Analysis of the conformation and stability of Escherichia coli derived recombinant human interleukin 4 by circular dichroism.
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.
Windsor
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Department of Biotechnology-Biochemistry, Schering-Plough Research, Bloomfield, New Jersey 07003.
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D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D004926:Escherichia coli; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D015847:Interleukin-4 / Q000737:chemistry / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000648:ultrastructure; D013601:T-Lymphocytes / Q000187:drug effects; D013816:Thermodynamics
D006146:Guanidines; D011994:Recombinant Proteins; D015847:Interleukin-4; D003553:Cystine; D019791:Guanidine; D003545:Cysteine