Convenient site-selective protein coupling from bacterial raw lysates to coenzyme A-modified tobacco mosaic virus (TMV) by Bacillus subtilis Sfp phosphopantetheinyl transferase.

A facile enzyme-mediated strategy enables site-specific covalent one-step coupling of genetically tagged luciferase molecules to coenzyme A-modified tobacco mosaic virus (TMV-CoA) both in solution and on solid supports. Bacillus subtilis surfactin phosphopantetheinyl transferase Sfp produced in E. coli mediated the conjugation of firefly luciferase N-terminally extended by eleven amino acids forming a 'ybbR tag' as Sfp-selective substrate, which even worked in bacterial raw lysates. The enzymes displayed on the protein coat of the TMV nanocarriers exhibited high activity. As TMV has proven a beneficial high surface-area adapter template stabilizing enzymes in different biosensing layouts in recent years, the use of TMV-CoA for fishing ybbR-tagged proteins from complex mixtures might become an advantageous concept for the versatile equipment of miniaturized devices with biologically active proteins. It comes along with new opportunities for immobilizing multiple functionalities on TMV adapter coatings, as desired, e.g., in handheld systems for point-of-care detection.

Impaired integrin α5 /ß1 -mediated hepatocyte growth factor release by stellate cells of the aged liver.

Hepatic blood flow and sinusoidal endothelial fenestration decrease during aging. Consequently, fluid mechanical forces are reduced in the space of Disse where hepatic stellate cells (HSC) have their niche. We provide evidence that integrin α5 /ß1 is an important mechanosensor in HSC involved in shear stress-induced release of hepatocyte growth factor (HGF), an essential inductor of liver regeneration which is impaired during aging. The expression of the integrin subunits α5 and ß1 decreases in liver and HSC from aged rats. CRISPR/Cas9-mediated integrin α5 and ß1 knockouts in isolated HSC lead to lowered HGF release and impaired cellular adhesion. Fluid mechanical forces increase integrin α5 and laminin gene expression whereas integrin ß1 remains unaffected. In the aged liver, laminin ß2 and γ1 protein chains as components of laminin-521 are lowered. The integrin α5 knockout in HSC reduces laminin expression via mechanosensory mechanisms. Culture of HSC on nanostructured surfaces functionalized with laminin-521 enhances Hgf expression in HSC, demonstrating that these ECM proteins are critically involved in HSC function. During aging, HSC acquire a senescence-associated secretory phenotype and lower their growth factor expression essential for tissue repair. Our findings suggest that impaired mechanosensing via integrin α5 /ß1 in HSC contributes to age-related reduction of ECM and HGF release that could affect liver regeneration.

Laminin-521 promotes quiescence in isolated stellate cells from rat liver.

The laminin α5 protein chain is an element of basement membranes and important to maintain stem cells. Hepatic stellate cells (HSC) are liver-resident mesenchymal stem cells, which reside in a quiescent state on a basement membrane-like structure in the space of Dissé. In the present study, laminin α5 chain was detected in the space of Dissé of normal rat liver. Since HSC are critical for liver regeneration and can contribute to fibrosis in chronic liver diseases, the effect of laminins on HSC maintenance was investigated. Therefore, isolated rat HSC were seeded on uncoated polystyrene (PS) or PS coated with either laminin-521 (PS/LN-521) or laminin-211 (PS/LN-211). PS/LN-521 improved HSC adhesion and better preserved their retinoid stores as well as quiescence- and stem cell-associated phenotype, whereas HSC on PS/LN-211 or PS developed into myofibroblasts-like cells. To improve the homogeneity as well as the presentation of laminin molecules on the culture surface to HSC, laminin-functionalized, gold-nanostructured glass surfaces were generated. This approach further enhanced the expression of quiescence-associated genes in HSC. In conclusion, the results indicate that LN-521 supports the quiescent state of HSC and laminin α5 can be regarded as an important element of their niche in the space of Dissé.

Dual Functionalization of Rod-Shaped Viruses on Single Coat Protein Subunits.

Plant viruses are emerging as versatile tools for nanotechnology applications since it is possible to modify their multivalent protein surfaces and thereby introduce and display new functionalities. In this chapter, we describe a tobacco mosaic virus (TMV) variant that exposes two selectively addressable amino acid moieties on each of its 2130 coat protein (CP) subunits. A lysine as well as a cysteine introduced at accessible sites of every CP can be modified with amino- and/or thiol-reactive chemistry such as N-hydroxysuccinimide esters (NHS ester) and maleimide containing reagents alone or simultaneously. This enables the pairwise immobilization of distinct molecules in close vicinity to each other on the TMV surface by simple standard conjugation protocols. We describe the generation of the mutations, the virus propagation and isolation as well as the dual functionalization of the TMV variant with two fluorescent dyes. The labeling is evaluated by SDS-PAGE and spectrophotometry and the degree of labeling (DOL) calculated.

