Advertisement

Journal of Protein Chemistry

, Volume 14, Issue 8, pp 665–678 | Cite as

Extensive modifications for methionine enhancement in the β-barrels do not alter the structural stability of the bean seed storage protein phaseolin

  • John M. Dyer
  • Jeffrey W. Nelson
  • Norimoto Murai
Article

Abstract

Common beans are widely utilized as a food source, yet are low in the essential amino acid methionine. As an initial step to overcome this defect the methionine content of the primary bean seed storage protein phaseolin was increased by replacing 20 evolutionarily variant hydrophobic residues with methionine and inserting short, methionine-rich sequences into turn and loop regions of the protein structure. Methionine enhancement ranged from 5 to 30 residues. AnEscherichia coli expression system was developed to characterize the structural stability of the mutant proteins. Proteins of expected sizes were obtained for all constructs except for negative controls, which were rapidly degraded inE. coli. Thermal denaturation of the purified proteins demonstrated that both wild-type and mutant phaseolin proteins denatured reversibly at approximately 61°C. In addition, urea denaturation experiments of the wild-type and a mutant protein (with 30 additional methionines) confirmed that the structural stability of the proteins was very similar. Remarkably, these results indicate that the phaseolin protein tolerates extensive modifications, including 20 substitutions and two loop inserts for methionine enhancement in theβ-barrel and loop structures, with extremely small effects on protein stability.

