4.1 Structural stability in high temperatures and extreme pH
Despite the successes in coating TMV, BSMV and their VLPs with metals such as gold, palladium and platinum, many industrially-desirable metals are not readily deposited due to unfavorable electrochemistries (Table 2) [30, 63]. This issue may be mitigated somewhat through the use of buffering and reducing agents, activation by primary deposition with another metal such as Pt, and higher temperatures to create more favorable processing conditions. These strategies ultimately increase metal-template interactions via altering residue ionization state, changing the reduction potentials of the metal ions and amino acid side chains, and/or create nucleation sites to drive metal mineralization. However, these conditions may destabilize the CP interactions that drive self-assembly of the template leading to biotemplate loss and low yields of metal mineralization. Biotemplates are only stable within fixed pH and temperature ranges (Table 1). To address this challenge, TMV and VLPs have been engineered to increase their structural stability to resist disassembly.
The stability of TMV biotemplates has been increased by control of the intermolecular forces that drive self-assembly (Figure 1), enabling more rapid biotemplating of a wider range of materials [64–66]. For example, single point mutations within the Caspar carboxylate cluster can enhance viral assembly. Neutralization of a negatively-charged residue (E50Q) or replacing a negatively-charged residue with a positively-charged residue in the Caspar carboxylate center (E50R or D77R) has been shown to produce longer virions that spontaneously self-assemble without an RNA that contain an OAS or other nucleic acids in transgenic plants [67]. In similar fashion, neutralizing the negative-charged residues with site-specific mutations, E50Q/D77N, was sufficient to rescue TMV-VLP assembly in E.coli even in the absence of RNA with an OAS, which typically results in viral disks alone [21]. In combination with cysteine engineering, E50Q/D77N mutants are able to attach to a gold-coated plate [21]. These mutations within the Caspar carboxylate cluster are sufficient to overcome the poor stability of nucleic acid-free VLPs and recover resistance to pH, attaining similar stability to wildtype virions. In so doing, they create nucleic acid-free VLPs that have a free internal channel for synthesis of thin nanowires [64]. Engineering of the hydrophobic interactions between CPs (Figure 1) may further improve the stability of VLPs; however, modifications of this nature have yet to be evaluated.
OAS-containing nucleic acids that initiate the assembly of wildtype virus and VLPs also act as a molecular ruler that sets the length of produced VLPs [20]. While VLPs that have been engineered appropriately (e.g. at the Caspar carboxylate cluster) can self-assemble without this molecular ruler, the resulting VLPs are more heterogenous in size than OAS-containing viral particles with sizes as small as 20 nm [23]. Thus, future work should include efforts to control the precise dimensions of VLPs or development of efficient separation technologies to isolate VLPs with specific dimensions.