Novel roles for well-known players: from tobacco mosaic virus pests to enzymatically active assemblies.

The rod-shaped nanoparticles of the widespread plant pathogen tobacco mosaic virus (TMV) have been a matter of intense debates and cutting-edge research for more than a hundred years. During the late 19th century, their behavior in filtration tests applied to the agent causing the 'plant mosaic disease' eventually led to the discrimination of viruses from bacteria. Thereafter, they promoted the development of biophysical cornerstone techniques such as electron microscopy and ultracentrifugation. Since the 1950s, the robust, helically arranged nucleoprotein complexes consisting of a single RNA and more than 2100 identical coat protein subunits have enabled molecular studies which have pioneered the understanding of viral replication and self-assembly, and elucidated major aspects of virus-host interplay, which can lead to agronomically relevant diseases. However, during the last decades, TMV has acquired a new reputation as a well-defined high-yield nanotemplate with multivalent protein surfaces, allowing for an ordered high-density presentation of multiple active molecules or synthetic compounds. Amino acid side chains exposed on the viral coat may be tailored genetically or biochemically to meet the demands for selective conjugation reactions, or to directly engineer novel functionality on TMV-derived nanosticks. The natural TMV size (length: 300 nm) in combination with functional ligands such as peptides, enzymes, dyes, drugs or inorganic materials is advantageous for applications ranging from biomedical imaging and therapy approaches over surface enlargement of battery electrodes to the immobilization of enzymes. TMV building blocks are also amenable to external control of in vitro assembly and re-organization into technically expedient new shapes or arrays, which bears a unique potential for the development of 'smart' functional 3D structures. Among those, materials designed for enzyme-based biodetection layouts, which are routinely applied, e.g., for monitoring blood sugar concentrations, might profit particularly from the presence of TMV rods: Their surfaces were recently shown to stabilize enzymatic activities upon repeated consecutive uses and over several weeks. This review gives the reader a ride through strikingly diverse achievements obtained with TMV-based particles, compares them to the progress with related viruses, and focuses on latest results revealing special advantages for enzyme-based biosensing formats, which might be of high interest for diagnostics employing 'systems-on-a-chip'.

Peptide-equipped tobacco mosaic virus templates for selective and controllable biomineral deposition.

The coating of regular-shaped, readily available nanorod biotemplates with inorganic compounds has attracted increasing interest during recent years. The goal is an effective, bioinspired fabrication of fiber-reinforced composites and robust, miniaturized technical devices. Major challenges in the synthesis of applicable mineralized nanorods lie in selectivity and adjustability of the inorganic material deposited on the biological, rod-shaped backbones, with respect to thickness and surface profile of the resulting coating, as well as the avoidance of aggregation into extended superstructures. Nanotubular tobacco mosaic virus (TMV) templates have proved particularly suitable towards this goal: Their multivalent protein coating can be modified by high-surface-density conjugation of peptides, inducing and governing silica deposition from precursor solutions in vitro. In this study, TMV has been equipped with mineralization-directing peptides designed to yield silica coatings in a reliable and predictable manner via precipitation from tetraethoxysilane (TEOS) precursors. Three peptide groups were compared regarding their influence on silica polymerization: (i) two peptide variants with alternating basic and acidic residues, i.e. lysine-aspartic acid (KD) x motifs expected to act as charge-relay systems promoting TEOS hydrolysis and silica polymerization; (ii) a tetrahistidine-exposing polypeptide (CA4H4) known to induce silicification due to the positive charge of its clustered imidazole side chains; and (iii) two peptides with high ZnO binding affinity. Differential effects on the mineralization of the TMV surface were demonstrated, where a (KD) x charge-relay peptide (designed in this study) led to the most reproducible and selective silica deposition. A homogenous coating of the biotemplate and tight control of shell thickness were achieved.

Modified TMV Particles as Beneficial Scaffolds to Present Sensor Enzymes.

