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, \u3b22-microglobulin (\u3b22m), 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 \u3b22m 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 \u3b22m 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 \u3b22m 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

Topologically non-trivial metal-organic assemblies inhibit \u3b22-microglobulin amyloidogenesis

Inhibiting amyloid aggregation through high-turnover dynamic interactions could be an efficient strategy that is already used by small heat-shock proteins in different biological contexts. We report the interactions of three topologically non-trivial, zinc-templated metal-organic assemblies, a [2]catenane, a trefoil knot (TK), and Borromean rings, with two \u3b22-microglobulin (\u3b22m) variants responsible for amyloidotic pathologies. Fast exchange and similar patterns of preferred contact surface are observed by NMR, consistent with molecular dynamics simulations. In vitro fibrillation is inhibited by each complex, whereas the zinc-free TK induces protein aggregation and does not inhibit fibrillogenesis. The metal coordination imposes structural rigidity that determines the contact area on the \u3b22m surface depending on the complex dimensions, ensuring in vitro prevention of fibrillogenesis. Administration of TK, the best protein-contacting species, to a disease-model organism, namely a Caenorhabditis elegans mutant expressing the D76N \u3b22m variant, confirms the bioactivity potential of the knot topology and suggests new developments

Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function

Missense mutations in p53 are severely deleterious and occur in over 50% of all human cancers. The majority of these mutations are located in the inherently unstable DNA-binding domain (DBD), many of which destabilize the domain further and expose its aggregation-prone hydrophobic core, prompting self-assembly of mutant p53 into inactive cytosolic amyloid-like aggregates. Screening an oligopyridylamide library, previously shown to inhibit amyloid formation associated with Alzheimer\u2019s disease and type II diabetes, identified a tripyridylamide, ADH-6, that abrogates self-assembly of the aggregation-nucleating subdomain of mutant p53 DBD. Moreover, ADH-6 targets and dissociates mutant p53 aggregates in human cancer cells, which restores p53\u2019s transcriptional activity, leading to cell cycle arrest and apoptosis. Notably, ADH-6 treatment effectively shrinks xenografts harboring mutant p53, while exhibiting no toxicity to healthy tissue, thereby substantially prolonging survival. This study demonstrates the successful application of a bona fide small-molecule amyloid inhibitor as a potent\ua0anticancer agent

NMR-Based Analysis of Nanobodies to SARS-CoV-2 Nsp9 Reveals a Possible Antiviral Strategy Against COVID-19

Following the entry into the host cell, SARS-CoV-2 replication is mediated by the replication transcription complex (RTC) assembled through a number of nonstructural proteins (Nsps). A monomeric form of Nsp9 is particularly important for RTC assembly and function. In the present study, 136 unique nanobodies targeting Nsp9 are generated. Several nanobodies belonging to different B-cell lineages are expressed, purified, and characterized. Results from immunoassays applied to purified Nsp9 and neat saliva from coronavirus disease (COVID-19) patients show that these nanobodies effectively and specifically recognize both recombinant and endogenous Nsp9. Nuclear magnetic resonance analyses supported by molecular dynamics reveal a composite Nsp9 oligomerization pattern and demonstrate that both nanobodies stabilize the tetrameric form of wild-type Nsp9 also identifying the epitopes on the tetrameric assembly. These results can have important implications in the potential use of these nanobodies to combat viral replication

Molecular electrostatics and pKa shifts calculations with the Generalized Born model. A tutorial through examples with Bluues2

Biomolecular electrostatics is of key importance for biological function and recognition. The continuum electrostatic model based on the Poisson-Boltzmann (PB) equation has been widely used to study biomolecular electrostatics. The solution of the PB equation gives the electrostatic potential over the space enclosing the molecule(s) of interest, typically obtained at points of a suitable grid. The Generalized Born (GB) model has been used to provide a useful approximation to the solution obtained by solving numerically the PB equation. The main advantage of the GB approach is to express the electrostatic energy of the system as the sum of pairwise interactions. The latter depend on geometric parameters (the GB radii) which depend on all atoms, and their radii, of the system. In this work we present a tutorial through examples for a more efficient and general version of the program Bluues which is able to compute: 1) the generalized Born radius of each atom; 2) the electrostatic potential at the surface of the molecule mapped to solvent accessible atoms; 3) the solvent accessible surface in a PDB formatted file; 4) the electrostatic potential in the volume surrounding the molecule; 5) the electrostatic free energy and different contributions to it; 6) the pH-dependent properties of proteins (total charge and pH-dependent free energy of folding) in the pH range −4 to 18; 7) the pKa shifts due to molecular structure of all ionizable groups. Program summary: Program Title: bluues2 CPC Library link to program files: https://doi.org/10.17632/bx98gcrzbg.1 Developer's repository link: https://github.com/federico-fogolari/bluues2 Code Ocean capsule: https://codeocean.com/capsule/9338710 Licensing provisions: GPLv3 Programming language: C Nature of problem: Biomolecular electrostatics is of key importance for biological function and recognition. Computation of electrostatic effects and their dissection in atomic contributions is a general problem in molecular modeling. Solution method: The GBR6 version of the Generalized Born (GB) model is implemented through surface integrals and all common molecular electrostatic analyses are performed by a single command line program. Additional comments including restrictions and unusual features: The program requires, as input, files in pqr format, which may be generated by independent software (e.g. babel, pdb2pqr). For the optional generation of the solvent excluded surface, the software msms must be installed and present in the path

The interaction of β2-microglobulin with gold nanoparticles: Impact of coating, charge and size

Gold nanoparticles (AuNPs) proved to be ideal scaffolds to build nanodevices whose performance can be tuned by changing their coating. In particular, their interaction with proteins 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, \u3b22-microglobulin (\u3b22m), whose clinical relevance in dialysis-related amyloidosis (DRA) and structural properties are well known. To this 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 \u3b22m 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 \u3b22m unfolding, MUTAB-AuNPs 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 a fine control of NP properties to master protein-NP interactions

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

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 β2-microglobulin (β2m) preserving the protein native structure. Moreover, Cit-AuNPs are able to hinder in vitro fibrillogenesis of a β2m pathologic variant, namely D76N, by reducing the oligomeric association of the protein in solution. Here, we clarify the characteristics of the interaction between β2m 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 β2m and Cit-AuNPs is dominated by electrostatic forces which can be tuned by modifying the ionic strength. This journal i

Exopolysaccharide produced by the potential probiotic Lactococcus garvieae C47: Structural characteristics, rheological properties, bioactivities and impact on fermented camel milk

Fermented camel milk possesses a weak (liquid-like) gel structure. We aimed to 1) investigate the characteristics, bioactivities and rheological properties of the exopolysaccharide (EPS) produced by Lactococcus garvieae-C47 (exopolysaccharide-C47 product), a potential probiotic bacterium, on milk extracted from camels and 2) examine the rheological properties of the fermented camel milk produced by L. garvieae-C47. Exopolysaccharide-C47 product (molecular weight: 7.3 7 106 Da) was composed of the following monosaccharides: glucose (82.51%), arabinose (5.32%) and xylose (12.17%). The antioxidant, antitumor and \u3b1-amylase inhibitory activities of exopolysaccharide-C47 product reached up to 67.52, 59.35 and 91.0%, respectively. The apparent viscosity of exopolysaccharide-C47 product decreased with the increase in shear rate and declined by increasing the temperature up to 50 \ub0C. The rheological properties of exopolysaccharide-C47 product are influenced by the salt type and pH value. The exopolysaccharide product produced by L. garvieae C47 possesses valuable health benefits and has the ability to improve the weak structure of fermented camel milk