Analysis of the conformation and stability of Escherichia coli derived-recombinant human interleukin 4 by circular dichroism

Abstract

The conformation and stability of Escherichia coli derived recombinant human interleukin 4 (rhuIL-4) have been examined by circular dichroism (CD). Protein unfolding was detected by ellipticity changes at 222 nm with increasing concentrations of guanidine hydrochloride (GdnHCl). The unfolding midpoint ([GdnHCl]1/2) was 3.7 M, the free energy of unfolding, (delta GDH2O), was 5.9 kcal/mol and the dependence of delta GD on the GdnHCl concentration (m) was 1.6 (kcal/mol)/M. This unfolding was demonstrated to be reversible upon removal of the GdnHCl by dialysis. Analysis of the far-UV CD spectrum indicated the presence of a high percentage of alpha-helical structure (ca. 73%). A small change in ellipticity was noted over the pH range 1.9-9.6, suggesting that the protein undergoes a minor conformational change with an apparent pKa of 4.17. Virtually complete biological activity, measured in vitro in a T-cell proliferation assay, was recovered following exposure to extreme values of pH (i.e., pH 3 and 10). An analysis of the near-UV CD spectrum indicated that the single tryptophan residue at position 91 was unconstrained and most likely exposed to the solvent. Titration with 4,4'-dithiodipyridine and 2-nitrothiosulfobenzoate established that the six cysteine residues in rhuIL-4 were involved in intramolecular disulfide linkages. These data support that rhuIL-4 has a highly stable three-dimensional structure.

Thiols decrease cytokine levels and down-regulate the expression of CD30 on human allergen-specific T helper (Th) 0 and Th2 cells

“…As shown here, IL‐4, IL‐5 and sCD30 all decreased, while IFN‐ γ was not as strongly affected by GSH/NAC treatment as determined by ELISA. Interestingly, it has previously been shown that the structural disulphides in IL‐4 and IL‐5 [39, 46] are necessary for their biological activity [47, 48]. Indeed, when we incubated rhIFN‐ γ with different concentrations of NAC, the levels of IFN‐ γ did not change.…”

Secondary structure and topology of human interleukin 4 in solution

“…As yet no X-ray structure is available for IL-4, although preliminary studies have shown that the molecule forms well-ordered crystals that diffract to beyond 2.8-A resolution and so are suitable for detailed diffraction analysis (Cook et al, 1991). In addition, CD studies of the protein in solution have shown that it has a high percentage of a-helical structure and has a high stability over a wide range of solvent conditions (Windsor et al, 1991).NMR spectroscopy is a powerful technique that can now be used to give detailed descriptions of protein structures (Wiithrich, 1989). As NMR provides the structure of proteins in solution, the method complements the use of X-ray diffraction for the determination of protein structures.…”

Site‐directed mutagenesis reveals the importance of disulfide bridges and aromatic residues for structure and proliferative activity of human Interleukin‐4

“…• • . Recently, biophysical and protein-chemical studies .on the, aromatic side chains of human IL4 have been reported [10,12]. We have singly• replaced the .two tyrosines by aspartic acid and the • tryptophane by • arginfne, yielding the muteins YS6D, 1240 and W91R.…”

“…2). According to drcular dichroism rn:easurements the tryptophane is largely accessible to the solvent , [12]. It.…”

“…It. is • • also easily modified by N-bromosuccinimide [10]. ln mouse IL4 the position of tryptophane 91 appears tobe…”