Gerald Stubbs

Inorganic–Organic Nanotube Composites from Template Mineralization of Tobacco Mosaic Virus

The use of biological molecules, assemblies and systems in the development of inorganic materials synthesis continues to offer new and exciting alternatives to conventional synthetic strategies. Biological templates, such as protein cages, viroid capsules, bacterial rhapidosomes, S-layers, multicellular superstructures, biolipid cylinders, and DNA, have been utilized to direct the deposition, assembly, and patterning of inorganic nanoparticles and microstructures. In this paper, we report a new approach to the template-directed synthesis of inorganic±organic nanotubes using tobacco mosaic virus (TMV). TMV is a remarkably stable virion, remaining intact at temperatures up to 60 C and at pH values between 2 and 10. Each viral particle consists of 2130 identical protein subunits arranged in a helical motif around a single strand of RNA to produce a hollow protein tube, 300 18 nm in size, with a 4 nm-wide central channel. The internal and external surfaces of the protein consist of repeated patterns of charged amino acid residues, such as glutamate, aspartate, arginine, and lysine. In principle, these functionalities should offer a wide variety of nucleation sites for surface-controlled inorganic deposition, which, in association with the high thermal and pH stability, could be exploited in the synthesis of unusual materials such as high-aspect-ratio composites and protein-confined inorganic nanowires. Here we show that TMV is a suitable template for reactions such as co-crystallization (CdS and PbS), oxidative hydrolysis (iron oxides), and sol-gel condensation (SiO2) (Fig. 1).

Visualization of protein-nucleic acid interactions in a virus. Refined structure of intact tobacco mosaic virus at 2.9 A resolution by X-ray fiber diffraction.

The structure of tobacco mosaic virus (TMV) has been determined by fiber diffraction methods at 2.9 A resolution, and refined by restrained least-squares to an R-factor of 0.096. Protein-nucleic acid interactions are clearly visible. The final model contains all of the non-hydrogen atoms of the RNA and the protein, 71 water molecules, and two calcium-binding sites. Viral disassembly is driven by electrostatic repulsions between the charges in two carboxyl-carboxylate pairs and a phosphate-carboxylate pair. The phosphate-carboxylate pair and at least one of the carboxyl-carboxylate pairs appear to be calcium-binding sites. Nucleotide specificity, enabling TMV to recognize its own RNA by a repeating pattern of guanine residues, is provided by two guanine-specific hydrogen bonds in one of the three base-binding sites.

Solid-state NMR structure of a pathogenic fibril of full-length human alpha-synuclein.

Misfolded α-synuclein amyloid fibrils are the principal components of Lewy bodies and neurites, hallmarks of Parkinsons disease (PD). We present a high-resolution structure of an α-synuclein fibril, in a form that induces robust pathology in primary neuronal culture, determined by solid-state NMR spectroscopy and validated by EM and X-ray fiber diffraction. Over 200 unique long-range distance restraints define a consensus structure with common amyloid features including parallel, in-register β-sheets and hydrophobic-core residues, and with substantial complexity arising from diverse structural features including an intermolecular salt bridge, a glutamine ladder, close backbone interactions involving small residues, and several steric zippers stabilizing a new orthogonal Greek-key topology. These characteristics contribute to the robust propagation of this fibril form, as supported by the structural similarity of early-onset-PD mutants. The structure provides a framework for understanding the interactions of α-synuclein with other proteins and small molecules, to aid in PD diagnosis and treatment.

Natural and synthetic prion structure from X-ray fiber diffraction

A conformational isoform of the mammalian prion protein (PrPSc) is the sole component of the infectious pathogen that causes the prion diseases. We have obtained X-ray fiber diffraction patterns from infectious prions that show cross-β diffraction: meridional intensity at 4.8 Å resolution, indicating the presence of β strands running approximately at right angles to the filament axis and characteristic of amyloid structure. Some of the patterns also indicated the presence of a repeating unit along the fiber axis, corresponding to four β-strands. We found that recombinant (rec) PrP amyloid differs substantially from highly infectious brain-derived prions, both in structure as demonstrated by the diffraction data, and in heterogeneity as shown by electron microscopy. In addition to the strong 4.8 Å meridional reflection, the recPrP amyloid diffraction is characterized by strong equatorial intensity at approximately 10.5 Å, absent from brain-derived prions, and indicating the presence of stacked β-sheets. Synthetic prions recovered from transgenic mice inoculated with recPrP amyloid displayed structural characteristics and homogeneity similar to those of naturally occurring prions. The relationship between the structural differences and prion infectivity is uncertain, but might be explained by any of several hypotheses: only a minority of recPrP amyloid possesses a replication-competent conformation, the majority of recPrP amyloid has to undergo a conformational maturation to acquire replication competency, or inhibitory forms of recPrP amyloid interfere with replication during the initial transmission.

