Imma Ratera

Bacterial inclusion bodies: making gold from waste

Many protein species produced in recombinant bacteria aggregate as insoluble protein clusters named inclusion bodies (IBs). IBs are discarded from further processing or are eventually used as a pure protein source for in vitro refolding. Although usually considered as waste byproducts of protein production, recent insights into the physiology of recombinant bacteria and the molecular architecture of IBs have revealed that these protein particles are unexpected functional materials. In this Opinion article, we present the relevant mechanical properties of IBs and discuss the ways in which they can be explored as biocompatible nanostructured materials, mainly, but not exclusively, in biocatalysis and tissue engineering.

A simple and robust reversible redox–fluorescence molecular switch based on a 1,4-disubstituted azine with ferrocene and pyrene units

Taking advantage of the properties of the ferrocene as a redox and electron donor active unit and the pyrene as a fluorescent unit, dyad 2 shows a fast and reversible redox-switchable fluorescence emission.

The nanoscale properties of bacterial inclusion bodies and their effect on mammalian cell proliferation.

The chemical and mechanical properties of bacterial inclusion bodies, produced in different Escherichia coli genetic backgrounds, have been characterized at the nanoscale level. In regard to wild type, DnaK(-) and ClpA(-) strains produce inclusion bodies with distinguishable wettability, stiffness and stiffness distribution within the proteinaceous particle. Furthermore it was possible to observe how cultured mammalian cells respond differentially to inclusion body variants when used as particulate materials to engineer the nanoscale topography, proving that the actual range of referred mechanical properties is sensed and discriminated by biological systems. The data provide evidence of the mechanistic activity of the cellular quality control network and the regulation of the stereospecific packaging of partially folded protein species in bacteria. This inclusion body nanoscale profiling offers possibilities for their fine genetic tuning and the resulting macroscopic effects when applied in biological interfaces.

Protein nanodisk assembling and intracellular trafficking powered by an arginine-rich (R9) peptide

AIMS Arginine(R)-rich cationic peptides are powerful tools in drug delivery since, alone or when associated with polyplexes, proteins or chemicals, they confer DNA condensation, membrane translocation and blood-brain barrier crossing abilities. The unusual stability and high in vivo performance of their associated drugs suggest a particulate organization or R(n) complexes, which this study aimed to explore. MATERIALS & METHODS We have analyzed the particulate organization and biological performance in DNA delivery of a model, R9-containing green fluorescent protein by dynamic light scattering, transmission electron microscopy, atomic force microscopy, single cell confocal microscopy and flow cytometry. RESULTS A deep nanoscale examination of R9-powered constructs reveals a novel and promising feature of R9, that when fused to a scaffold green fluorescent protein, promote its efficient self-assembling as highly stable, regular disk-shaped nanoparticles of 20 x 3 nm. These constructs are efficiently internalized in mammalian cells and rapidly migrate through the cytoplasm towards the nucleus in a fully bioactive form. Besides, such particulate platforms accommodate, condense and deliver plasmid DNA to the nucleus and promote plasmid-driven transgene expression. CONCLUSION The architectonic properties of arginine-rich peptides at the nanoscale reveal a new category of protein nanoparticles, namely nanodisks, and provide novel strategic concepts and architectonic tools for the tailored construction of new-generation artificial viruses for gene therapy and drug delivery.

Bistability in Fc-PTM crystals: the role of intermolecular electrostatic interactions.

Fc-PTM is a valence tautomeric radical, where the ferrocene (Fc) group, a good electron donor, is linked by an ethylenic spacer to a perchlorotriphenylmethyl radical (PTM(*)), a good electron acceptor. In solution this compound exists mainly in the neutral Fc-PTM(*) form which can be photoexcited through an intramolecular electron transfer to the zwitterionic Fc(+*)-PTM(-) form. By contrast, in crystals of Fc-PTM at room temperature both the neutral and the zwitterionic forms coexist, pointing to a true bistability phenomenon. We rationalize these findings accounting for the role of intermolecular electrostatic interactions in Fc-PTM crystals. In fact the energy of the zwitterionic Fc(+*)-PTM(-) form is lowered in the crystal by attractive electrostatic intermolecular interactions and the cooperative nature of these interactions explains the observed coexistence of neutral Fc-PTM(*) and zwitterionic Fc(+*)-PTM(-) species. The temperature evolution of Mossbauer spectra of Fc-PTM is quantitatively reproduced adopting a bottom-up modeling strategy that combines a molecular model, derived from optical spectra of Fc-PTM in solution, with a model for intermolecular electrostatic interactions, supported by quantum-chemical calculations. Fc-PTM then offers the first experimental demonstration of bistability induced by electrostatic interactions in crystals of valence tautomeric donor-acceptor molecules.

