Unfolding and Conformational Distributions during Protein Precipitation

Authors

  • Stephen T. Chang,

    1. Department of Chemical Engineering, 102 Engineers' Way, PO Box 400741, Charlottesville, Virginia 22904–4741
    Current affiliation:
    1. MedImmune, 35 West Watkins Mill Road, Gaithersburg, MD 20878.
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  • Scott A. Tobler,

    1. Department of Chemical Engineering, 102 Engineers' Way, PO Box 400741, Charlottesville, Virginia 22904–4741
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  • Erik J. Fernandez

    Corresponding author
    1. Department of Chemical Engineering, 102 Engineers' Way, PO Box 400741, Charlottesville, Virginia 22904–4741
    • Department of Chemical Engineering, 102 Engineers' Way, PO Box 400741, Charlottesville, Virginia 22904–4741. Ph: 804–924–1351. Fx: 804–982–2658
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Abstract

The association of misfolded proteins, or aggregation, is a critical problem in a number of human diseases as well during the expression, refolding, formulation, and delivery of therapeutic proteins. In this study, we investigate lysozyme precipitation with hydrogen exhange using nuclear magnetic resonance (NMR) and mass spectrometry (MS). We show that MS can reveal the presence of conformational distributions, albeit without the detailed structural information afforded by NMR. Further, we find that increases in precipitant concentration alter the structure and composition of precipitates. The selective unfolding of one portion of the protein in these precipitates is correlated with hydrogen exchange patterns observed under nonprecipitating conditions and in other studies of lysozyme.

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