Visualization of alpha-helices in tobacco mosaic virus by cryo-electron microscopy

We have used tobacco mosaic virus (TMV) as a test specimen, in order to develop techniques for the analysis of high-resolution structural detail in electron micrographs of biological assemblies with helical symmetry. It has previously been shown that internal details of protein structure can be visualized by processing electron micrographs of unstained specimens of extended two-dimensional crystalline arrays. However, the techniques should in principle be applicable to other periodic specimens, such as assemblies with helical symmetry. We show here that data to spacings better than 10 A can be retrieved from electron images of frozen hydrated TMV. The three-dimensional computed map agrees well with that derived from X-ray diffraction and shows the two pairs of alpha-helices forming the core of the coat subunit, the C alpha-helix and the viral RNA. The results demonstrate that it is possible to determine detailed internal structure in helical particles.

Structure of Flexible Filamentous Plant Viruses

ABSTRACT Flexible filamentous viruses make up a large fraction of the known plant viruses, but in comparison with those of other viruses, very little is known about their structures. We have used fiber diffraction, cryo-electron microscopy, and scanning transmission electron microscopy to determine the symmetry of a potyvirus, soybean mosaic virus; to confirm the symmetry of a potexvirus, potato virus X; and to determine the low-resolution structures of both viruses. We conclude that these viruses and, by implication, most or all flexible filamentous plant viruses share a common coat protein fold and helical symmetry, with slightly less than 9 subunits per helical turn.

Preparation of high quality nanowires by tobacco mosaic virus templating of gold nanoparticles

A protocol for the preparation of stable suspensions of well-defined metallized tobacco mosaic virus (TMV) nanorods in high yields and with uniform coatings is reported. Inorganic growth on the virion surface was controlled by adding aliquots of gold precursor (HAuCl4) and reducing agent (NaBH4) incrementally over five addition–reduction cycles to produce hybrid anisotropic nanostructures consisting of densely packed gold nanoparticles. Several novel steps, including addition of ethanol after the first addition–reduction cycle and wrapping of the TMV/Au nanohybrids with poly-L-lysine, were introduced into the protocol to ensure high homogeneity and stability in the nanowire suspension. Control of the interparticle spacing in the metallized nanostructures was achieved by restricting the number of reaction cycles used to less than four iterations. The importance of bio-mediated reduction was probed using laser light scattering microscopy. The results indicated that short incubation times were consistent with increased nanoparticle monodispersity and high fidelity replication.

Microtubule structure at 18 A resolution.

A model for the structure of microtubules at a resolution of 18 A (1 A = 0.1 nm) is described, based on X-ray fiber diffraction data from hydrated reassembled calf brain microtubules. The model was derived by an iterative solvent flattening refinement procedure, with initial phases based on those determined by electron microscopy. The major microtubule surface grooves are those defining the protofilaments, which form a hollow cylinder of maximum diameter 300 A. Strong electron density fluctuations in the microtubule wall are interpreted as evidence for a domain structure within the tubulin subunit. The arrangement of domains is such that the tubulin molecule could be quite flexible at the domain connections; thus, slight changes in this arrangement could account for the unusual polymorphism of tubulin assemblies.

Structure of the U2 strain of tobacco mosaic virus refined at 3.5 A resolution using X-ray fiber diffraction.

The structure of the U2 strain of tobacco mosaic virus (TMV) has been determined by fiber diffraction methods at 3.5 A resolution, and refined by a combination of restrained least-squares and molecular dynamics methods to an R-factor of 0.096. The structure is extremely similar to that of the common strain of TMV, with the largest differences being in the protein loop that makes up the inner surface of the virus, and in the C-terminal region on the outer surface. Differences in the inner loop can be correlated with differences in the properties of the two viruses.

Site-directed mutagenesis confirms the involvement of carboxylate groups in the disassembly of tobacco mosaic virus

Electrostatic repulsion between carboxylate groups across subunit interfaces has for many years been recognized as important in the disassembly of simple plant viruses. In the coat protein of tobacco mosaic virus (TMV), the amino acids Glu50 and Asp77 have been proposed as examples of such carboxylate groups. Site-directed mutagenesis has been used to replace these amino acids by Gln and Asn, respectively. Increased virion stability, together with reduced infectivity and reduced capacity for long-distance transport within the host plant confirms that the negative charges on the side chains of these amino acids are involved in the disassembly of TMV. Mixing purified mutant coat proteins with wild-type virions under appropriate conditions stabilizes the virions to alkaline disassembly and reduces their infectivity. It is suggested that transgenic plants expressing such mutant coat proteins could have enhanced resistance to virus infection.