title: Aggregation of granulocyte-colony stimulating factor in vitro involves a conformationally altered monomeric state.
abstract: 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 degrees 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.
authors.affiliation: Department of Pharmaceutics, MS 2-2-A, Amgen, Inc., 1 Amgen Center Drive, Thousand Oaks, CA 91320, USA.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Raso
authors.forename: Jeff
authors.initials: J
authors.lastname: Abel
authors.forename: Jesse M
authors.initials: JM
authors.lastname: Barnes
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Maloney
authors.forename: Gary
authors.initials: G
authors.lastname: Pipes
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Treuheit
authors.forename: Jonathan
authors.initials: J
authors.lastname: King
authors.forename: David N
authors.initials: DN
authors.lastname: Brems
mesh_terms: D055592:Biophysical Phenomena; D001703:Biophysics; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D005453:Fluorescence; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins; D014364:Tryptophan / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D014364:Tryptophan; D019791:Guanidine; D003545:Cysteine