The effects of disulfide bonds on the denatured state of barnase

Authors

  • Jane Clarke,

    1. Centre for Protein Engineering, University Chemical Laboratories, Lensfield Rd., Cambridge CB2 1EW, United Kingdom
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  • Andrea M. Hounslow,

    1. Centre for Protein Engineering, University Chemical Laboratories, Lensfield Rd., Cambridge CB2 1EW, United Kingdom
    Current affiliation:
    1. Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom
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  • Alan R. Fersht,

    1. Centre for Protein Engineering, University Chemical Laboratories, Lensfield Rd., Cambridge CB2 1EW, United Kingdom
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  • Chris J. Bond,

    1. Biomolecular Structure and Design Program, University of Washington, Seattle, Washington 98195-7610
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  • Valerie Daggett

    Corresponding author
    1. Biomolecular Structure and Design Program, University of Washington, Seattle, Washington 98195-7610
    2. Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195-7610
    • Department of Medicinal Chemistry, University of Washington, H-174B, Box 357610, Seattle, Washington 98195-7610
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Abstract

The effects of engineered disulfide bonds on protein stability are poorly understood because they can influence the structure, dynamics, and energetics of both the native and denatured states. To explore the effects of two engineered disulfide bonds on the stability of barnase, we have conducted a combined molecular dynamics and NMR study of the denatured state of the two mutants. As expected, the disulfide bonds constrain the denatured state. However, specific extended β-sheet structure can also be detected in one of the mutant proteins. This mutant is also more stable than would be predicted. Our study suggests a possible cause of the very high stability conferred by this disulfide bond: the wild-type denatured ensemble is stabilized by a nonnative hydrophobic cluster, which is constrained from occurring in the mutant due to the formation of secondary structure.

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