Thermophilic Ferritin 24mer Assembly and Nanoparticle Encapsulation Modulated by Interdimer Electrostatic Repulsion

Abstract

Protein cage self-assembly enables encapsulation and sequestration of small molecules, macromolecules, and nanomaterials for many applications in bionanotechnology. Notably, wild-type thermophilic ferritin from Archaeoglobus fulgidus (AfFtn) exists as a stable dimer of four-helix bundle proteins at low ionic strength, and the protein forms a hollow assembly of 24 protomers at high ionic strength (∼800 mM NaCl). This assembly process can also be initiated by highly charged gold nanoparticles (AuNPs) in solution, leading to encapsulation. These data suggest that salt solutions or charged AuNPs can shield unfavorable electrostatic interactions at AfFtn dimer-dimer interfaces, but specific “hot-spot” residues controlling assembly have not been identified. To investigate this further, we computationally designed three AfFtn mutants (E65R, D138K, A127R) that introduce a single positive charge at sites along the dimer-dimer interface. These proteins exhibited different assembly kinetics and thermodynamics, which were ranked in order of increasing 24mer propensity: A127R < WT < D138K ≪ E65R. E65R assembled to the 24mer across a wide range of ionic strengths (0 – 800 mM NaCl), and the dissociation temperature for the 24mer was 98 °C. X-ray crystal structure analysis of the E65R mutant identified a more compact, closed-pore cage geometry. A127R and D138K mutants exhibited wild-type ability to encapsulate and stabilize 5-nm AuNPs, whereas E65R gained ability to remain assembled in apo-form. This work illustrates designed protein cages with distinct assembly and encapsulation properties.

A protein–protein host–guest complex: Thermostable ferritin encapsulating positively supercharged green fluorescent protein

“…The experiments conducted by Tetter et al raised several questions regarding the encapsulation process, such as whether cargo can be encapsulated within fully assembled AfFtn cages or whether the encapsulation process must start with disassembled AfFtn subunits. To address these questions, we use a designed AfFtn mutant which we recently characterized that remains predominantly assembled in low ionic strength solutions . We show that AfFtn and its more stably assembled mutant can encapsulate GFP(+36), demonstrating that unlike other ferritins, AfFtn is capable of encapsulating large cargo without complete prior disassembly.…”

Spatially controlled assembly of affinity ligand and enzyme cargo enables targeting ferritin nanocarriers to caveolae

“…This enables site-specific conjugation of cargo inside the Ft nanocage. Assembly and disassembly of tF is controlled by ionic strength [37], which enables facile encapsulation of useful enzyme cargo within the protein interior in physiological conditions [31,38,39]. …”

Infinite Assembly of Folded Proteins in Evolution, Disease, and Engineering

“…Key to the design of agglomerated state materials are the assembly rules illustrated in Figure c and also described in other reviews . Lessons learned from the evolution and from the design of soluble closed‐symmetry assemblies can be applied to constructing materials by designing outward‐facing interfaces, thus creating assemblies with open symmetries.…”