. [DOI] [PubMed] [Google Scholar]
  15. Lim W. A., Sauer R. T. The role of internal packing interactions in determining the structure and stability of a protein. J Mol Biol. 1991 May 20;219(2):359–376. doi: 10.1016/0022-2836(91)90570-v. [DOI] [PubMed] [Google Scholar]
  16. Pille S., Chuat J. C., Breton A. M., Clément-Métral J. D., Galibert F. Cloning, nucleotide sequence, and expression of the Rhodobacter sphaeroides Y thioredoxin gene. J Bacteriol. 1990 Mar;172(3):1556–1561. doi: 10.1128/jb.172.3.1556-1561.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Russel M., Model P. The role of thioredoxin in filamentous phage assembly. Construction, isolation, and characterization of mutant thioredoxins. J Biol Chem. 1986 Nov 15;261(32):14997–15005. [PubMed] [Google Scholar]
  18. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  19. Slaby I., Holmgren A. Thioredoxin reductase-dependent insulin disulfide reduction by phage T7 DNA polymerase reflects dissociation of the enzyme into subunits. J Biol Chem. 1989 Oct 5;264(28):16502–16506. [PubMed] [Google Scholar]
  20. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  21. Wynn R., Richards F. M. Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques. Protein Sci. 1993 Mar;2(3):395–403. doi: 10.1002/pro.5560020311. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES