Disruption of the disulfide bonds of recombinant murine interleukin-6 induces formation of a partially unfolded state.

Abstract

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.