EFFECTS OF MODIFICATIONS NEAR THE 2-FOLD, 3-FOLD AND 4-FOLD SYMMETRY AXES AN HUMAN FERRITIN RENATURATION

Ferritin is a protein of 24 subunits which assemble into a shell with 432 point symmetry. It can be denatured reversibly in acidic guanidine hydrochloride, with the formation of poorly populated renaturation in termediates. In order to increase the accumulation of intermediates an d to study the mechanism of ferritin renaturation, we analysed variant s of the human ferritin H-chain altered at the N-terminus (Delta(1-13) ), near the 4-fold axis (Leu-169 --> Arg), the 3-fold axis (Asp-131 -- > IIe + Glu-134 --> Phe) or the 2-fold axis (Ile-85 --> Cys), We also carried out specific chemical modifications of Cys-130 (near the 3-fol d axis) and Cys-85 (near the 2-fold axis). Renaturation of the modifie d ferritins yielded assembly intermediates that differed in size and p hysical properties. Alterations of residues around the 2-, 4- and 3-fo ld axes produced subunit monomers, dimers and higher oligomers respect ively. All these intermediates could be induced to assemble into ferri tin 24-mers by concentrating them or by co-renaturing them with wild-t ype H-ferritin. The results support the hypothesis that the symmetric subunit dimers are the building blocks of ferritin assembly, and are c onsistent with a reassembly pathway involving the coalescence of dimer s, probably around the 4-fold axis, followed by stepwise addition of d imers until the 24-mer cage is completed. In addition they show that a ssembly interactions are responsible for the large hysteresis of foldi ng and unfolding plots. The implications of the studies for in vivo he teropolymer formation in vertebrates, which have two types of ferritin chain (H and L), are discussed.