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


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 


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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

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