Key words

Fusion protein expression methionine enhancement phaseolin protein engineering structural stability 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bollini, R., and Chrispeels, M. J. (1978).Planta 142, 291–298.Google Scholar
  2. Bollini, R., and Chrispeels, M. J. (1979).Planta 146, 487–501.Google Scholar
  3. Bollini, R., Van der Wilden, W., and Chrispeels, M. J. (1982).Physiol. Plant. 55, 82–92.Google Scholar
  4. Bradford, M. M. (1976).Anal. Biochem. 72, 248–254.PubMedGoogle Scholar
  5. Bustos, M. M., Luckow, V. A., Griffing, L. R., Summers, M. D., and Hall, T. C. (1988).Plant Mol. Biol. 10, 475–488.Google Scholar
  6. Bustos, M. M., Kalkan, F. A., VandenBosch, K. A., and Hall, T. C. (1991).Plant Mol. Biol. 16, 381–395.PubMedGoogle Scholar
  7. Ceriotti, A., Pedrazzini, E., Fabbrini, M. S., Zoppe, M., Bollini, R., and Vitale, A. (1991).Eur. J. Biochem. 202, 959–968.PubMedGoogle Scholar
  8. Chrispeels, M. J., Higgins, T. J. V., and Spencer, D. (1982).J. Cell Biol. 93, 306–313.PubMedGoogle Scholar
  9. De Clercq, A., Vandewiele, M., Van Damme, J., Guerche, P., Van Montagu, M., Vanderkerckhove, J., and Krebbers, E. (1990).Plant Physiol. 94, 970–979.Google Scholar
  10. Dickinson, C. D., Scoot, M. P., Hussein, E. H. A., Argos, P., and Nielsen, N. C. (1990).Plant Cell 2, 403–413.PubMedGoogle Scholar
  11. Doyle, J. J., Schuler, M. A., Godette, W. D., Zenger, V., Beachy, R. N., and Slightom, J. L. (1986).J. Biol. Chem. 261, 9228–9238.PubMedGoogle Scholar
  12. Dyer, J. M. (1993). Structural Analysis and Methionine Enhancement of the Bean Seed Storage Protein Phaseolin, Ph.D. thesis, Louisiana State University, Baton Rouge, Louisiana.Google Scholar
  13. Dyer, J. M., Nelson, J. W., and Murai, N. (1992).J. Protein Chem. 11, 281–288.PubMedGoogle Scholar
  14. Dyer, J. M., Nelson, J. W., and Murai, N. (1993).J. Protein Chem. 12, 545–560.PubMedGoogle Scholar
  15. Ferrer, A., Aparicio, C., and Noques, N. (1990).FEES Lett. 266, 67–71.Google Scholar
  16. Gepts, P., and Bliss, F. A. (1984).Theor. Appl. Genet. 69, 47–53.Google Scholar
  17. Greenwood, J. S., and Chrispeels, M. J. (1985).Plant Physiol. 79, 65–71.Google Scholar
  18. Hoffman, L. M., Donaldson, D. D., and Herman, E. M. (1988).Plant Mol. Biol. 11, 717–729.Google Scholar
  19. Ko, T.-P., Ng, J. D., and McPherson, A. (1993).Plant Physiol. 101, 729–744.PubMedGoogle Scholar
  20. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987).Meth. Enzymol. 154, 367–382.PubMedGoogle Scholar
  21. Lawrence, M. C., Suzuki, E., Varghese, J. N., Davis, P. C., Donkelaar, A. V., Tulloch, P. A., and Colman, P. M. (1990).EMBO J. 9, 9–15.PubMedGoogle Scholar
  22. Lawrence, M. C., Izard, T., Beuchat, M., Blagrove, R. J., and Colman, P. M. (1944).J. Mol. Biol. 238, 748–776.Google Scholar
  23. Mertz, E. T., Bates, L. S., and Nelson, O. E. (1964).Science 45, 279–280.Google Scholar
  24. Mutschier, M. A., Bliss, F. A., and Hall, T. C. (1980).Plant Physiol. 65, 627–630.Google Scholar
  25. Ng, J. D., Ko, T.-P., and McPherson, A. (1993).Plant Physiol. 101, 713–728.PubMedGoogle Scholar
  26. Pace, C. N. (1986).Meth. Enzymol. 131, 266–280.PubMedGoogle Scholar
  27. Pace, C. N. (1990).Trends Biochem. Sci. 15, 14–17.PubMedGoogle Scholar
  28. Pace, C. N., Shirley, A., and Thomson, J. A. (1989). InProtein Structure: A Practical Approach (Creighton, T. E., ed.), IRL Press, Oxford, pp. 311–330.Google Scholar
  29. Parsell, D. A., and Sauer, R. T. (1989).J. Biol. Chem. 264, 7590–7595.PubMedGoogle Scholar
  30. Sanger, F., Nicklen, S., and Coulson, A. R. (1977).Proc. Natl. Acad. Sci. USA 74, 5463–5467.PubMedGoogle Scholar
  31. Santoro, M. M., and Bolen, D. W. (1988).Biochemistry 27, 8063–8068.PubMedGoogle Scholar
  32. Slightom, J. L., Sun, S. M., and Hall, T. C. (1983).Proc. Natl. Acad. Sci. USA 80, 1897–1901.Google Scholar
  33. Sturm, A., Van Kuik, J. A., Vliegenthart, J. F. G., and Chrispeels, M. J. (1987).J. Biol. Chem. 262, 13392–13403.PubMedGoogle Scholar
  34. Sun, S. M., McLeester, R. C., Bliss, F. A., and Hall, T. C. (1974).J. Biol. Chem. 249, 2118–2121.PubMedGoogle Scholar
  35. Sun, S. M., Mutschler, M. A., Bliss, F. A., and Hall, T. C. (1978).Plant Physiol. 61, 918–923.Google Scholar
  36. Talbot, D. R., Adang, M. J., Slightom, J. L., and Hall, T. C. (1984).Mol. Gen. Genet. 198, 42–49.Google Scholar
  37. Wallace, J. C., Galili, G., Kawata, E. E., Cuellar, R. E., Shotwell, M. A., and Larkins, B. A. (1988).Science 240, 662–664.PubMedGoogle Scholar
  38. Zheng, Z., Tanake, K., and Murai, N. (1994).Plant Physiol. 105, 321.PubMedGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1995

Authors and Affiliations

  • John M. Dyer
    • 1
  • Jeffrey W. Nelson
    • 1
  • Norimoto Murai
    • 1
    • 2
  1. 1.Department of BiochemistryLouisiana State UniversityBaton Rouge
  2. 2.Departments of Plant Pathology and Crop PhysiologyLouisiana State UniversityBaton Rouge

Personalised recommendations