Calcium Binds to Transthyretin with Low Affinity.

The plasma protein transthyretin (TTR), a transporter for thyroid hormones and retinol in plasma and cerebrospinal fluid, is responsible for the second most common type of systemic (ATTR) amyloidosis either in its wild type form or as a result of destabilizing genetic mutations that increase its aggregation propensity. The association between free calcium ions (Ca2+) and TTR is still debated, although recent work seems to suggest that calcium induces structural destabilization of TTR and promotes its aggregation at non-physiological low pH in vitro. We apply high-resolution NMR spectroscopy to investigate calcium binding to TTR showing the formation of labile interactions, which leave the native structure of TTR substantially unaltered. The effect of calcium binding on TTR-enhanced aggregation is also assessed at physiological pH through the mechano-enzymatic mechanism. Our results indicate that, even if the binding is weak, about 7% of TTR is likely to be Ca2+-bound in vivo and therefore more aggregation prone as we have shown that this interaction is able to increase the protein susceptibility to the proteolytic cleavage that leads to aggregation at physiological pH. These events, even if involving a minority of circulating TTR, may be relevant for ATTR, a pathology that takes several decades to develop.

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 ß-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.

The corona of protein-gold nanoparticle systems: the role of ionic strength.

The nature of the nanoparticle-protein corona is emerging as a key aspect in determining the impact of nanomaterials on proteins and in general on the biological response. We previously demonstrated that citrate-stabilized gold nanoparticles (Cit-AuNPs) interact with ß2-microglobulin (ß2m) preserving the protein native structure. Moreover, Cit-AuNPs are able to hinder in vitro fibrillogenesis of a ß2m pathologic variant, namely D76N, by reducing the oligomeric association of the protein in solution. Here, we clarify the characteristics of the interaction between ß2m and Cit-AuNPs by means of different techniques, i.e. surface enhanced Raman spectroscopy, NMR and quartz crystal microbalance with dissipation monitoring. All the results obtained clearly show that by simply changing the ionic strength of the medium it is possible to switch from a labile and transient nature of the protein-NP adduct featuring the so-called soft corona, to a more "hard" interaction with a layer of proteins having a longer residence time on the NP surface. This confirms that the interaction between ß2m and Cit-AuNPs is dominated by electrostatic forces which can be tuned by modifying the ionic strength.

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.

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, ß2-microglobulin (ß2m), 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 ß2m 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 ß2m 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 ß2m 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.

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: .

Polyhydroxybutyrate Bioresins with High Thermal Stability by Cross-linking with Resorcinol Diglycidyl Ether.

The development of sustainable materials by employing natural and nontoxic resources has been attracting much attention over the previous years. In this work, we discuss for the first time the chemical combination between resorcinol diglycidyl ether (RDGE), an aromatic biobased thermosetting monomer, and polyhydroxybutyrate (PHB), a bioderived and biodegradable thermoplastic polyester. By this combination, we aimed to associate the high thermal stability of RDGE with a toughening effect by the aliphatic chains of PHB. The investigations on the mechanism of the cross-linking reaction and on the structural connectivity between the two components were realized by Fourier transform infrared (FTIR) and NMR spectroscopies. We found that the epoxide polymerization catalyzed by tertiary amines triggers the formation of crotonyl species by polyhydroxybutyrate cleavage. Two-dimensional NMR experiments show that polyhydroxybutyrate fragments covalently bind as side chains to the rigid aromatic network of the epoxide frame. The cross-linking between the two systems entails the formation of new ester and ether bonds. The obtained structures show a network homogeneity confirmed by a single Tg, from 85 to 47 °C, as a function of the formulation, and tan δ values from 87 to 53 °C. The combination of the two comonomers showed a positive effect. The PHB increased the toughness of RDGE-based thermosets, improving the material elasticity by increasing the chain length between the cross-links. An important result of this study is the high thermal stability of RDGE/PHB bioresins, with the T5% varying between 330 and 310 °C as a function of the PHB ratio.

