Disruption of the disulfide bonds of recombinant murine interleukin-6 induces formation of a partially unfolded state.
A chemical modification approach was used to investigate the role of the two disulfide bonds of recombinant murine interleukin-6 (mIL-6) in terms of biological activity and conformational stability. Disruption of the disulfide bonds of mIL-6 by treatment with iodoacetic acid (IAA-IL-6) or iodoacetamide (IAM-IL-6) reduced the biological activity, in the murine hybridoma growth factor assay, by 500- and 200-fold, respectively. Both alkylated derivatives as well as the fully reduced (but not modified) molecule (DTT-IL-6) retained a high degree of alpha-helical structure as measured by far-UV CD (37-51%) when compared to the mIL-6 (59%). However, the intensity of the near-UV CD signal of the S-alkylated derivatives was very low relative to that of mIL-6, suggesting a reduction in fixed tertiary interactions. Both IAA-IL-6 and IAM-IL-6 exhibit native-like unfolding properties at pH 4.0, characteristic of a two-state unfolding mechanism, and are destabilized relative to mIL-6, by 0.3 +/- 1.6 and 2.4 +/- 1.2 kcal/mol, respectively. At pH 7.4, however, both modified proteins display stable unfolding intermediates. These intermediates are stable over a wide range of GdnHCl concentrations (0.5-2 M) and are characterized by increased fluorescence quantum yield and a blue shift of lambda(max) from 345 nm, for wild-type recombinant mIL-6, to 335 nm. These properties were identical to those observed for DTT-IL-6 in the absence of denaturant. DTT-IL-6 appears to form a partially unfolded and highly aggregated conformation under all conditions studied, as showed by a high propensity to self-associate (demonstrated using a biosensor employing surface plasmon resonance), and an increased ability to bind the hydrophobic probe 8-anilino-1-naphthalenesulfonic acid. The observed protein concentration dependence of the fluorescence characteristics of these mIL-6 derivatives is consistent with the aggregation of partially folded forms of DTT-IL-6, IAM-IL-6, and IAA-IL-6 during denaturant-induced unfolding. For all forms of the protein studied here, the aggregated intermediates unfold at similar denaturant concentrations (2.1-2.9 M GdnHCl), suggesting that the alpha-helical structure and nonspecific hydrophobic interprotein interactions are of similar strength in all cases.
Zhang
J G
JG
Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research, Parkville, Victoria, Australia.
Matthews
J M
JM
Ward
L D
LD
Simpson
R J
RJ
D000478:Alkylation; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D015850:Interleukin-6 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D051379:Mice; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D004220:Disulfides; D015850:Interleukin-6; D011994:Recombinant Proteins; C039256:6-anilino-1-naphthalenesulfonic acid; D014508:Urea; D003545:Cysteine