Nanoparticles have repeatedly been shown to enhance fibril formation when assayed with amyloidogenic proteins. Recently, however, evidence casting some doubt about the generality of this conclusion started to emerge. Therefore, to investigate further the influence of nanoparticles on the fibrillation process, we used a naturally occurring variant of the paradigmatic amyloidogenic protein β-microglobulin (β2m), namely D76N β2m where asparagine replaces aspartate at position 76. This variant is responsible for aggressive systemic amyloidosis. After characterizing the interaction of the variant with citrate-stabilized gold nanoparticles (Cit-AuNPs) by NMR and modeling, we analyzed the fibril formation by three different methods: thioflavin T fluorescence, native agarose gel electrophoresis and transmission electron microscopy. The NMR evidence indicated a fast-exchange interaction involving preferentially specific regions of the protein that proved, by subsequent modeling, to be consistent with a dimeric adduct interacting with Cit-AuNPs. The fibril detection assays showed that AuNPs are able to hamper D76N β2m fibrillogenesis through an effective interaction that competes with protofibril formation or recruitment. These findings open promising perspectives for the optimization of the nanoparticle surface to design tunable interactions with proteins.
The effects induced by extrinsic paramagnetic probes on protein NMR spectra, widely used for surface mapping, can also be exploited to detect the sites of slow and intermediate exchange due to structural or intermolecular interaction dynamics.
a b s t r a c tPristine (as prepared) carbon nanotube (CNT) based substrates have been widely used to grow and interface neurons in culture. Nerve cells normally differentiate on CNTs and the synaptic networks, newly formed at the interface with this material, usually show an improved robustness in signal transfer. However manipulation of pristine CNTs is often prevented by their low dispersibility and tendency to aggregate in most solvents. This issue can be at least partially solved by adding solubilizing groups to the surface of CNT, which also helps improving their biocompatibility. It becomes therefore of crucial importance to determine whether chemically manipulated CNTs may maintain their performance in improving nerve signaling. Here we study and compare the impact in vitro on neuronal signaling of two classes of chemically modified multiwalled CNTs in reference to pristine CNTs, which are known to be a substrate able to boost neuronal growth and communication. We found that the extent of functionalization and the nature of the functional groups on MWNT sidewalls affect the conductivity and the biological effects of the final derivatives. This information is important for the future design of biointegrated devices.
Background: The interaction between proteins and nanoparticles is a very relevant subject because of the potential applications in medicine and material science in general. Further interest derives from the amyloidogenic character of the considered protein, β2-microglobulin (β2m), which may be regarded as a paradigmatic system for possible therapeutic strategies. Previous evidence showed in fact that gold nanoparticles (AuNPs) are able to inhibit β2m fibril formation in vitro. Methods: NMR (Nuclear Magnetic Resonance) and ESR (Electron Spin Resonance) spectroscopy are employed to characterize the paramagnetic perturbation of the extrinsic nitroxide probe Tempol on β2m in the absence and presence of AuNPs to determine the surface accessibility properties and the occurrence of chemical or conformational exchange, based on measurements conducted under magnetization equilibrium and non-equilibrium conditions. Results: The nitroxide perturbation analysis successfully identifies the protein regions where protein-protein or protein-AuNPs interactions hinder accessibility or/and establish exchange contacts. These information give interesting clues to recognize the fibrillation interface of β2m and hypothesize a mechanism for AuNPs fibrillogenesis inhibition. Conclusions: The presented approach can be advantageously applied to the characterization of the interface in protein-protein and protein-nanoparticles interactions.
