Biodistribution, pharmacokinetics, and blood compatibility of native and PEGylated tobacco mosaic virus nano-rods and -spheres in mice.

Understanding the pharmacokinetics, blood compatibility, biodistribution and clearance properties of nanoparticles is of great importance to their translation to clinical application. In this paper we report the biodistribution and pharmacokinetic properties of tobacco mosaic virus (TMV) in the forms of 300×18nm(2) rods and 54nm-sized spheres. The availability of rods and spheres made of the same protein provides a unique scaffold to study the effect of nanoparticle shape on in vivo fate. For enhanced biocompatibility, we also considered a PEGylated formulation. Overall, the versions of nanoparticles exhibited comparable in vivo profiles; a few differences were noted: data indicate that rods circulate longer than spheres, illustrating the effect that shape plays on circulation. Also, PEGylation increased circulation times. We found that macrophages in the liver and spleen cleared the TMV rods and spheres from circulation. In the spleen, the viral nanoparticles trafficked through the marginal zone before eventually co-localizing in B-cell follicles. TMV rods and spheres were cleared from the liver and spleen within days with no apparent changes in histology, it was noted that spheres are more rapidly cleared from tissues compared to rods. Further, blood biocompatibility was supported, as none of the formulations induced clotting or hemolysis. This work lays the foundation for further application and tailoring of TMV for biomedical applications.

Potato virus X as a novel platform for potential biomedical applications.

We demonstrate that nanoparticles formed from the rod-shaped plant virus Potato virus X (PVX) can serve as a novel platform for biomedical applications. Bioconjugation protocols including amine modification and "click" chemistry allowed the efficient functionalization of PVX with biotins, dyes, and PEGs. Fluorescent-labeled and PEGylated PVX particles revealed that different fluorescent labels have a profound effect on PVX-cell interactions. Applying bioconjugation chemistries to PVX opens the door for chemical functionalization with targeting and therapeutic molecules.

Synergy of NMR, computation, and X-ray crystallography for structural biology.

NMR spectroscopy and X-ray crystallography are currently the two most widely applied methods for the determination of macromolecular structures at high resolution. More recently, significant advances have been made in algorithms for the de novo prediction of protein structure, and, in favorable cases, the predicted models agree extremely well with experimentally determined structures. Here, we demonstrate a synergistic combination of NMR spectroscopy, de novo structure prediction, and X-ray crystallography in an effective overall strategy for rapidly determining the structure of the coat protein C-terminal domain from the Sulfolobus islandicus rod-shaped virus (SIRV). This approach takes advantage of the most accessible aspects of each structural technique and may be widely applicable for structure determination.

Synergistic, random sequential binding of substrates in cobalamin-independent methionine synthase.

Cobalamin-independent methionine synthase (MetE) catalyzes the transfer of the N5-methyl group of methyltetrahydrofolate (CH(3)-H(4)folate) to the sulfur of homocysteine (Hcy) to form methionine and tetrahydrofolate (H(4)folate) as products. This reaction is thought to involve a direct methyl transfer from one substrate to the other, requiring the two substrates to interact in a ternary complex. The crystal structure of a MetE.CH(3)-H(4)folate binary complex shows that the methyl group is pointing away from the Hcy binding site and is quite distant from the position where the sulfur of Hcy would be, raising the possibility that this binary complex is nonproductive. The CH(3)-H(4)folate must either rearrange or dissociate before methyl transfer can occur. Therefore, determining the order of substrate binding is of interest. We have used kinetic and equilibrium measurements in addition to isotope trapping experiments to elucidate the kinetic pathway of substrate binding in MetE. These studies demonstrate that both substrate binary complexes are chemically and kinetically competent for methyl transfer and suggest that the conformation observed in the crystal structure is indeed on-pathway. Additionally, the substrates are shown to bind synergistically, with each substrate binding 30-fold more tightly in the presence of the other. Methyl transfer has been determined to be slow compared to ternary complex formation and dissociation. Simulations indicate that nearly all of the enzyme is present as the ternary complex under physiological conditions.

Activation of methyltetrahydrofolate by cobalamin-independent methionine synthase.

Cobalamin-independent methionine synthase (MetE) catalyzes the final step of de novo methionine synthesis using the triglutamate derivative of methyltetrahydrofolate (CH(3)-H(4)PteGlu(3)) as methyl donor and homocysteine (Hcy) as methyl acceptor. This reaction is challenging because at physiological pH the Hcy thiol is not a strong nucleophile and CH(3)-H(4)PteGlu(3) provides a very poor leaving group. Our laboratory has previously established that Hcy is ligated to a tightly bound zinc ion in the MetE active site. This interaction activates Hcy by lowering its pK(a), such that the thiolate is stabilized at neutral pH. The remaining chemical challenge is the activation of CH(3)-H(4)PteGlu(3). Protonation of N5 of CH(3)-H(4)PteGlu(3) would produce a better leaving group, but occurs with a pK(a) of 5 in solution. We have taken advantage of the sensitivity of the CH(3)-H(4)PteGlu(3) absorption spectrum to probe its protonation state when bound to MetE. Comparison of free and MetE-bound CH(3)-H(4)PteGlu(3) absorbance spectra indicated that the N5 is not protonated in the binary complex. Rapid reaction studies have revealed changes in CH(3)-H(4)PteGlu(3) absorbance that are consistent with protonation at N5. These absorbance changes show saturable dependence on both Hcy and CH(3)-H(4)PteGlu(3), indicating that protonation of CH(3)-H(4)PteGlu(3) occurs upon formation of the ternary complex and prior to methyl transfer. Furthermore, the tetrahydrofolate (H(4)PteGlu(3)) product appears to remain bound to MetE, and in the presence of excess Hcy a MetE.H(4)PteGlu(3).Hcy mixed ternary complex forms, in which H(4)PteGlu(3) is protonated.