Understanding the self-assembly mechanism of E2 protein cage and exploring its potential applications

Abstract

Dihydrolipoamide acetyltransferase (E2 protein) is part of pyruvate dehydrogenase multi-enzyme complex from Bacillus stearothermophilus. However, we don't consider its enzymatic activity in this work. E2 protein used in this work is a protein cage derived and is composed of 60 subunits. Structurally, the 60 subunits selfassemble to form a 25-nm hollow caged structure. Previous works have demonstrated its potential application as nanocapsule in drug delivery to encapsulate small molecules in its inner cavity, i.e. cancer drugs. The researches that we have been done in this work are summarized as followings: To better control the release of the encapsulated drugs at target sites during drug delivery, modulating the self-assembly process of E2 protein under different environmental stimuli is desirable. Therefore, understanding of the self-assembly mechanism of E2 protein is required. By truncating the C-terminal α-helix of E2 protein, the association between trimeric structures are eliminated and the selfassembly was halted at the trimer intermediate state. Molecular dynamic simulation reaffirms that the E2 protein adopts trimer intermediate prior to its fully assembled caged structure and that the C-terminus plays a critical role in mediating the selfassembly from monomer to trimer and to 60-mer. Upon identifying the importance of the C-terminus in the trimer and subsequently the cage assembly, other functionalities were introduced to enhance the release of molecular cargos (e.g. cancer drugs) from the cavity of the E2 protein cage. To this goal, amino acid histidines are introduced at critical sites at the interfaces between trimer structures. Histidine has a unique property that it is charged at acidic pH while VI remains uncharged at neutral and basic pH-s. The proximity of two or more histidines at the subunit interfaces induces repulsion and hence their separation at acidic pH, subsequently leading to the soluble and non-denatured disassembly of the E2 protein. Incorporation of a unique GALA peptide (peptide with repeating amino acids: Glu-Ala-Leu-Ala) with pH-sensitive helix-to-coil transition at low and high pH, respectively, to substitute the original C-terminal α-helix induces an interesting assembly characteristic. As a result, the self-assembly of E2 protein become pHresponsive and present in inversed direction. At neutral or high pH-s, the extended random coil state of GALA peptide dissociates the E2 protein cage. As the pH is lowered, E2 protein reassembled due to the formation of α-helix from GALA peptide. Moreover, the pH-inducible self-assembly of this engineered E2 protein is reversible. The well-defined inner cavity provides the possibility to utilize the E2 protein cage as a nanoreactor for inorganic nanoparticle synthesis for potential application as an imaging contrast agent. Iron-binding peptides were incorporated to the interior surface of E2 protein without jeopardizing its self-assembly and caged structure. The engineered proteins are able to nucleate irons. The subsequent oxidation of the ...