Protein aggregation including the formation of dimers and multimers in solution, underlies an array of human diseases such as systemic amyloidosis which is a fatal disease caused by misfolding of native globular proteins damaging the structure and function of affected organs. Different kind of interactors can interfere with the formation of protein dimers and multimers in solution. A very special class of interactors are nanoparticles thanks to the extremely efficient extension of their interaction surface. In particular citrate-coated gold nanoparticles (cit-AuNPs) were recently investigated with amyloidogenic protein β2-microglobulin (βm). Here we present the computational studies on two challenging models known for their enhanced amyloidogenic propensity, namely ΔN6 and D76N βm naturally occurring variants, and disclose the role of cit-AuNPs on their fibrillogenesis. The proposed interaction mechanism lies in the interference of the cit-AuNPs with the protein dimers at the early stages of aggregation, that induces dimer disassembling. As a consequence, natural fibril formation can be inhibited. Relying on the comparison between atomistic simulations at multiple levels (enhanced sampling molecular dynamics and Brownian dynamics) and protein structural characterisation by NMR, we demonstrate that the cit-AuNPs interactors are able to inhibit protein dimer assembling. As a consequence, the natural fibril formation is also inhibited, as found in experiment.
The use of binary blends of hydrogenated and fluorinated alkanethiolates represents an interesting approach to the construction of anisotropic hybrid organic-inorganic nanoparticles since the fluorinated and hydrogenated components are expected to self-sort on the nanoparticle surface because of their reciprocal phobicity. These mixed monolayers are therefore strongly non-ideal binary systems. The synthetic routes we explored to achieve mixed monolayer gold nanoparticles displaying hydrogenated and fluorinated ligands clearly show that the final monolayer composition is a non-linear function of the initial reaction mixture. Our data suggest that, under certain geometrical constraints, nucleation and growth of fluorinated domains could be the initial event in the formation of these mixed monolayers. The onset of domain formation depends on the structure of the fluorinated and hydrogenated species. The solubility of the mixed monolayer nanoparticles displayed a marked discontinuity as a function of the monolayer composition. When the fluorinated component content is small, the nanoparticle systems are fully soluble in chloroform, at intermediate content the nanoparticles become soluble in hexane and eventually they become soluble in fluorinated solvents only. The ranges of monolayer compositions in which the solubility transitions are observed depend on the nature of the thiols composing the monolayer.
The oriented immobilization of proteins, key for the development of novel responsive biomaterials, relies on the availability of effective probes. These are generally provided by standard approaches based on in vivo maturation and in vitro selection of antibodies and/or aptamers. These techniques can suffer technical problems when a non-immunogenic epitope needs to be targeted. Here we propose a strategy to circumvent this issue by in silico design. In our method molecular binders, in the form of cyclic peptides, are computationally evolved by stochastically exploring their sequence and structure space to identify high-affinity peptides for a chosen epitope of a target globular protein: here a solvent-exposed site of β2-microglobulin (β2m). Designed sequences were screened by explicit solvent molecular dynamics simulations (MD) followed by experimental validation. Five candidates gave dose-response surface plasmon resonance signals with dissociation constants in the micromolar range. One of them was further analyzed by means of isothermal titration calorimetry, nuclear magnetic resonance, and 250 ns of MD. Atomic-force microscopy imaging showed that this peptide is able to immobilize β2m on a gold surface. In short, we have shown by a variety of experimental techniques that it is possible to capture a protein through an epitope of choice by computational design.
The need for thermosets from renewable resources is continuously increasing to find eco-friendly alternatives to petroleum-derived materials. Products obtained from biomass have shown to play an important role in this challenge. Here, we present the structural characterization of new biobased thermosets made of humins, a byproduct of lignocellulosic biorefinery, and glycidylated phloroglucinol coming from the biomass phenolic fraction. By employing attenuated total reflection-Fourier transform infrared and NMR spectroscopies, we elucidated the connections between these two systems, contributing to clarify their molecular structures and their reactivities. We demonstrated that the resin curing takes place through ether bond formation between humin hydroxyl functions and phloroglucinol epoxides. Besides cross-linking, humins show a complex rearrangement of their furanic structure through different concomitant chemical pathways depending on the reaction conditions.
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