EFFECT OF PH AND DENATURANTS ON THE FOLDING AND STABILITY OF MURINE INTERLEUKIN-6
- Author(s)
- Ward, LD; Zhang, JG; CHECKLEY, G; PRESTON, B; Simpson, RJ;
- Details
- Publication Year 1993-08,Volume 2,Issue #8,Page 1291-1300
- Journal Title
- PROTEIN SCIENCE
- Publication Type
- Journal Article
- 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 HI was 30% in the random coil conformation and 60% in the presence of alpha-helix-promoting solvents. Replacement of His-3 3 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.
- Publisher
- CAMBRIDGE UNIV PRESS
- Keywords
- AMINO-ACIDS; HUMAN IL-6; DELETION; RESIDUES; MUTANTS; BIOLOGY; HELIX
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- Refer to copyright notice on published article.
Creation Date: 1993-08-01 12:00:00