Stable Disk Assemblies of a Tobacco Mosaic Virus Mutant as Nanoscale Scaffolds for Applications in Drug Delivery.

Current approaches to nanoscale therapeutic delivery rely on the attachment of a drug of interest to a nanomaterial scaffold that is capable of releasing the drug selectively in a tumor environment. One class of nanocarriers receiving significant attention is protein nanomaterials, which are biodegradable and homogeneous in morphology and can be equipped with multiple functional handles for drug attachment. Although most protein-based nanocarriers are spherical in morphology, recent research has revealed that nonspherical nanomaterials may have favorable tumor uptake in comparison to their spherical counterparts. It is therefore important to expand the number of nonspherical protein-based nanocarriers that are available. Herein, we report the development of a self-assembling nanoscale disk derived from a double arginine mutant of recombinantly expressed tobacco mosaic virus coat protein (RR-TMV). RR-TMV disks display highly stable double-disk assembly states. These RR-TMV disks were functionalized with the chemotherapy drug doxorubicin (DOX) and further modified with polyethylene glycol (PEG) for improved solubility. RR-TMVDOX-PEG displayed cytotoxic properties similar to those of DOX alone when incubated with U87MG glioblastoma cells, but unmodified RR-TMV did not cause any cytotoxicity. The RR-TMV disk assembly represents a promising protein-based nanomaterial for applications in drug delivery.

Dramatic thermal stability of virus-polymer conjugates in hydrophobic solvents.

We have developed a method for integrating the self-assembling tobacco mosaic virus capsid into hydrophobic solvents and hydrophobic polymers. The capsid was modified at tyrosine residues to display an array of linear poly(ethylene glycol) chains, allowing it to be transferred into chloroform. In a subsequent step, the capsids could be transferred to a variety of hydrophobic solvents, including benzyl alcohol, o-dichlorobenzene, and diglyme. The thermal stability of the material against denaturation increased from 70 °C in water to at least 160 °C in hydrophobic solvents. With a view toward material fabrication, the polymer-coated TMV rods were also incorporated into solid polystyrene and thermally cast at 110 °C. Overall, this process significantly expands the range of processing conditions for TMV-based materials, with the goal of incorporating these templated nanoscale systems into conductive polymer matrices.

Molecular Mechanics Simulations and Improved Tight-Binding Hamiltonians for Artificial Light Harvesting Systems: Predicting Geometric Distributions, Disorder, and Spectroscopy of Chromophores in a Protein Environment.

We present molecular mechanics and spectroscopic calculations on prototype artificial light harvesting systems consisting of chromophores attached to a tobacco mosaic virus (TMV) protein scaffold. These systems have been synthesized and characterized spectroscopically, but information about the microscopic configurations and geometry of these TMV-templated chromophore assemblies is largely unknown. We use a Monte Carlo conformational search algorithm to determine the preferred positions and orientations of two chromophores, Coumarin 343 together with its linker and Oregon Green 488, when these are attached at two different sites (104 and 123) on the TMV protein. The resulting geometric information shows that the extent of disorder and aggregation properties and therefore the optical properties of the TMV-templated chromophore assembly are highly dependent on both the choice of chromophores and the protein site to which they are bound. We use the results of the conformational search as geometric parameters together with an improved tight-binding Hamiltonian to simulate the linear absorption spectra and compare with experimental spectral measurements. The ideal dipole approximation to the Hamiltonian is not valid because the distance between chromophores can be very small. We found that using the geometries from the conformational search is necessary to reproduce the features of the experimental spectral peaks.

Manipulating Excited-State Dynamics of Individual Light-Harvesting Chromophores through Restricted Motions in a Hydrated Nanoscale Protein Cavity.

Manipulating the photophysical properties of light-absorbing units is a crucial element in the design of biomimetic light-harvesting systems. Using a highly tunable synthetic platform combined with transient absorption and time-resolved fluorescence measurements and molecular dynamics simulations, we interrogate isolated chromophores covalently linked to different positions in the interior of the hydrated nanoscale cavity of a supramolecular protein assembly. We find that, following photoexcitation, the time scales over which these chromophores are solvated, undergo conformational rearrangements, and return to the ground state are highly sensitive to their position within this cavity and are significantly slower than in a bulk aqueous solution. Molecular dynamics simulations reveal the hindered translations and rotations of water molecules within the protein cavity with spatial specificity. The results presented herein show that fully hydrated nanoscale protein cavities are a promising way to mimic the tight protein pockets found in natural light-harvesting complexes. We also show that the interplay between protein, solvent, and chromophores can be used to substantially tune the relaxation processes within artificial light-harvesting assemblies in order to significantly improve the yield of interchromophore energy transfer and extend the range of excitation transport. Our observations have implications for other important, similarly sized bioinspired materials, such as nanoreactors and biocompatible targeted delivery agents.

Viral capsids as self-assembling templates for new materials.

The self-assembling protein shells of viruses have provided convenient scaffolds for the construction of many new materials with well-defined nanoscale architectures. In some cases, the native amino acid functional groups have served as nucleation sites for the deposition of metals and semiconductors, leading to organic-inorganic composites with interesting electronic, magnetic, optical, and catalytic properties. Other approaches have involved the covalent modification of the protein monomers, typically with the goal of generating targeting delivery vehicles for drug and imaging cargo. Covalently modified capsid proteins have also been used to generate periodic arrays of chromophores for use in light harvesting and photocatalytic applications. All of these research areas have taken advantage of the low polydispersity, high chemical stability, and intrinsically multivalent properties that are uniquely offered by these biological building blocks.