Engineered disulfide bonds in recombinant human interferon-γ: the impact of the N-terminal helix A and the AB-loop on protein stability

Insertion sites for cysteines with optimal stereochemistry for the formation of unstrained disulfide bridges were identified in recombinant human interferon-γ (rhu-IFN-γ) by computer modelling. We have engineered two different disulfide cross-linked mutants, containing a pair of symmetry-related dis...

Full description

Saved in:
Bibliographic Details
Published in:Protein engineering 1996-10, Vol.9 (10), p.905-912
Main Authors: Waschütza, Gero, Li, Volkhart, Schäfer, Thomas, Schomburg, Dietmar, Villmann, Carmen, Zakaria, Hayssam, Otto, Bernd
Format: Article
Language:English
Subjects:
Circular Dichroism
computer modelling
Computer Simulation
disulfide bridges
Disulfides - chemistry
DNA Primers - chemistry
Electrophoresis, Polyacrylamide Gel
Guanidine
Guanidines - chemistry
Helix-Loop-Helix Motifs
Humans
interferon
Interferon-gamma - biosynthesis
Interferon-gamma - chemistry
Interferon-gamma - genetics
Interferon-gamma - metabolism
Models, Molecular
Mutagenesis, Site-Directed - genetics
Osmolar Concentration
Protein Conformation
Protein Denaturation
Protein Folding
Protein Structure, Secondary
Recombinant Proteins
site-directed mutagenesis
Temperature
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Insertion sites for cysteines with optimal stereochemistry for the formation of unstrained disulfide bridges were identified in recombinant human interferon-γ (rhu-IFN-γ) 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 homodimenc protein. Mutations E7C and S69C allow the formation of an intramonomer disuffide 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 ΔTm of mutant IFN-γ E7C/S69C was 15°C. All measured biological activities of this mutant were equal to wild type. In the case of the other mutant IFN-γ A17C/H111C, the ΔTm value was 25°C. This mutation abolishes nearly the entire biological activity (
ISSN:1741-0126
0269-2139
1741-0134
1460-213X
DOI:10.1093/protein/9.10.905