Assessing nanobody interaction with SARS-CoV-2 Nsp9.

The interaction between SARS-CoV-2 non-structural protein Nsp9 and the nanobody 2NSP90 was investigated by NMR spectroscopy using the paramagnetic perturbation methodology PENELOP (Paramagnetic Equilibrium vs Nonequilibrium magnetization Enhancement or LOss Perturbation). The Nsp9 monomer is an essential component of the replication and transcription complex (RTC) that reproduces the viral gRNA for subsequent propagation. Therefore preventing Nsp9 recruitment in RTC would represent an efficient antiviral strategy that could be applied to different coronaviruses, given the Nsp9 relative invariance. The NMR results were consistent with a previous characterization suggesting a 4:4 Nsp9-to-nanobody stoichiometry with the occurrence of two epitope pairs on each of the Nsp9 units that establish the inter-dimer contacts of Nsp9 tetramer. The oligomerization state of Nsp9 was also analyzed by molecular dynamics simulations and both dimers and tetramers resulted plausible. A different distribution of the mapped epitopes on the tetramer surface with respect to the former 4:4 complex could also be possible, as well as different stoichiometries of the Nsp9-nanobody assemblies such as the 2:2 stoichiometry suggested by the recent crystal structure of the Nsp9 complex with 2NSP23 (PDB ID: 8dqu), a nanobody exhibiting essentially the same affinity as 2NSP90. The experimental NMR evidence, however, ruled out the occurrence in liquid state of the relevant Nsp9 conformational change observed in the same crystal structure.

Molecular basis of ß-lactam antibiotic resistance of ESKAPE bacterium E. faecium Penicillin Binding Protein PBP5.

Penicillin-binding proteins (PBPs) are essential for the formation of the bacterial cell wall. They are also the targets of ß-lactam antibiotics. In Enterococcus faecium, high levels of resistance to ß-lactams are associated with the expression of PBP5, with higher levels of resistance associated with distinct PBP5 variants. To define the molecular mechanism of PBP5-mediated resistance we leveraged biomolecular NMR spectroscopy of PBP5 - due to its size (>70 kDa) a challenging NMR target. Our data show that resistant PBP5 variants show significantly increased dynamics either alone or upon formation of the acyl-enzyme inhibitor complex. Furthermore, these variants also exhibit increased acyl-enzyme hydrolysis. Thus, reducing sidechain bulkiness and expanding surface loops results in increased dynamics that facilitates acyl-enzyme hydrolysis and, via increased ß-lactam antibiotic turnover, facilitates ß-lactam resistance. Together, these data provide the molecular basis of resistance of clinical E. faecium PBP5 variants, results that are likely applicable to the PBP family.

Approaching Protein Aggregation and Structural Dynamics by Equilibrium and Nonequilibrium Paramagnetic Perturbation.

PENELOP (Paramagnetic Equilibrium vs Nonequilibrium magnetization Enhancement or LOss Perturbation) is the presented nuclear magnetic resonance (NMR) approach to identify at once the location of proteins' exposed surface, hindered accessibility, and exchange processes occurring on a µs-ms time scale. In addition to mapping the protein surface accessibility, the application of this method under specific conditions makes it possible to distinguish conformational mobility and chemical exchange processes, thereby providing an alternative to characterization by more demanding techniques (transverse relaxation dispersion, saturation transfer, and high-pressure NMR). Moreover, its high sensitivity enables studying samples at low, physiologically more relevant concentrations. Association, dynamics, and oligomerization are addressed by PENELOP for a component of SARS-CoV-2 replication transcription complex and an amyloidogenic protein.

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.

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.

Structure of Nanobody Nb23.

BACKGROUND: Nanobodies, or VHHs, are derived from heavy chain-only antibodies (hcAbs) found in camelids. They overcome some of the inherent limitations of monoclonal antibodies (mAbs) and derivatives thereof, due to their smaller molecular size and higher stability, and thus present an alternative to mAbs for therapeutic use. Two nanobodies, Nb23 and Nb24, have been shown to similarly inhibit the self-aggregation of very amyloidogenic variants of ß2-microglobulin. Here, the structure of Nb23 was modeled with the Chemical-Shift (CS)-Rosetta server using chemical shift assignments from nuclear magnetic resonance (NMR) spectroscopy experiments, and used as prior knowledge in PONDEROSA restrained modeling based on experimentally assessed internuclear distances. Further validation was comparatively obtained with the results of molecular dynamics trajectories calculated from the resulting best energy-minimized Nb23 conformers. METHODS: 2D and 3D NMR spectroscopy experiments were carried out to determine the assignment of the backbone and side chain hydrogen, nitrogen and carbon resonances to extract chemical shifts and interproton separations for restrained modeling. RESULTS: The solution structure of isolated Nb23 nanobody was determined. CONCLUSIONS: The structural analysis indicated that isolated Nb23 has a dynamic CDR3 loop distributed over different orientations with respect to Nb24, which could determine differences in target antigen affinity or complex lability.

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's 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's 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 anticancer agent.

The Redox-Active Conopeptide Derived from the Venom Duct Transcriptome of Conus lividus Assists in the Oxidative Folding of Conotoxin.

