Weak self-association of human growth hormone investigated by nitrogen-15 NMR relaxation

Authors

  • Malene Ringkjøbing Jensen,

    1. Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark
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    • Malene Ringkjøbing Jensen and Søren M. Kristensen contributed equally to this work

    • Institut de Biologie Structurale Jean-Pierre Ebel, 41 Rue Jules Horowitz, 38027 Grenoble, France.

  • Søren M. Kristensen,

    1. Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark
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    • Malene Ringkjøbing Jensen and Søren M. Kristensen contributed equally to this work

  • Camille Keeler,

    1. Department of Laboratory Medicine, Yale University School of Medicine, P.O. Box 208035, New Haven, Connecticut 06520-8035
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  • Hans E. M. Christensen,

    1. Department of Chemistry, The Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark
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  • Michael E. Hodsdon,

    1. Department of Laboratory Medicine, Yale University School of Medicine, P.O. Box 208035, New Haven, Connecticut 06520-8035
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  • Jens J. Led

    Corresponding author
    1. Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark
    • Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK–2100 Copenhagen Ø, Denmark
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Abstract

The self-association of human growth hormone (hGH) was investigated using 15N NMR relaxation. The investigation relies on the 15N R1 and R2 relaxation rates and the heteronuclear {1H}-15N NOEs of the backbone amide groups at multiple protein concentrations. It is shown that the rotational correlation time of hGH in solution depends strongly on its concentration, indicating a significant degree of self-association. The self-association is reversible and the monomers in the aggregates are noncovalently linked. Extrapolation of the relaxation data to zero concentration predicts a correlation time of 13.4 ns and a rotational diffusion anisotropy of 1.26 for monomeric hGH, in agreement with the rotational diffusion properties estimated by hydrodynamic calculations. Moreover, the extrapolation allows characterization of the backbone dynamics of monomeric hGH without interference from self-association phenomena, and it is found that hGH is considerably more flexible than originally thought. A concerted least-squares analysis of the 15N relaxations and their concentration dependence reveals that the self-association goes beyond a simple monomer-dimer equilibrium, and that tetramers or other multimeric states co-exist in fast exchange with the monomeric and dimeric hGH at sub-millimolar concentrations. Small changes in the 1H and 15N amide chemical shifts suggest that a region around the C-terminus is involved in the oligomer formation. Proteins 2008. © 2008 Wiley-Liss, Inc.

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