title: Effect of pH and denaturants on the folding and stability of murine interleukin-6.
abstract: The conformation and stability of a recombinant mouse interleukin-6 (mIL-6) has been investigated by analytical ultracentrifugation, fluorescence spectroscopy, urea-gradient gel electrophoresis, and near- and far-ultraviolet circular dichroism. On decreasing the pH from 8.0 to 4.0, the tryptophan fluorescence of mIL-6 was quenched 40%, the midpoint of the transition occurring at pH 6.9. The change in fluorescence quantum yield was not due to unfolding of the molecule because the conformation of mIL-6, as judged by both urea-gradient gel electrophoresis and CD spectroscopy, was stable over the pH range 2.0-10.0. Sedimentation equilibrium experiments indicated that mIL-6 was monomeric, with a molecular mass of 22,500 Da over the pH range used in these physicochemical studies. Quenching of tryptophan fluorescence (20%) also occurred in the presence of 6 M guanidine hydrochloride upon going from pH 7.4 to 4.0 suggesting that an amino acid residue vicinal in the primary structure to one or both of the two tryptophan residues, Trp-36 and Trp-160, may be partially involved in the quenching of endogenous fluorescence. In this regard, similar results were obtained for a 17-residue synthetic peptide, peptide H1, which corresponds to an N-terminal region of mIL-6 (residues Val-27-Lys-43). The pH-dependent acid quenching of endogenous tryptophan fluorescence of peptide H1 was 30% in the random coil conformation and 60% in the presence of alpha-helix-promoting solvents. Replacement of His-33 with Ala-33 in peptide H1 alleviated a significant portion of the pH-dependent quenching of fluorescence suggesting that the interaction of the imidazole ring of His-33 with the indole ring of Trp-36 is a major determinant responsible for the quenching of the endogenous protein fluorescence of mIL-6.
authors.affiliation: Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research, Parkville, Victoria, Australia.
authors.forename: L D
authors.initials: LD
authors.lastname: Ward
authors.forename: J G
authors.initials: JG
authors.lastname: Zhang
authors.forename: G
authors.initials: G
authors.lastname: Checkley
authors.forename: B
authors.initials: B
authors.lastname: Preston
authors.forename: R J
authors.initials: RJ
authors.lastname: Simpson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D015850:Interleukin-6 / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014508:Urea / Q000494:pharmacology*
chemical_list: D015850:Interleukin-6; D011994:Recombinant Proteins; D014508:Urea