The tetrapeptides Li504 and Li520, differing in the modification of the 4-trans-hydroxylation of proline, are novel conopeptides derived from the venom duct transcriptome of the marine cone snail Conus lividus. These predicted mature peptides are homologous to the active site motif of oxidoreductases that catalyze the oxidation, reduction, and rearrangement of disulfide bonds in peptides and proteins. The estimated reduction potential of the disulfide of Li504 and Li520 is within the range of disulfide reduction potentials of oxidoreductases, indicating that they may catalyze the oxidative folding of conotoxins. Conformational features of Li504 and Li520 include the trans configuration of the Cys1-Pro2/Hyp2 peptide bond with a type 1 turn that is similar to the active site motif of glutaredoxin that regulates the oxidation of cysteine thiols to disulfides. Li504- and Li520-assisted oxidative folding of α-conotoxin ImI confirms that Li520 improves the yield of the natively folded peptide by concomitantly decreasing the yield of the non-native disulfide isomer and thus acts as a miniature disulfide isomerase. The geometry of the Cys1-Hyp2 peptide bond of Li520 shifts between the trans and cis configurations in the disulfide form and thiol/thiolate form, which regulates the deprotonation of the N-terminal cysteine residue. Hydrogen bonding of the hydroxyl group of 4-trans-hydroxyproline with the interpeptide chain unit in the mixed disulfide form may play a vital role in shifting the geometry of the Cys1-Hyp2 peptide bond from cis to trans configuration. The Li520 conopeptide together with similar peptides derived from other species may constitute a new family of "redox-active" conopeptides that are integral components of the oxidative folding machinery of conotoxins.

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: .

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 × 106 Da) was composed of the following monosaccharides: glucose (82.51%), arabinose (5.32%) and xylose (12.17%). The antioxidant, antitumor and α-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 °C. 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.

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.

Characterization, bioactivities, and rheological properties of exopolysaccharide produced by novel probiotic Lactobacillus plantarum C70 isolated from camel milk.

Various industries highly regard the functionalities and bioactivities of bacterial polysaccharides. We aimed to characterize the exopolysaccharide (EPS) produced by novel probiotic Lactobacillus plantarum C70 (accession number KX881779) isolated from camel milk and to investigate its bioactivities and rheological properties. EPS-C70 had a weight-average molecular weight (Mw) of 3.8 × 105 Da. Arabinose (13.3%), mannose (7.1%), glucose (74.6%), and galactose (5.0%) were the major monosaccharides constituents. EPS-C70 had two endothermic peaks at 76.95 °C and 158.76 °C corresponding to glass transition (Tg) and melting point (Tm), respectively. Zeta potential and particle size of EPS-C70 were -330.71 mV and 525.5 nm, respectively. DPPH and ABTS of EPS-C70 were 75.91% and 49.42% at 10 mg/mL concentration, respectively. The cytotoxic activities against colon cancer and breast cancer lines were 88.1% and 73.1% at concentration 10 mg/mL, respectively. EPS-C70 exhibited shear-thinning behaviour. Salts and pH values had a significant impact on the rheological properties of EPS-C70.

Disulfide engineering on temporin-SHf: Stabilizing the bioactive conformation of an ultra-short antimicrobial peptide.

In Silico searching for short antimicrobial peptides has revealed temporin-SHf as the short (8AA), hydrophobic, broad spectrum, and natural antimicrobial peptide. Important drawback associated with temporin-SHf is the susceptibility of its bioactive conformation for denaturation and proteolytic degradation. In the current report, disulfide engineering strategy has been adopted to improve the stability of bioactive conformation of temporin-SHf. The functionally non-critical Leu4 and Ile7 residues at i and i + 3 position of helical conformation of temporin-SHf were mutated with cysteine disulfide. Designed [L4C, I7C]temporin-SHf was synthesized, characterized using NMR spectroscopy, and accessed for antimicrobial activity. [L4C, I7C]Temporin-SHf adopts helical conformation from Phe3 to Phe8 in the absence of membrane-mimetic environment and retains broad spectrum antimicrobial activity. The reduction potential of cysteine disulfide of [L4C, I7C]temporin-SHf is -289 mV. Trypsin-induced digestion and serum-induced digestion have confirmed the advantage of cysteine disulfide in imparting proteolytic stability to temporin-SHf. Disulfide-stabilized temporin-SHf may serve as a good model for the rational design of temporin-SHf based antibiotics for treatment of infectious diseases.

Electrical Conductivity, Selective Adhesion, and Biocompatibility in Bacteria-Inspired Peptide-Metal Self-Supporting Nanocomposites.

Bacterial type IV pili (T4P) are polymeric protein nanofibers that have diverse biological roles. Their unique physicochemical properties mark them as a candidate biomaterial for various applications, yet difficulties in producing native T4P hinder their utilization. Recent effort to mimic the T4P of the metal-reducing Geobacter sulfurreducens bacterium led to the design of synthetic peptide building blocks, which self-assemble into T4P-like nanofibers. Here, it is reported that the T4P-like peptide nanofibers efficiently bind metal oxide particles and reduce Au ions analogously to their native counterparts, and thus give rise to versatile and multifunctional peptide-metal nanocomposites. Focusing on the interaction with Au ions, a combination of experimental and computational methods provides mechanistic insight into the formation of an exceptionally dense Au nanoparticle (AuNP) decoration of the nanofibers. Characterization of the thus-formed peptide-AuNPs nanocomposite reveals enhanced thermal stability, electrical conductivity from the single-fiber level up, and substrate-selective adhesion. Exploring its potential applications, it is demonstrated that the peptide-AuNPs nanocomposite can act as a reusable catalytic coating or form self-supporting immersible films of desired shapes. The films scaffold the assembly of cardiac cells into synchronized patches, and present static charge detection capabilities at the macroscale. The study presents a novel T4P-inspired biometallic material.

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.