Molten globule intermediate of recombinant human growth hormone: stabilization with surfactants.
We demonstrate that a surfactant-stabilized molten globule intermediate exists for recombinant human growth hormone (rhGH), is very hydrophobic, and tends to form aggregates. Characterization of this intermediate included equilibrium denaturation measured by electron paramagnetic resonance (EPR) and CD spectroscopy, assessment of aggregation during refolding, and fluorescence studies of its binding to the hydrophobic probe, 1-anilinonapthalene-8-sulfonate (1,8-ANS). We have found that at 4.5 M guanidinium hydrochloride (GuHCl), a molten globule intermediate of rhGH is stabilized and results in significant aggregation upon refolding. This intermediate is populated by the addition of the nonionic surfactant, Tween. This surfactant also reduces the extent of aggregation during refolding of rhGH from 4.5 M GuHCl. Overall, our studies reveal that rhGH forms a molten globule-like intermediate during folding and this intermediate self-associates. This self-association is reduced upon formation of a Tween-rhGH complex. Tween also binds to the native protein. Thus, nonionic surfactants such as Tween may act like molecular chaperones in facilitating protein folding while not altering the native conformation.
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Department of Chemical Engineering, Yale University, New Haven, Connecticut 06520, USA.
Cleland
J L
JL
Randolph
T W
TW
D000817:Anilino Naphthalenesulfonates; D002942:Circular Dichroism; D004355:Drug Stability; D004578:Electron Spin Resonance Spectroscopy; D005456:Fluorescent Dyes; D019382:Human Growth Hormone / Q000737:chemistry*; D006801:Humans; D008875:Middle Aged; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013501:Surface-Active Agents / Q000494:pharmacology*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D013501:Surface-Active Agents; D019382:Human Growth Hormone; C027132:1-anilino-8-naphthalenesulfonate