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Rapid Analysis of Single-Cysteine Variants of Recombinant Proteins

  • Thomas W. Keough
  • Yiping Sun
  • Bobby L. Barnett
  • Martin P. Lacey
  • Mark D. Bauer
  • Ellen S. Wang
  • Christopher R. Erwin
Part of the Methods in Molecular Biology™ book series (MIMB, volume 61)

Abstract

Protein engineering methods have been widely used to study individual structural factors that contribute to protein stability (1,2). An important goal of that research is to enhance the commercial or medicinal utility of wild-type (WT) proteins by increasing then stability in a rational, step-by-step fashion (3). We recently characterized a series of single-cysteine variants of subtilisin BPN′, a proteolytic enzyme used in commercial laundry formulations. They had been prepared (4) in part because random mutagenesis experiments indicated that some of these variants were more stable than the WT enzyme (3). We wanted to compare the stabilities of the mutants with those observed after engineering single disulfide bonds into the subtilisin BPN′ backbone (5,6).

Keywords

Tryptic Peptide Sinapinic Acid MALDI Mass Spectrum Surface Accessibility Tryptic Fragment 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Alber, T. (1989) Mutational effects on protein stability. Annu. Rev. Biochem. 58, 765–798.PubMedCrossRefGoogle Scholar
  2. 2.
    Pantoliano, M. W., Ladner, R. C., Bryan, P. N., Rollence, M. L., Wood, J. F., and Poulos, T. L. (1987) Protein engineering of subtilisin BPN′: enhanced stabilization through the introduction of two cysteines to form a disulfide bond. Biochemistry 26, 2077–2082.PubMedCrossRefGoogle Scholar
  3. 3.
    Pantoliano, M. W., Whitlow, M., Wood, J. F., Dodd, S. W., Hardman, K. D., Rollence, M. L., and Bryan, P. N. (1989) Large increases in general stability of subtilisin BPN′ through incremental changes in the free energy of unfolding. Biochemistry 28, 7205–7212.PubMedCrossRefGoogle Scholar
  4. 4.
    Brode, P. F., Erwin, C. R., Rauch, D. S., Lucas, D. S., and Rubingh, D. N. (1994) Site-specific variants of subtilisin BPN′ with enhanced surface stability. J. Biol. Chem. 269, 23,538–23,543.PubMedGoogle Scholar
  5. 5.
    Katz, B. A. and Kossiakoff, A. (1986) The crystallographically determined structures of atypical strained disulfides engineered into subtilisin. J. Biol Chem. 261, 15,480–15,485.PubMedGoogle Scholar
  6. 6.
    Mitchinson, C. and Wells, J. A. (1989) Protein engineering disulfide bonds in subtilisin BPN′. Biochemistry, 28, 4807–4815.PubMedCrossRefGoogle Scholar
  7. 7.
    Falke, J. J. and Koshland, D., Jr (1987) Global flexibility in a sensory receptor a sate-directed cross-linking approach. Science 237, 1596–1600.PubMedCrossRefGoogle Scholar
  8. 8.
    Flitsch, S. L. and Khorana, H. G. (1989) Structure studies on transmembrane proteins. 1. Model study using bacteriorhodopsin mutants containing single cysteine residues. Biochemistry 28, 7800–7805.PubMedCrossRefGoogle Scholar
  9. 9.
    Sarsawat, L. D., Pastra-Landis, C., and Lowey, S. (1992) Mapping single cysteine mutants of light chain 2 in chicken skeletal myosin. J. Biol. Chem. 29, 21,112–21,118.Google Scholar
  10. 10.
    Ling, R. and Luckey, M. (1994) Use of single-cysteine mutants to probe the location of the disulfide bond in LamB protein from Escherichia coli. Biochem. Biophys. Res. Commun. 201, 242–247.PubMedCrossRefGoogle Scholar
  11. 11.
    Beavis, R. C. and Chait, B. T. (1990) Rapid, sensitive analysis of protein mixtures by mass spectrometry. Proc. Natl. Acad. Sci. USA 87, 6873–6877.PubMedCrossRefGoogle Scholar
  12. 12.
    Hillenkamp, F., Karas, M., Beavis, R. C., and Chait, B. T. (1991) Matrix-assisted laser desorption/ionization mass spectrometry of biopolymers. Anal. Chem. 63, 1193A–1203A.PubMedCrossRefGoogle Scholar
  13. 13.
    Carr, S. A., Hemling, M. E., Bean, M. F., and Roberts, G. D. (1991) Integration of mass spectrometry in analytical biotechnology. Anal. Chem. 63, 2802–2824.PubMedCrossRefGoogle Scholar
  14. 14.
    Briggs, R. G. and Fee, J. A. (1978) Sulfhydryl reactivity of human erythrocyte superoxide dismutase, on the origin of the unsual spectral properties of the protein when prepared by a procedure utilizing chloroform and ethanol for the precipitation of hemoglobin. Biochim. Biophys. Acta 537, 100–109.PubMedGoogle Scholar
  15. 15.
    Beavis, R. C. and Chait, B. T. (1990) High accuracy mass determination of proteins using matrix-assisted laser desorption mass spectrometry. Anal. Chem. 62, 1836–1840.PubMedCrossRefGoogle Scholar
  16. 16.
    Markland, F. S. and Smith, E. L. (1967) Subtilisin BPN′ VIII. Isolation of CNBr peptides and the complete amino acid sequence. J. Biol. Chem. 242, 5198–5211.PubMedGoogle Scholar
  17. 17.
    Wells, J. A., Ferrari, E., Henner, D. J., Estell, D. A., and Chen, E. Y. (1983) Cloning sequencing and secretion of Bacillus amyloliquefaciens subtilisin in Bacillus subtilis. Nucleic Acids Res. 11, 7911–7925.PubMedCrossRefGoogle Scholar
  18. 18.
    Lee, B. and Richards, F. M. (1971) The Interpretation of protein structures: estimation of static accessibility. J. Mol. Biol. 55, 379–400.PubMedCrossRefGoogle Scholar
  19. 19.
    Bott, R., Ultsch, M., Kossiakoff, A., Graycar, T., Katz, B., and Power, S. (1988) The three dimensional structure of Bacillus amyloliquefaciens subtilisin at 1.8 Å and an analysis of the structural consequences of peroxide inactivation. J. Biol. Chem. 263, 7895–7906.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

  • Thomas W. Keough
    • 1
  • Yiping Sun
    • 1
  • Bobby L. Barnett
    • 1
  • Martin P. Lacey
    • 1
  • Mark D. Bauer
    • 1
  • Ellen S. Wang
    • 1
  • Christopher R. Erwin
    • 2
  1. 1.Miami Valley LaboratoriesThe Procter and Gamble CompanyCincinnati
  2. 2.The Children’s Hospital Medical Research FoundationCincinnati

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