Structural basis for cyclodextrins' suppression of human growth hormone aggregation

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DOI

  • Daniel Erik Otzen
  • Benjamin Raerup Knudsen
  • ,
  • Finn Aachmann
  • ,
  • Kim Lambertsen Larsen, Department of Life Sciences
  • ,
  • Reinhard Wimmer

Many therapeutic proteins require storage at room temperature for extended periods of time. This can lead to aggregation and loss of function. Cyclodextrins (CDs) have been shown to function as aggregation suppressors for a wide range of proteins. Their potency is often ascribed to their affinity for aromatic amino acids, whose surface exposure would otherwise lead to protein association. However, no detailed structural studies are available. Here we investigate the interactions between human growth hormone (hGH) and different CDs at low pH. Although hGH aggregates readily at pH 2.5 in 1 M NaCl to form amorphous aggregates, the presence of 25 to 50 mM of various β-CD derivatives is sufficient to completely avoid this. α- and γ-CD are considerably less effective. Stopped-flow data on the aggregation reaction in the presence of β-CD are analyzed according to a minimalist association model to yield an apparent hGH-β-CD dissociation constant of -6 mM. This value is very similar to that obtained by simple fluorescence-based titration of hGH with β-CD. Nuclear magnetic resonance studies indicate that β-CD leads to a more unfolded conformation of hGH at low pH and predominantly binds to the aromatic side-chains. This indicates that aromatic amino acids are important components of regions of residual structure that may form nuclei for aggregation.

Original languageEnglish
JournalProtein Science
Volume11
Issue7
Pages (from-to)1779-1787
Number of pages9
ISSN0961-8368
DOIs
Publication statusPublished - 2 Jul 2002

    Research areas

  • Aggregation, Aromatic amino acids, Cyclodextrins, Human growth hormone, Stopped-flow kinetics

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