Effective charge saturation in ferritin cages

Ferritins are proteins consisting of 24 subunits that make a spherical cage structure. We prepared a series of net-charge mutants of Escherichia coli and Pseudo-nitzschia multiseries ferritins (EcFtn and PmFtn, respectively) in which glutamate (Glu) and aspartate (Asp) residues located at the outer surface of the cage were substituted with glutamine and asparagine or vice versa. The effective negative charges of mutant proteins were evaluated by nondenaturing polyacrylamide gel electrophoresis and ζ-potential measurement. Surprisingly, the effective negative charges of mutants did not increase when the number of Glu and Asp residues increased, while the effective negative charges decreased as the number of Glu and Asp residues decreased. These results suggest that there is a physical net-charge limit in ferritins, and that EcFtn and PmFtn bear maximally possible negative charges. Amino acid sequence analysis has shown that many bacteria and plant ferritins seem to have nearly maximum negative charges, but animal ferritins have fewer negative charges.