Stability Study of Engineered Ferritin Nano-vaccines by Combined Molecular simulation and Experiments

Abstract

Ferritins are a type of protein that is present in almost all living things. They have a symmetric spherical shape with strong physical and chemical stabilities. Human heavy-chain ferritins (HFns) have an inner cavity of 12 nm and 24 identical subunits. Ferritins are known to be assembled and stabilised by hydrogen bonds, salt bridges, and hydrophobic interactions at 2-3-4 symmetry. Previous research has indicated that it is possible to create vaccines using human heavy-chain ferritin with a portion of EBNA1 inserted at the C-terminus. Four mutant ferritins with short C-terminal amino acid mutations and the letters C1 through C4 are used in this project. C1 was neutrally charged (Gln 167 and 171), C2 was positively charged (Arg 167 and 171), and C3 and C4 were hydrophobic (Val 165 and 169, Ile 166,168, and 170) and hydrophilic (Gln 165 and 169, Asn 166,168, and 170). The changes were done in order to comprehend the relationship between the general stability of the structures and E-helices mutations. The majority of past research has focused on the mutations and stability changes of ferritin. To study the effects of the mutations, this project, however, analysed the ferritin carrier, linker, and antigen as a unified system. As a result, this study is more useful for demonstrating the safety of the developed vaccine. In this project, experiments and MD simulations on Gromacs were used to examine the hydrophobic, thermal, and pH stability of four genetically altered human heavy-chain ferritins (C1, C2, C3, and C4). By working on this project, we can determine how the mutation will impact the HFns' stability performance. Additionally, to employ simulation in the project to examine the viability of using simulation for vaccine development research. E. coli was used to express the four mutant HFns. On the basis of recently released data, the purification process for the 4 mutants was examined and improved. C1 was satisfactorily purified by heat-acid precipitation, followed by HIC utilising a prepacked butyl FF column to remove the nucleic acids. C2, C3, and C4 each contained an extra IEC stage with a Sartobind Q column to remove the remaining HCPs. The final SEC flow through samples of C1 and C2 had purity levels of over 90% and were suitable for characterisation tests. Unexpectedly, C3 and C4 were unable to be purified to an extremely high purity throughout this process. Following purification, the self-assembly of these proteins was verified using SEC-MALS, and the purity and recovery rate were measured using the Bradford assay and SDS-PAGE. A prepacked Butyl FF column was utilised to compare the hydrophobicity of the proteins, and fluorescence was employed to evaluate the changes in tertiary structure over a volume of temperature and pH. Additionally, Gromacs was used to conduct MD simulations at varied pH and temperature levels. For analysis, it was necessary to calculate the solvent accessible surface area (SASA), root mean square deviation (RMSD), radius of gyration (Rg), root mean square of fluctuation (RMSF), and the distance between subunits. While C1 and C2 were correctly assembled into a symmetric spherical shape, according to SEC-MALS and MD simulation, they had differing hydrodynamic diameters. C1 is extremely close to the unmutated HFn (F1L3E1), while C2 is larger. The results demonstrate that these helices E mutations would not prevent ferritin from self-assembling. However, the positively charged amino acids of C2 strengthen the electrostatic interaction at the C-terminus, which may result in the larger structure of C2. Based on experiments, C2 had a higher hydrophobicity, while C1 was similar to F1L3E1. And the computed SASA confirmed these findings, since C2 had a larger SASA than C1 and F1L3E1, and a larger SASA theoretically implied a higher hydrophobicity. The repelling power of the altered amino acids may contribute to additional hydrophobic interactions as the E-helices create the hydrophobic fourfold symmetries. The SASA of C3 and C4 was used to compare their hydrophobicity. The SASA in C3 was the smallest, while C4 had the largest. Therefore, the hydrophobic interactions at the C-terminus contribute a greater contribution to the final structure and consequently influence the hydrophobicity. The fluorescence results demonstrated that the modification of C1 increased its pH stability while having little effect on its thermal resistance. And C1 had demonstrated a more stable structure than others in all of the simulation results. The C2, C3, and C4 thermal and pH experiments were carried out using MD simulations. The most unstable compounds were C3, C4, and C2. Due to the mutation of C2 into positively charged amino acids, the initial electrostatic bond has been destroyed, increasing interactions in acidic environments, resulting in lower thermal and pH stability. As a result of changing the hydrophobic channels, C3 and C4 displayed greater fluctuation and instability in simulations, potentially leading to significant changes in their structural stability. However, the simulation failed to indicate the denature of all proteins at the experimentally determined temperature and pH. This study showed that the C-terminal E-helices can significantly affect stability through mutations. Overall, C-terminus mutation research is essential for the security and effectiveness of this ferritin-based vaccine. And MD simulation is a useful method for developing studies, but more research is required to advance this method.