ENGINEERED DISULFIDE BONDS IN RECOMBINANT HUMAN INTERFERON-GAMMA - THE IMPACT OF THE N-TERMINAL HELIX-A AND THE AB-LOOP ON PROTEIN STABILITY

Insertion sites for cysteines with optimal stereochemistry for the for mation of unstrained disulfide bridges were identified in recombinant human interferon-gamma (rhu-IFN-gamma) by computer modelling, We have engineered two different disulfide cross-linked mutants, containing a pair of symmetry-related disulfide bonds, which stabilize the N-termin i ;If both monomers of the homodimeric protein, Mutations E7C and S69C allow the formation of an intramonomer disulfide bond between helices A and D. In contrast, the A17C and H111C mutations lead to a covalent cross-link between both monomers. The AB-loop is linked to helix F. T he fluorescence properties of native and disulfide cross-linked protei ns were studied as a function of guanidine hydrochloride concentration . Melting temperatures (T-m) were calculated from the decrease in CD e llipticity at 220 nm, The induction of the antiviral effect was measur ed using A549 fibroblast cells infected with encephalomyocarditis viru s. The ability to induce the expression of the HLA-DR antigen in Cole 205 cells was determined by fluorescence-activated cell scanning analy sis, The stability of both mutants was strongly enhanced against tempe rature- and cosolvent-induced unfolding, The Delta T-m, of mutant IFN- gamma E7C/S69C was 15 degrees C. All measured biological activities of this mutant were equal to wild type. In the case of the other mutant IFN-gamma A17C/H111C the Delta T-m, value was 25 degrees C. This mutat ion abolishes nearly the entire biological activity (<1%) with no dete ctable changes of secondary structure in the CD spectrum. Our results illustrate the importance of the N-terminal helix A and the AB-loop fo r the unfolding pathway and thermodynamic stability of rhu-IFN-gamma.