Mechanisms of ferritin assembly studied by time-resolved small-angle X-ray scattering

Abstract

The assembly reaction of Escherichia coli ferritin A (EcFtnA) was studied using time-resolved small-angle X-ray scattering (SAXS). EcFtnA forms a cage-like structure that consists of 24 identical subunits and dissociates into dimers at acidic pH. The dimer maintains native-like secondary and tertiary structures and can reassemble into a 24-mer when the pH is increased. The time-dependent changes in the SAXS profiles of ferritin during its assembly were roughly explained by a simple model in which only tetramers, hexamers, and dodecamers were considered intermediates. The rate of assembly increased with increasing ionic strength and decreased with increasing pH (from pH 6 to pH 8). These tendencies might originate from repulsion between assembly units (dimers) with the same net charge sign. To test this hypothesis, ferritin mutants with different net charges (net-charge mutants) were prepared. In buffers with low ionic strength, the rate of assembly increased with decreasing net charge. Thus, repulsion between the assembly unit net charges was an important factor influencing the assembly rate. Although the differences in the assembly rate among net-charge mutants were not significant in buffers with an ionic strength higher than 0.1, the assembly rates increased with increasing ionic strength, suggesting that local electrostatic interactions are also responsible for the ionic-strength dependence of the assembly rate and are, on average, repulsive.