A point mutation that decreases the thermal stability of human interferon gamma.
We have identified a mutation of human gamma-interferon (IFN gamma) causing a temperature-sensitive phenotype. We used a randomized oligonucleotide to mutagenize a synthetic human IFN gamma gene, then screened the resulting mutants produced in Escherichia coli for proteins with altered biological activity. One mutant protein selected for detailed characterization exhibited less than 0.3% of the specific biological activity of native IFN gamma in an antiviral activity assay performed at 37 degrees C. However, the protein bound the human IFN gamma receptor with native efficiency at 4 degrees C. Sequencing the plasmid DNA encoding this protein showed that the mutation changed the lysine residue at amino acid 43 to glutamic acid (IFN gamma/K43E). Site-specific mutagenesis at amino acid 43 showed that this protein's phenotype resulted from positioning a negative charge at position 43. Structural characterization of IFN gamma/K43E using CD demonstrated that the protein had native conformation at 25 degrees C, but assumed an altered conformation at 37 degrees C. IFN gamma/K43E in this altered conformation bound poorly to the IFN gamma receptor at 37 degrees C, providing a rationale for the mutant's decreased antiviral activity.
Lunn
C A
CA
Department of Biotechnology/Molecular Biology, Schering-Plough Research Institute, Bloomfield, NJ 07003.
Fossetta
J
J
Murgolo
N
N
Zavodny
P J
PJ
Lundell
D
D
Narula
S K
SK
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003588:Cytopathogenic Effect, Viral; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000235:genetics; D010641:Phenotype; D011487:Protein Conformation; D015202:Protein Engineering; D017471:Receptors, Interferon / Q000378:metabolism; D011994:Recombinant Proteins; D013696:Temperature
D009841:Oligonucleotides; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D007371:Interferon-gamma