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Isolation of point mutations that affect the folding of the H chain of human ferritin in E.coli.
Author(s) -
Luzzago A.,
Cesareni G.
Publication year - 1989
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1989.tb03411.x
Subject(s) - ferritin , point mutation , folding (dsp implementation) , recombinant dna , mutant , polypeptide chain , protein folding , biology , chain (unit) , computational biology , genetics , biochemistry , gene , physics , engineering , electrical engineering , amino acid , astronomy
We have approached the problem of folding and assembly of the heavy (H) chain of human ferritin by isolating point mutations that affect this process. Apoferritin is an ideal model system to approach the problem of protein folding and assembly into multimeric structures. We have developed a recombinant hybrid molecule that allows us to select for ferritin mutants in which the folding‐assembly process is altered or completely impaired. The selection procedure is based on a recombinant protein which consists of a fusion between the H chain of human ferritin and the alpha‐peptide of beta‐galactosidase. In the wild type situation, the alpha‐peptide domain is segregated inside the apoferritin shell upon assembly and is unable to interact with the substrate and perform its enzymic function. We show that by selecting for mutations that restore beta‐galactosidase activity we are able to identify ferritin mutations that affect the folding‐assembly process. The selective procedure was applied to the analysis of the amino acid side chains that are important for the attainment of the correct conformation of the carboxy‐terminal E helix in the 4‐fold axis.