Characterizing the Oligomers Distribution along the Aggregation Pathway of Amyloid Aß1-40 by NMR.

This study delves into the early aggregation process of the Aß1-40 amyloid peptide, elucidating the associated oligomers distribution. Motivated by the acknowledged role of small oligomers in the neurotoxic damage linked to Alzheimer's disease, we present an experimental protocol for preparing 26-O-acyl isoAß1-40, a modified Aß1-40 peptide facilitating rapid isomerization to the native amide form at neutral pH. This ensures seed-free solutions, minimizing experimental variability. Additionally, we demonstrate the efficacy of coupling NMR diffusion ordered spectroscopy (DOSY) with the Inverse Laplace Transform (ILT) reconstruction method, for effective characterization of early aggregation processes. This innovative approach efficiently maps oligomers distributions across a wide spectrum of initial peptide concentrations offering unique insights into the evolution of oligomers relative populations. As a proof of concept, we demonstrate the efficacy of our approach assessing the impact of Epigallocathechin gallate, a known remodeling agent of amyloid fibrils, on the oligomeric distributions of aggregated Aß1-40. The DOSY-ILT proposed approach stands as a robust and discriminating asset, providing a powerful strategy for rapidly gaining insight into potential inhibitors' impact on the aggregation process.

Identification of a novel extracellular inhibitor of FGF2/FGFR signaling axis by combined virtual screening and NMR spectroscopy approach.

The aberrant activation of the fibroblast growth factor 2 (FGF2)/fibroblast growth factor receptor (FGFR) signalling pathway drives severe pathologies, including cancer development and angiogenesis-driven pathologies. The perturbation of the FGF2/FGFR axis via extracellular allosteric small inhibitors is a promising strategy for developing FGFR inhibitors with improved safety and efficacy for cancer treatment. We have previously investigated the role of new extracellular inhibitors, such as rosmarinic acid (RA), which bind the FGFR-D2 domain and directly compete with FGF2 for the same binding site, enabling the disruption of the functional FGF2/FGFR interaction. To select ligands for the previously identified FGF2/FGFR RA binding site, NMR data-driven virtual screening has been performed on an in-house library of non-commercial small molecules and metabolites. A novel drug-like compound, a resorcinol derivative named RBA4 has been identified. NMR interaction studies demonstrate that RBA4 binds the FGF2/FGFR complex, in agreement with docking prediction. Residue-level NMR perturbations analysis highlights that the mode of action of RBA4 is similar to RA in terms of its ability to target the FGF2/FGFR-D2 complex, inducing perturbations on both proteins and triggering complex dissociation. Biological assays proved that RBA4 inhibited FGF2 proliferative activity at a level comparable to the previously reported natural product, RA. Identification of RBA4 chemical groups involved in direct interactions represents a starting point for further optimization of drug-like extracellular inhibitors with improved activity.

Inhibition of FGFR Signaling by Targeting FGF/FGFR Extracellular Interactions: Towards the Comprehension of the Molecular Mechanism through NMR Approaches.

NMR-based approaches play a pivotal role in providing insight into molecular recognition mechanisms, affording the required atomic-level description and enabling the identification of promising inhibitors of protein-protein interactions. The aberrant activation of the fibroblast growth factor 2 (FGF2)/fibroblast growth factor receptor (FGFR) signaling pathway drives several pathologies, including cancer development, metastasis formation, resistance to therapy, angiogenesis-driven pathologies, vascular diseases, and viral infections. Most FGFR inhibitors targeting the intracellular ATP binding pocket of FGFR have adverse effects, such as limited specificity and relevant toxicity. A viable alternative is represented by targeting the FGF/FGFR extracellular interactions. We previously identified a few small-molecule inhibitors acting extracellularly, targeting FGFR or FGF. We have now built a small library of natural and synthetic molecules that potentially act as inhibitors of FGF2/FGFR interactions to improve our understanding of the molecular mechanisms of inhibitory activity. Here, we provide a comparative analysis of the interaction mode of small molecules with the FGF2/FGFR complex and the single protein domains. DOSY and residue-level NMR analysis afforded insights into the capability of the potential inhibitors to destabilize complex formation, highlighting different mechanisms of inhibition of FGF2-induced cell proliferation.

Molecular Basis of the Antiangiogenic Action of Rosmarinic Acid, a Natural Compound Targeting Fibroblast Growth Factor-2/FGFR Interactions.

