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.