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).
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References
Alber, T. (1989) Mutational effects on protein stability. Annu. Rev. Biochem. 58, 765–798.
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.
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.
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.
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.
Mitchinson, C. and Wells, J. A. (1989) Protein engineering disulfide bonds in subtilisin BPN′. Biochemistry, 28, 4807–4815.
Falke, J. J. and Koshland, D., Jr (1987) Global flexibility in a sensory receptor a sate-directed cross-linking approach. Science 237, 1596–1600.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Lee, B. and Richards, F. M. (1971) The Interpretation of protein structures: estimation of static accessibility. J. Mol. Biol. 55, 379–400.
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.
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© 1996 Humana Press Inc., Totowa, NJ
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Keough, T.W. et al. (1996). Rapid Analysis of Single-Cysteine Variants of Recombinant Proteins. In: Chapman, J.R. (eds) Protein and Peptide Analysis by Mass Spectrometry. Methods in Molecular Biology™, vol 61. Humana Press. https://doi.org/10.1385/0-89603-345-7:171
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DOI: https://doi.org/10.1385/0-89603-345-7:171
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Print ISBN: 978-0-89603-345-0
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