Exploring exchange processes in proteins by paramagnetic perturbation of NMR spectra.

The effect of extrinsic paramagnetic probes on NMR relaxation rates for surface mapping of proteins and other biopolymers is a widely investigated and powerful NMR technique. Here we describe a new application of those probes. It relies on the setting of the relaxation delay to generate magnetization equilibrium and off-equilibrium conditions, in order to tailor the extent of steady state signal recovery with and without the water-soluble nitroxide Tempol. With this approach it is possible to identify signals whose relaxation is affected by exchange processes and, from the relative assignments, to map the protein residues involved in association or conformational interconversion processes on a micro-to-millisecond time scale. This finding is confirmed by the comparison with the results obtained from relaxation dispersion measurements. This simple and convenient method allows preliminary inspection to highlight regions where structural or chemical exchange events are operative, in order to focus on quantitative subsequent determinations by transverse relaxation dispersion experiments or analogous NMR relaxation studies, and/or to gain insights into the predictions of calculations.

Biorefinery Byproducts and Epoxy Biorenewable Monomers: A Structural Elucidation of Humins and Triglycidyl Ether of Phloroglucinol Cross-Linking.

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.

Interference of citrate-stabilized gold nanoparticles with ß2-microglobulin oligomeric association.

Protein fibrillation is involved in many serious diseases, and protein oligomers are proved to be precursors of amyloid fibrils. NMR and QCMD experiments allowed us to establish that the interaction between citrate-stabilized gold nanoparticles and a paradigmatic amyloidogenic protein, ß2-microglobulin, is able to interfere with protein association into oligomers.

Citrate stabilized gold nanoparticles interfere with amyloid fibril formation: D76N and ΔN6 ß2-microglobulin variants.

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 ß2-microglobulin (ß2m). Here we present the computational studies on two challenging models known for their enhanced amyloidogenic propensity, namely ΔN6 and D76N ß2m 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 interaction of ß2-microglobulin with gold nanoparticles: impact of coating, charge and size.

Gold nanoparticles (AuNPs) have been proved to be ideal scaffolds to build nanodevices whose performance can be tuned by changing their coating. In particular, the interaction of AuNPs with proteins was revealed to be highly dependent on the physico-chemical properties of the gold cluster protecting monolayer. In this work we studied the behavior of three different alkanethiolate-coated AuNPs (AT-AuNPs) when they are incubated with a model amyloidogenic protein, ß2-microglobulin (ß2m), whose clinical relevance in dialysis-related amyloidosis (DRA) and structural properties are well known. To the aim we synthesized 6-mercaptohexanoic acid-coated AuNPs (MHA-AuNPs) and (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide-coated AuNPs (MUTAB-AuNPs) of 7.5 nm diameter and 3-mercaptopropionic acid-coated AuNPs (MPA-AuNPs) of 3.6 nm diameter. To study the effects of the incubation with ß2m of these NPs that differ in charge and dimension, we employed NMR, UV-vis and fluorescence spectroscopy, along with transmission electron microscopy (TEM). The three tested AuNP systems gave different results. We found that MHA-AuNPs precipitate with the protein into large agglomerates inducing ß2m unfolding, MUTAB-AuNP precipitation is triggered by the protein that remains unchanged in solution, at least at the higher considered protein/NP ratio, and MPA-AuNPs interact preferentially with a localized region of the protein that stays essentially stably dissolved. These results stress the complexity of the bio-nano interface and the relevance and viability of the fine control of NP properties to master protein-NP interactions.

Citrate-stabilized gold nanoparticles hinder fibrillogenesis of a pathological variant of ß2-microglobulin.

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 ß2-microglobulin (ß2m), namely D76N ß2m 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 ß2m 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.

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 ß2-microglobulin (ß2m). 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 ß2m 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.

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.