In vivo architectonic stability of fully de novo designed protein-only nanoparticles.

The fully de novo design of protein building blocks for self-assembling as functional nanoparticles is a challenging task in emerging nanomedicines, which urgently demand novel, versatile, and biologically safe vehicles for imaging, drug delivery, and gene therapy. While the use of viruses and virus-like particles is limited by severe constraints, the generation of protein-only nanocarriers is progressively reachable by the engineering of protein-protein interactions, resulting in self-assembling functional building blocks. In particular, end-terminal cationic peptides drive the organization of structurally diverse protein species as regular nanosized oligomers, offering promise in the rational engineering of protein self-assembling. However, the in vivo stability of these constructs, being a critical issue for their medical applicability, needs to be assessed. We have explored here if the cross-molecular contacts between protein monomers, generated by end-terminal cationic peptides and oligohistidine tags, are stable enough for the resulting nanoparticles to overcome biological barriers in assembled form. The analyses of renal clearance and biodistribution of several tagged modular proteins reveal long-term architectonic stability, allowing systemic circulation and tissue targeting in form of nanoparticulate material. This observation fully supports the value of the engineered of protein building blocks addressed to the biofabrication of smart, robust, and multifunctional nanoparticles with medical applicability that mimic structure and functional capabilities of viral capsids.

Intra- and Intermolecular Charge Transfer in Aggregates of Tetrathiafulvalene-Triphenylmethyl Radical Derivatives in Solution

An extensive investigation of aggregation phenomena occurring in solution for a family of electron donor-acceptor derivatives, based on polychlorotriphenylmethyl radicals (PTM) linked via a vinylene-bridge to tetrathiafulvalene (TTF) units, is presented. A large set of temperature and/or concentration dependent optical absorption and electron spin resonance (ESR) spectra in a solution of dyads bearing different number of electrons and/or with a hydrogenated PTM residue offer reliable information on the formation of homo dimers and mixed valence dimers. The results shed light on the reciprocal influence of intramolecular electron transfer (IET) within a dyad and the intermolecular charge transfer (CT) occurring in a dimer between the TTF residues and are rationalized based on a theoretical model that describes both interactions.

Supramolecular organization of protein-releasing functional amyloids solved in bacterial inclusion bodies

Slow protein release from amyloidal materials is a molecular platform used by nature to control protein hormone secretion in the endocrine system. The molecular mechanics of the sustained protein release from amyloids remains essentially unexplored. Inclusion bodies (IBs) are natural amyloids that occur as discrete protein nanoparticles in recombinant bacteria. These protein clusters have been recently explored as protein-based functional biomaterials with diverse biomedical applications, and adapted as nanopills to deliver recombinant protein drugs into mammalian cells. Interestingly, the slow protein release from IBs does not significantly affect the particulate organization and morphology of the material, suggesting the occurrence of a tight scaffold. Here, we have determined, by using a combined set of analytical approaches, a sponge-like supramolecular organization of IBs combining differently folded protein versions (amyloid and native-like), which supports both mechanical stability and sustained protein delivery. Apart from offering structural clues about how amyloid materials release their monomeric protein components, these findings open exciting possibilities for the tailored development of smart biofunctional materials, adapted to mimic the functions of amyloid-based secretory glands of higher organisms.

Essential State Models for Solvatochromism in Donor-Acceptor Molecules : The Role of the Bridge

Essential state models are presented to discuss absorption spectra of two related donor-acceptor (DA) chromophores that show two solvatochromic bands in the near-infrared spectral region. The two-state model only accounts for the lowest energy band and results in a very small value of mu(0), the dipole moment associated with the D(+)A(-) state. The model is then extended to account for the active role of the bridge: the resulting three-state model satisfactorily reproduces the double solvatochromism, leading at the same time to a roughly doubled estimate of mu(0). This result, supported by a detailed analysis of an N-state model that explicitly accounts for bridge states, rationalizes the well-known discrepancy between the geometrical DA distance and the dipole length extracted from the analysis of optical spectra of DA chromophores as reflecting the active role of bridge states, not explicitly accounted for in essential state models.

Naked-eye and Selective Detection of Mercury (II) Ions in Mixed Aqueous Media Using a Cellulose-based Support

A test paper for high-selectivity detecting Hg2+ ions in mixed acetonitrile-water solutions has been achieved using a bis(ferrocenyl) azine, as chromogenic chemosensor molecule, and a solid cellulose fibre, as a substrate. Depending on the amount of mercury ions in contact with the detecting molecule a spectacular color change in the cellulose indicator is produced, being possible to determine the concentration of Hg2+ ions either by naked eye or spectroscopically.