Challenging protein-nanoparticle interactions. Results with gold nanoparticle and \u3b22-microglobulin system.

The work presented here deals with the investigation of the interaction of gold nanoparticles (AuNPs) with a paradigmatic amyloidogenic protein, namely \u3b22-microglobulin (\u3b22m). The interaction between proteins and nanoparticles is currently a topic of great interest concerning both nanomedical applications and nano-safty issues. However, a deep knowledge about the molecular mechanisms that drive these interactions is still lacking. To contribute improving the understanding of these interactions, several techniques (dynamic light scattering, microscopy, nuclear magnetic resonance, fluorescence and UV-Vis spectroscopy and quartz crystal microbalance) were exploited to elucidate the characteristics of protein-NP systems. \u3b22m was chosen as a convenient model for amyloidogenic proteins that may be quite unstable polypeptides with a high tendency to misfold and aggregate into fibrils. Among the nanoparticles, AuNPs were selected for their versatility and their widespread application. AuNPs with different organic shell composition were synthesized, characterized and tested mainly with \u3b22m wild-type and in some cases also with two more amyloidogenic variants, i.e. \u394N6 and D76N. The obtained results showed that the nature of the organic shell and the dimensions of AuNPs play a critical role in determining the nature of the interaction. It has been found that 7.5 nm citrate-stabilized AuNPs (Cit-AuNPs) form transient adducts with \u3b22m that proved capable of reducing protein association and aggregation and, as far as \u394N6 variant is concerned, to avoid intrinsic protein partial unfolding. Furthermore, Cit-AuNPs were able to partially hamper D76N fibrillogenesis in vitro. AuNPs coated with 6-mercaptohexanoic acid (MHA), (11-mercaptoundecyl)-N,N,N-trimethylammonium (MUTAB) were prepared starting from Cit-AuNPs and smaller 3-mercaptopropionic acid (MPA) AuNPs were obtained through a direct synthesis. It was found that MHA-AuNPs caused \u3b22m unfolding and its precipitation in protein-NP large agglomerates. On the other hand, MUTAB-AuNPs in presence of \u3b22m irreversibly aggregate while the overall protein structure was preserved except for some minimal conformational changes. With the smaller MPA-AuNPs \u3b22m formed dynamic complexes through a highly localized patch maintaining mainly its native structure. These results underline once again the complexity and the versatility of protein-NP systems, but also suggest the possibility to exploit these interactions to interfere with physiological or pathological processes. Regardless the specific results obtained with the particular protein-NP system considered, in this thesis the inadequacy of some techniques to describe quantitatively protein-NP interactions and the care that should be taken in the interpretation of the results are highlighted

Routes to the preparation of mixed monolayers of fluorinated and hydrogenated alkanethiolates grafted on the surface of gold nanoparticles

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

Correction: Exploring exchange processes in proteins by paramagnetic perturbation of NMR spectra

Correction for 'Exploring exchange processes in proteins by paramagnetic perturbation of NMR spectra' by Yamanappa Hunashal et al., Phys. Chem. Chem. Phys., 2020, 22, 6247–6259, DOI: 10.1039/c9cp06950j

Conversion of the Native N-Terminal Domain of TDP-43 into a Monomeric Alternative Fold with Lower Aggregation Propensity.

TAR DNA-binding protein 43 (TDP-43) forms intraneuronal cytoplasmic inclusions associated with amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. Its N-terminal domain (NTD) can dimerise/oligomerise with the head-to-tail arrangement, which is essential for function but also favours liquid-liquid phase separation and inclusion formation of full-length TDP-43. Using various biophysical approaches, we identified an alternative conformational state of NTD in the presence of Sulfobetaine 3-10 (SB3-10), with higher content of α-helical structure and tryptophan solvent exposure. NMR shows a highly mobile structure, with partially folded regions and β-sheet content decrease, with a concomitant increase of α-helical structure. It is monomeric and reverts to native oligomeric NTD upon SB3-10 dilution. The equilibrium GdnHCl-induced denaturation shows a cooperative folding and a somewhat lower conformational stability. When the aggregation processes were compared with and without pre-incubation with SB3-10, but at the identical final SB3-10 concentration, a slower aggregation was found in the former case, despite the reversible attainment of the native conformation in both cases. This was attributed to protein monomerization and oligomeric seeds disruption by the conditions promoting the alternative conformation. Overall, the results show a high plasticity of TDP-43 NTD and identify strategies to monomerise TDP-43 NTD for methodological and biomedical applications

Computational design of cyclic peptides for the customized oriented immobilization of globular proteins

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

Insights on peptide topology in the computational design of protein ligands: the example of lysozyme binding peptides

Herein, we compared the ability of linear and cyclic peptides generated in silico to target different protein sites: internal pockets and solvent-exposed sites. We selected human lysozyme (HuL) as a model target protein combined with the computational evolution of linear and cyclic peptides. The sequence evolution of these peptides was based on the PARCE algorithm. The generated peptides were screened based on their aqueous solubility and HuL binding affinity. The latter was evaluated by means of scoring functions and atomistic molecular dynamics (MD) trajectories in water, which allowed prediction of the structural features of the protein-peptide complexes. The computational results demonstrated that cyclic peptides constitute the optimal choice for solvent exposed sites, while both linear and cyclic peptides are capable of targeting the HuL pocket effectively. The most promising binders found in silico were investigated experimentally by surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS) techniques. All tested peptides displayed dissociation constants in the micromolar range, as assessed by SPR; however, both NMR and ESI-MS suggested multiple binding modes, at least for the pocket binding peptides. A detailed NMR analysis confirmed that both linear and cyclic pocket peptides correctly target the binding site they were designed for

Design of Sustainable Materials by Cross-linking a Biobased Epoxide with Keratin and Lignin

Awareness of the environmental impact of using polymeric materials obtained from petroleum is causing increased interest in sustainable materials manufacturing. Here, we present the elaboration of fully biobased materials using an aromatic epoxy resin matrix coming from wood biomass and two natural by-products, namely, keratin from chicken feathers and lignin. In situ FTIR kinetic studies show that the two natural fillers increase the conversion of the epoxide during cross-linking. This result, together with DSC studies, proves the chemical interactions between the keratin or lignin and the epoxide network. Up to 30% of these natural components could be reacted and incorporated into the epoxide with good compatibility. The thermomechanical properties of the elaborated materials are comparable to those of commercial ones

Biorefinery by-products and epoxy biorenewable monomers: a structural elucidation of humins and triglycidyl ether of phloroglucinol crosslinking

International audienceTThe need for thermosets from renewable resources is permanently increasing in order 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 bio-based thermosets made by humins, a by-product of lignocellulosic biorefinery, and glycidylated phloroglucinol coming from biomass phenolic fraction. By employing ATR-FTIR and NMR spectroscopies, we elucidated the connections between these two systems contributing to clarify their molecular structure and their reactivity. We demonstrated that the resin curing takes place through ether bond formation between humins hydroxyl functions and phloroglucinol epoxides. Besides crosslinking, humins show a complex rearrangement of their furanic structure through different concomitant chemical pathways depending on the reaction conditions

Insights into a Protein-Nanoparticle System by Paramagnetic Perturbation NMR Spectroscopy

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