Fibroblast growth factor (FGF2)/fibroblast growth factor receptor (FGFR) signalling plays a major role both in physiology and in several pathologies, including cancer development, metastasis formation and resistance to therapy. The development of small molecules, acting extracellularly to target FGF2/FGFR interactions, has the advantage of limiting the adverse effects associated with current intracellular FGFR inhibitors. Herein, we discuss the ability of the natural compound rosmarinic acid (RA) to induce FGF2/FGFR complex dissociation. The molecular-level description of the FGF2/FGFR/RA system, by NMR spectroscopy and docking, clearly demonstrates that RA binds to the FGFR-D2 domain and directly competes with FGF2 for the same binding site. Direct and allosteric perturbations combine to destabilise the complex. The proposed molecular mechanism is validated by cellular studies showing that RA inhibits FGF2-induced endothelial cell proliferation and FGFR activation. Our results can serve as the basis for the development of new extracellular inhibitors of the FGF/FGFR pathways.

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: .

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.

Rhodamine binds to silk fibroin and inhibits its self-aggregation.

Amyloid structures are universal structures, widely diffuse in nature. Silk, capable of forming some of the strongest tensile materials on earth represents an important example of formation of functional amyloid fibrils, a process reminiscent of the oligomerization of peptides involved in neurodegenerative diseases. The stability of silk fibroin solutions in different conditions and its transition from α-helix/random coil to ß-sheet structures, at the basis of gelation processes and fibril formation, have been here investigated and monitored employing different biophysical approaches. Silk fibroin aggregation state as a function of concentration, pH and aging has been characterized employing NMR ordered diffusion spectroscopy. The change of silk fibroin diffusion coefficient over time, which reflects the progress of oligomerization, has been monitored for silk fibroin alone and in the presence of a polycondensed aromatic dye, namely rhodamine 6G. NMR, UV and DLS measurements indicated that rhodamine specifically binds to silk fibroin with a micromolar KD. The reported data reveal, for the first time, that RHD is capable of inhibiting fibroin self-association, thus controlling ß-conformational transition at the basis of fibril formation. The described approach could be extended to further protein systems, allowing better control of the oligomerisation process.

Unsaturated Long-Chain Fatty Acids Are Preferred Ferritin Ligands That Enhance Iron Biomineralization.

Ferritin is a ubiquitous nanocage protein, which can accommodate up to thousands of iron atoms inside its cavity. Aside from its iron storage function, a new role as a fatty acid binder has been proposed for this protein. The interaction of apo horse spleen ferritin (HoSF) with a variety of lipids has been here investigated through NMR spectroscopic ligand-based experiments, to provide new insights into the mechanism of ferritin-lipid interactions, and the link with iron mineralization. 1D 1 H, diffusion (DOSY) and saturation-transfer difference (STD) NMR experiments provided evidence for a stronger interaction of ferritin with unsaturated fatty acids compared to saturated fatty acids, detergents, and bile acids. Mineralization assays showed that oleate c aused the most efficient increase in the initial rate of iron oxidation, and the highest formation of ferric species in HoSF. The comprehension of the factors inducing a faster biomineralization is an issue of the utmost importance, given the association of ferritin levels with metabolic syndromes, such as insulin resistance and diabetes, characterized by fatty acid concentration dysregulation. The human ferritin H-chain homopolymer (HuHF), featuring ferroxidase activity, was also tested for its fatty acid binding capabilities. Assays show that oleate can bind with high affinity to HuHF, without altering the reaction rates at the ferroxidase site.

The study of transient protein-nanoparticle interactions by solution NMR spectroscopy.

The rapid development of novel nanoscale materials for applications in biomedicine urges an improved characterization of the nanobio interfaces. Nanoparticles exhibit unique structures and properties, often different from the corresponding bulk materials, and the nature of their interactions with biological systems remains poorly characterized. Solution NMR spectroscopy is a mature technique for the investigation of biomolecular structure, dynamics, and intermolecular associations, however its use in protein-nanoparticle interaction studies remains scarce and highly challenging, particularly due to unfavorable hydrodynamic properties of most nanoscale assemblies. Nonetheless, recent efforts demonstrated that a number of NMR observables, such as chemical shifts, signal intensities, amide exchange rates and relaxation parameters, together with newly designed saturation transfer experiments, could be successfully employed to characterize the orientation, structure and dynamics of proteins adsorbed onto nanoparticle surfaces. This review provides the first survey and critical assessment of the contributions from solution NMR spectroscopy to the study of transient interactions between proteins and both inorganic (gold, silver, and silica) and organic (polymer, carbon and lipid based) nanoparticles. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions.

Searching for protein binding sites from Molecular Dynamics simulations and paramagnetic fragment-based NMR studies.

