Cooperativity of mutational effects within a six amino acid residues substitution that induces a major conformational change in human H ferritin

Ferritin is an iron-storage protein composed of 24 polypeptide chains which assemble into a hollow shell. Previously, we have shown that a multisubstituted ferritin mutant containing the peptide ES-VWNP in place of the wild-type sequence GAPESG in a short exposed loop directs the synthesis of a product that assembles in a conformation remarkably different from that of the normal molecule. We have further characterized this mutant and we have tried to determine which of the substituted residues causes the large conformational change. Reversion of the mutant conformation was obtained changing the three residues WNP back to the wild-type sequence ESG (DE loop: ESVESG). However, the converse three amino acid change GAPWNP produced insoluble and unassembled ferritin. Therefore, the substitutions of GAP by ESV together with ESG by WNP have a largely cooperative and hardly predictable effect.