title: Engineered disulfide bonds in recombinant human interferon-gamma: the impact of the N-terminal helix A and the AB-loop on protein stability.
abstract: Insertion sites for cysteines with optimal stereochemistry for the formation 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-termini of 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. The fluorescence properties of native and disulfide cross-linked proteins were studied as a function of guanidine hydrochloride concentration. Melting temperatures (Tm) were calculated from the decrease in CD ellipticity at 220 nm. The induction of the antiviral effect was measured using A549 fibroblast cells infected with encephalomyocarditis virus. The ability to induce the expression of the HLA-DR antigen in Colo 205 cells was determined by fluorescence-activated cell scanning analysis. The stability of both mutants was strongly enhanced against temperature- and cosolvent-induced unfolding. The delta Tm 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 Tm value was 25 degrees C. This mutation abolishes nearly the entire biological activity (< 1%) with no detectable changes of secondary structure in the CD spectrum. Our results illustrate the importance of the N-terminal helix A and the AB-loop for the unfolding pathway and thermodynamic stability of rhu-IFN-gamma.
authors.affiliation: Fraunhofer Institut für Grenzflächen- und Bioverfahrenstechnik, Hannover, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Waschütza
authors.forename: V
authors.initials: V
authors.lastname: Li
authors.forename: T
authors.initials: T
authors.lastname: Schäfer
authors.forename: D
authors.initials: D
authors.lastname: Schomburg
authors.forename: C
authors.initials: C
authors.lastname: Villmann
authors.forename: H
authors.initials: H
authors.lastname: Zakaria
authors.forename: B
authors.initials: B
authors.lastname: Otto
mesh_terms: D002942:Circular Dichroism; D003198:Computer Simulation; D017931:DNA Primers / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009994:Osmolar Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D013696:Temperature
chemical_list: D017931:DNA Primers; D004220:Disulfides; D006146:Guanidines; D011994:Recombinant Proteins; D007371:Interferon-gamma; D019791:Guanidine