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Waschutza Gero; Li Volkhart; Schafer Thomas; Schomburg Dietmar; Villmann Carmen; Zakaria Hayssam; Otto Bernd 《Protein engineering, design & selection : PEDS》1996,9(10):905-912
Insertion sites for cysteines with optimal stereochemistry forthe formation of unstrained disulfide bridges were identifiedin recombinant human interferon- (rhu-IFN-) by computer modelling.We have engineered two different disulfide cross-linked mutants,containing a pair of symmetry-related disulfide bonds, whichstabilize the N-termini of both monomers of the homodimenc protein.Mutations E7C and S69C allow the formation of an intramonomerdisuffide bond between helices A and D. In contrast, the A17Cand H111C mutations lead to a covalent cross-link between bothmonomers. The AB-loop is linked to helix F. The fluorescenceproperties of native and disulfide cross-linked proteins werestudied as a function of guanidine hydrochloride concentration.Melting temperatures (Tm) were calculated from the decreasein CD ellipticity at 220 nm. The induction of the antiviraleffect was measured using A549 fibroblast cells infected withencephalomyocarditis virus. The ability to induce the expressionof the HLA-DR antigen in Colo 205 cells was determined by fluorescence-activatedcell scanning analysis. The stability of both mutants was stronglyenhanced against temperature- and cosolvent-induced unfolding.The Tm of mutant IFN- E7C/S69C was 15°C. All measured biologicalactivities of this mutant were equal to wild type. In the caseof the other mutant IFN- A17C/H111C, the Tm value was 25°C.This mutation abolishes nearly the entire biological activity(<1%) with no detectable changes of secondary structure inthe CD spectrum. Our results illustrate the importance of theN-terminal helix A and the AB-loop for the unfolding pathwayand thermodynamic stability of rhu-IFN-. 相似文献
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