Hotspot delineation on protein surfaces represents a fundamental step for targeting protein-protein interfaces. Disruptors of protein-protein interactions can be designed provided that the sterical features of binding pockets, including the transient ones, can be defined. Molecular Dynamics, MD, simulations have been used as a reliable framework for identifying transient pocket openings on the protein surface. Accessible surface area and intramolecular H-bond involvement of protein backbone amides are proposed as descriptors for characterizing binding pocket occurrence and evolution along MD trajectories. TEMPOL induced paramagnetic perturbations on (1)H-(15)N HSQC signals of protein backbone amides have been analyzed as a fragment-based search for surface hotspots, in order to validate MD predicted pockets. This procedure has been applied to CXCL12, a small chemokine responsible for tumor progression and proliferation. From combined analysis of MD data and paramagnetic profiles, two CXCL12 sites suitable for the binding of small molecules were identified. One of these sites is the already well characterized CXCL12 region involved in the binding to CXCR4 receptor. The other one is a transient pocket predicted by Molecular Dynamics simulations, which could not be observed from static analysis of CXCL12 PDB structures. The present results indicate how TEMPOL, instrumental in identifying this transient pocket, can be a powerful tool to delineate minor conformations which can be highly relevant in dynamic discovery of antitumoral drugs.

The role of dynamics in modulating ligand exchange in intracellular lipid binding proteins.

Lipids are essential for many biological processes and crucial in the pathogenesis of several diseases. Intracellular lipid-binding proteins (iLBPs) provide mobile hydrophobic binding sites that allow hydrophobic or amphipathic lipid molecules to penetrate into and across aqueous layers. Thus iLBPs mediate the lipid transport within the cell and participate to a spectrum of tissue-specific pathways involved in lipid homeostasis. Structural studies have shown that iLBPs' binding sites are inaccessible from the bulk, implying that substrate binding should involve a conformational change able to produce a ligand entry portal. Many studies have been reported in the last two decades on iLBPs indicating that their dynamics play a pivotal role in regulating ligand binding and targeted release. The ensemble of reported data has not been reviewed until today. This review is thus intended to summarize and possibly generalize the results up to now described, providing a picture which could help to identify the missing notions necessary to improve our understanding of the role of dynamics in iLBPs' molecular recognition. Such notions would clarify the chemistry of lipid binding to iLBPs and set the basis for the development of new drugs.

Dynamics of a globular protein adsorbed to liposomal nanoparticles.

A solution-state NMR method is proposed to investigate the dynamics of proteins that undergo reversible association with nanoparticles (NPs). We applied the recently developed dark-state exchange saturation transfer experiment to obtain residue-level dynamic information on a NP-adsorbed protein in the form of transverse spin relaxation rates, R2bound. Based on dynamic light scattering, fluorescence, circular dichroism, and NMR spectroscopy data, we show that the test protein, human liver fatty acid binding protein, interacts reversibly and peripherally with liposomal NPs without experiencing significant structural changes. The significant but modest saturation transfer from the bound state observed at 14.1 and 23.5 T static magnetic fields, and the small determined R2bound values were consistent with a largely unrestricted global motion at the lipid surface. Amino acid residues displaying faster spin relaxation mapped to a region that could represent the epitope of interaction with an extended phospholipid chain constituting the protein anchor. These results prove that atomic-resolution protein dynamics is accessible even after association with NPs, supporting the use of saturation transfer methods as powerful tools in bionanoscience.

Chemical exchange saturation transfer (CEST): an efficient tool for detecting molecular information on proteins' behaviour.

A versatile method for assessing protein properties via magnetic resonance imaging (MRI) through chemical exchange saturation transfer (CEST) modality is described. The observed CEST signal changes allow monitoring of protein aggregation processes, protein folding/unfolding steps and interaction with lipid membranes.

NMR investigation of the equilibrium partitioning of a water-soluble bile salt protein carrier to phospholipid vesicles.

Membrane binding by cytosolic fatty acid binding proteins (FABP) appears to constitute a key step of intracellular lipid trafficking. We applied NMR spectroscopy to study the partitioning of a water-soluble bile acid binding protein (BABP), belonging to the FABP family, between its free and lipid-vesicle-bound states. As the lipid-bound protein was NMR-invisible, the signals of the free biomolecule were analyzed to obtain quantitative information on binding affinity and steady-state kinetics. The data indicated a reversible interaction of BABP with anionic vesicles occurring in a very slow exchange regime on the NMR time scale. The approximate binding epitope was demonstrated from results on BABP samples in which different positively charged lysine residues were mutated to neutral alanines. H/D exchange measurements indicated a higher exposure to solvent for the core amino acid residues in the liposome-bound state. Finally, the BABP-liposome interaction was also investigated for the first time through an MRI-chemical exchange saturation transfer experiment that has potential applications not only in the field of biology, but also in biomedicine, bioanalytical chemistry, and nanotechnology.

