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 (mlL-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 a-helix-promoting solvents. Replacement of His-33 with Ala-33 in peptide HI 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. Keywords: circular dichroism; denaturation; fluorescence studies; interleukin-6; protein structure Interleukin-6 is a multifunctional cytokine that acts on a wide variety of tissues to elicit a broad spectrum of biological functions including terminal differentiation of B cells, growth and differentiation of T cells, and regulation of the acute-phase responses. IL-6 is also capable of inducing the differentiation of hematopoietic progenitor cells and megakaryocytes (for reviews see Kishimoto [1989], Van Snick [19901, and Gordon& Hoffman [1992]). Abbreviutions: IL-6, interleukin-6; m-, murine; h-, human; GdnHC1, guanidine hydrochloride; SDS, sodium dodecyl sulfate; MES, 2-(N-morpho1ino)ethane sulfonic acid; S, weight average sedimentation coefficient; [ B ] M~~, mean residue ellipticity; HEPES, N-2-hydroxyethylpiperazine-N1-2-ethane sulfonic acid; Tris, Tris(hydroxymethy1)aminomethane; CAPS, 3-(cyclohexylamino)-I-propane sulfonic acid; TFE, trifluoroethanol; GM-CSF, granulocyte-macrophage colony-stimulating factor.

The Binding of Calcium to the B-repeat Segment of SdrD, a Cell Surface Protein of Staphylococcus aureus

“…The Tyr residues 2, 7, 52, 73, and 100 ( Fig. 1) are very well conserved in all of the B-repeats, and the spectra suggest that the rotation of several of these is restricted, as was also found in the case of wild-type and deletion mutants of interleukin-6 (28,29) and in the synthetic (Tyr-AlaGlu) n polypeptide (30). Removal of Ca 2ϩ changed the intensity of ellipticity only slightly, with a 2-nm blue shift, indicating that microenvironment of the Tyr residues in apoB1-B5 is asymmetric and therefore structured.…”

Use of a Biosensor with Surface Plasmon Resonance Detection for the Determination of Binding Constants: Measurement of Interleukin-6 Binding to the Soluble Interleukin-6 Receptor

“…Alternatively, because the purification of both constructs involved solubilization of "inclusion body" material with denaturants, subtle changes in refolding conditions may have influenced the pathway of refolding. In this regard, it may be relevant that pUC9-derived murine IL-6 which had been purified (Zhang et al, 1992) by an identical procedure to that adopted for human IL-6 also exhibited a tendency to aggregate under conditions of moderate ionic strength (Ward et al, 1993b). Although murine IL-6 is monomeric at low ionic strength ( I < 0.01), human IL-6 is essentially dimeric under all conditions (pH 4-7.4, I = 0.005-0.20), even at concentrations in the vicinity of 10 pg/mL: relatively strong interactions are thus responsible for the quaternary structure of human IL-6.…”

Solution structure of synthetic peptides corresponding to the C‐terminal helix of interleukin‐6

“…All of these structures have an 'up-up-down-down' helical topology, with long loops between the first and second helices and the third and fourth. Preliminary spectral analyses by circular dichroism (Kruttgen et al, 1990a;Ward et al, 1993a) and '33-NMR spectroscopy (Nishimura et al, 1990; Morton, C. J., Mabbot, B. C., Simpson, R. J. and Norton, R. S., unpublished results) of human and murine IL-6 support the prediction that IL-6 is largely helical in solution.…”