Tobacco mosaic virus (TMV) is a robust nanotubular nucleoprotein scaffold increasingly employed for the high density presentation of functional molecules such as peptides, fluorescent dyes, and antibodies. We report on its use as advantageous carrier for sensor enzymes. A TMV mutant with a cysteine residue exposed on every coat protein (CP) subunit (TMVCys) enabled the coupling of bifunctional maleimide-polyethylene glycol (PEG)-biotin linkers (TMVCys/Bio). Its surface was equipped with two streptavidin [SA]-conjugated enzymes: glucose oxidase ([SA]-GOx) and horseradish peroxidase ([SA]-HRP). At least 50% of the CPs were decorated with a linker molecule, and all thereof with active enzymes. Upon use as adapter scaffolds in conventional "high-binding" microtiter plates, TMV sticks allowed the immobilization of up to 45-fold higher catalytic activities than control samples with the same input of enzymes. Moreover, they increased storage stability and reusability in relation to enzymes applied directly to microtiter plate wells. The functionalized TMV adsorbed to solid supports showed a homogeneous distribution of the conjugated enzymes and structural integrity of the nanorods upon transmission electron and atomic force microscopy. The high surface-increase and steric accessibility of the viral scaffolds in combination with the biochemical environment provided by the plant viral coat may explain the beneficial effects. TMV can, thus, serve as a favorable multivalent nanoscale platform for the ordered presentation of bioactive proteins.

TMV nanorods with programmed longitudinal domains of differently addressable coat proteins.

The spacing of functional nanoscopic elements may play a fundamental role in nanotechnological and biomedical applications, but is so far rarely achieved on this scale. In this study we show that tobacco mosaic virus (TMV) and the RNA-guided self-assembly process of its coat protein (CP) can be used to establish new nanorod scaffolds that can be loaded not only with homogeneously distributed functionalities, but with distinct molecule species grouped and ordered along the longitudinal axis. The arrangement of the resulting domains and final carrier rod length both were governed by RNA-templated two-step in vitro assembly. Two selectively addressable TMV CP mutants carrying either thiol (TMVCys) or amino (TMVLys) groups on the exposed surface were engineered and shown to retain reactivity towards maleimides or NHS esters, respectively, after acetic acid-based purification and re-assembly to novel carrier rod types. Stepwise combination of CP(Cys) and CP(Lys) with RNA allowed fabrication of TMV-like nanorods with a controlled total length of 300 or 330 nm, respectively, consisting of adjacent longitudinal 100-to-200 nm domains of differently addressable CP species. This technology paves the way towards rod-shaped scaffolds with pre-defined, selectively reactive barcode patterns on the nanometer scale.

Superresolution imaging of biological nanostructures by spectral precision distance microscopy.

For the improved understanding of biological systems on the nanoscale, it is necessary to enhance the resolution of light microscopy in the visible wavelength range beyond the limits of conventional epifluorescence microscopy (optical resolution of about 200 nm laterally, 600 nm axially). Recently, various far-field methods have been developed allowing a substantial increase of resolution ("superresolution microscopy", or "lightoptical nanoscopy"). This opens an avenue to 'nano-image' intact and even living cells, as well as other biostructures like viruses, down to the molecular detail. Thus, it is possible to combine light optical spatial nanoscale information with ultrastructure analyses and the molecular interaction information provided by molecular cell biology. In this review, we describe the principles of spectrally assigned localization microscopy (SALM) of biological nanostructures, focusing on a special SALM approach, spectral precision distance/position determination microscopy (SPDM) with physically modified fluorochromes (SPDM(Phymod) . Generally, this SPDM method is based on high-precision localization of fluorescent molecules, which can be discriminated using reversibly bleached states of the fluorophores for their optical isolation. A variety of application examples is presented, ranging from superresolution microscopy of membrane and cytoplasmic protein distribution to dual-color SPDM of nuclear proteins. At present, we can achieve an optical resolution of cellular structures down to the 20-nm range, with best values around 5 nm (∼1/100 of the exciting wavelength).

Enhancing the magnetoviscosity of ferrofluids by the addition of biological nanotubes.

Applying a magnetic field to many ferrofluids leads to a significant increase in viscosity, but the phenomenon has yet to find technological exploitation because of the thinning caused by even weak shear flows. We have discovered that the addition of plant-virus-derived nanotubes to a commercial ferrofluid can give rise to a dramatic enhancement in magnetoviscosity and a suppression of shear thinning. The dependence of this effect on nanotube aspect ratio and surface charge, both of which were varied biotechnologically, is consistent with a "scaffolding" of magnetic particles into quasi-linear arrays. Direct support for this explanation is derived from transmission electron micrographs, which reveal a marked tendency for the magnetic nanoparticles to decorate the outside surface of the virus nanotubes.