Electrostatic Repulsion during Ferritin Assembly and Its Screening by Ions.

Escherichia coli non-heme-binding ferritin A (EcFtnA) is a spherical cagelike protein that is composed of 24 identical subunits. EcFtnA dissociates into 2-mers under acidic conditions and can reassemble into the native structure when the pH is increased. To understand how electrostatic interactions influence the assembly reaction, the dependence of the process on ionic strength and pH was investigated. The assembly reaction was initiated by stopped-flow mixing of the acid-dissociated EcFtnA solution and high-pH buffer solutions and monitored by time-resolved small-angle X-ray scattering. The rate of assembly increased with increasing ionic strength and decreased with increasing pH from 6 to 8. These dependences were thought to originate from repulsion between assembly units (2-mer in the case of EcFtnA) with the same net charge sign; therefore, to test this assumption, mutants with different net charges (net-charge mutants) were prepared. In buffers with a low ionic strength, the rate of assembly increased with a decreasing net charge. Thus, repulsion between the assembly unit net charges was demonstrated to be an important factor determining the rate of assembly. However, the difference in the assembly rate among net-charge mutants was not significant in buffers with an ionic strength of >0.1. Notably, under such high-ionic strength conditions, the assembly rate increased with an increasing ionic strength, suggesting that local electrostatic interactions are also responsible for the ionic strength dependence of the rate of assembly and are repulsive on average.

Ferritin Assembly Revisited: A Time-Resolved Small-Angle X-ray Scattering Study.

The assembly reaction of Escherichia coli ferritin A (EcFtnA) was studied using time-resolved small-angle X-ray scattering (TR-SAXS). EcFtnA forms a cagelike structure that consists of 24 identical subunits and dissociates into dimers at acidic pH. The dimer maintains nativelike secondary and tertiary structures and is able to reassemble into a 24-mer when the pH is increased. The reassembly reaction was induced by pH jump, and reassembly was followed by TR-SAXS. Time-dependent changes in the forward scattering intensity and in the gyration radius suggested the existence of a significant population of intermediate oligomers during the assembly reaction. The initial reaction was a mixture of second- and third-order reactions (formation of tetramers and hexamers) from the protein concentration dependence of the initial velocity. The time-dependent change in the SAXS profile was roughly explained by a simple model in which only tetramers, hexamers, and dodecamers were considered as intermediates.

A Physicochemical and Mutational Analysis of Intersubunit Interactions of Escherichia coli Ferritin A.

Ferritin A from Escherichia coli (EcFtnA) is 24-meric protein, which forms spherical cagelike structures called nanocages. The nanocage structure is stabilized by the interface around 4-, 3-, and 2-fold symmetric axes. The subunit structure of EcFtnA comprises a four-helix bundle (helices A-D) and an additional helix E, which forms a 4-fold axis. In this study, we examined the contribution of the interface around three symmetric axes. pH-induced dissociation experiments monitored by analytical ultracentrifugation and small-angle X-ray scattering showed that the dimer related by 2-fold symmetry is the most stable unit. Mutations located near the 3-fold axis revealed that the contribution of each interaction was small. A mutant lacking helix E at the 4-fold axis formed a nanocage, suggesting that helix E is not essential for nanocage formation. Further truncation of the C-terminus of helix D abrogated the formation of the nanocage, suggesting that a few residues located at the C-terminus of helix D are critical for this process. These properties are similar to those known for mammalian ferritins and seem to be common principles for nanocage formation. The difference between EcFtnA and mammalian ferritins was that helix E-truncated EcFtnA maintained an iron-incorporating ability, whereas mammalian mutants lost it.