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Aggregation of granulocyte-colony stimulating factor in vitro involves a conformationally altered monomeric state

¹ Biology Department, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
² Department of Pharmaceutics, Amgen, Inc., Thousand Oaks, California 91320, USA

(RECEIVED March 31, 2005; FINAL REVISION March 31, 2005; ACCEPTED June 17, 2005)

Aggregation of partially folded intermediates populated during protein folding processes has been described for many proteins. Likewise, partially unfolded chains, generated by perturbation of numerous proteins by heat or chemical denaturants, have also been shown to aggregate readily. However, the process of protein aggregation from native-state conditions is less well understood. Granulocyte-colony stimulating factor (G-CSF), a member of the four-helix bundle class of cytokines, is a therapeutically relevant protein involved in stimulating the growth and maturation of phagocytotic white blood cells. Under native-like conditions (37°C [pH 7.0]), G-CSF shows a significant propensity to aggregate. Our data suggest that under these conditions, native G-CSF exists in equilibrium with an altered conformation, which is highly aggregation prone. This species is enriched in 1–2 M GdmCl, as determined by tryptophan fluorescence and increased aggregation kinetics. In particular, specific changes in Trp58 fluorescence report a local rearrangement in the large loop region between helices A and B. However, circular dichroism, reactivity toward cyanylation, and ANS binding demonstrate that this conformational change is subtle, having no substantial disruption of secondary and tertiary structure, reactivity of the free sulfhydryl at Cys17 or exposure of buried hydrophobic regions. There is no indication that this altered conformation is important to biological activity, making it an attractive target for rational protein stabilization.

Keywords: GCSF; protein aggregation; protein folding intermediate; cysteine labeling

Abbreviations: ANS, 8-anilinonaphthalene-2-sulfonic acid • CD, circular dichroism • CDAP, 1-cyano-4-dimethylaminopyridinium • DTT, dithiothreitol • G-CSF, granulocyte-colony stimulating factor • GdmCl, guanidinium chloride • HPLC, high performance liquid chromatography • LC/MS, liquid chromatography/mass spectrometry • MOPS, 3-(N-morpholino) propanesulfonic acid • PBS, phosphate-buffered saline • PMT, photomultiplier tube • SDS, sodium dodecyl sulfate • SEC, size exclusion chromatography • Tris-HCl, tris(hydroxymethyl)-aminomethane hydrochloride • UV, ultraviolet

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051489405.

Reprint requests to: David N. Brems, Department of Pharmaceutics, MS 2–2-A, Amgen, Inc., 1 Amgen Center Drive, Thousand Oaks, CA 91320, USA; e-mail: dbrems{at}amgen.com; fax: (805) 375-5794.

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