Ligand binding promiscuity of human liver fatty acid binding protein: structural and dynamic insights from an interaction study with glycocholate and oleate.

Human liver fatty acid binding protein (hL-FABP) has been reported to act as an intracellular shuttle of lipid molecules, thus playing a central role in systemic metabolic homeostasis. The involvement of hL-FABP in the transport of bile salts has been postulated but scarcely investigated. Here we describe a thorough NMR investigation of glycocholate (GCA) binding to hL-FABP. The protein molecule bound a single molecule of GCA, in contrast to the 1:2 stoichiometry observed with fatty acids. GCA was found to occupy the large internal cavity of hL-FABP, without requiring major conformational rearrangement of the protein backbone; rather, this led to increased stability, similar to that estimated for the hL-FABP:oleate complex. Fast-timescale dynamics appeared not to be significantly perturbed in the presence of ligands. Slow motions (unlike for other proteins of the family) were retained or enhanced upon binding, consistent with a requirement for structural plasticity for promiscuous recognition.

Encapsulation of a rhodamine dye within a bile acid binding protein: toward water processable functional bio host-guest materials.

New strategies are requested for the preparation of bioinspired host-guest complexes to be employed in technologically relevant applications, as sensors and optoelectronic devices. We report here a new approach employing a single monomeric protein as host for the strongly fluorescent rhodamine dye. The selected protein, belonging to the intracellular lipid binding protein family, fully encapsulates one rhodamine molecule inside its cavity forming a host-guest complex stabilized by H and π-hydrogen bonds, a salt bridge, and favorable hydrophobic contacts, as revealed by the NMR derived structural model. The protein-dye solutions are easily processable and form homogeneous thin films exhibiting excellent photophysical and morphological properties, as derived from photoluminescence and AFM data. The obtained results represent the proof of concept of the viability of this bio host-guest system for the development of bioinspired optoelectronic devices.

Bacteria as sensors: Real-time NMR analysis of extracellular metabolites detects sub-lethal amounts of bactericidal molecules released from functionalized materials.

BACKGROUND: Cells exposed to stress factors experience time-dependent variations of metabolite concentration, acting as reliable sensors of the effective concentration of drugs in solution. NMR can detect and quantify changes in metabolite concentration, thus providing an indirect estimate of drug concentration. The quantification of bactericidal molecules released from antimicrobial-treated biomedical materials is crucial to determine their biocompatibility and the potential onset of drug resistance. METHODS: Real-time NMR measurements of extracellular metabolites produced by bacteria grown in the presence of known concentrations of an antibacterial molecule (irgasan) are employed to quantify the bactericidal molecule released from antimicrobial-treated biomedical devices. Viability tests assess their activity against E. coli and S. aureus planktonic and sessile cells. AFM and contact angle measurements assisted in the determination of the mechanism of antibacterial action. RESULTS: NMR-derived concentration kinetics of metabolites produced by bacteria grown in contact with functionalized materials allows for indirectly evaluating the effective concentration of toxic substances released from the device, lowering the detection limit to the nanomolar range. NMR, AFM and contact angle measurements support a surface-killing mechanism of action against bacteria. CONCLUSIONS: The NMR based approach provides a reliable tool to estimate bactericidal molecule release from antimicrobial materials. GENERAL SIGNIFICANCE: The novelty of the proposed NMR-based strategy is that it i) exploits bacteria as sensors of the presence of bactericidal molecules in solution; ii) is independent of the chemo-physical properties of the analyte; iii) establishes the detection limit to nanomolar concentrations.

Urine metabolic signature of pancreatic ductal adenocarcinoma by (1)h nuclear magnetic resonance: identification, mapping, and evolution.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis and is highly chemoresistant. Early detection is the only means to impact long-term survival, but screening methods are lacking. Given the complex and heterogeneous nature of pancreatic cancer, unbiased analytical methods such as metabolomics by nuclear magnetic resonance (NMR) spectroscopy show promise to identify disease-specific molecular fingerprints. NMR profiles constitute a fingerprint of the biofluid, reporting quantitatively on all detectable small biomolecules. NMR spectroscopy was applied to investigate the urine metabolome of PDAC patients (n = 33) and to detect altered metabolic profiles in comparison with healthy matched controls (n = 54). The spectral data were analyzed using multivariate statistical techniques. Statistically significant differences were found between urine metabolomic profiles of PDAC and control individuals (p < 10(-5)). Group discrimination was possible due to average concentration differences of several metabolite signals, pointing to a multimolecular signature of the disease. The robustness of the determined statistical model is confirmed by its predictive performance (sensitivity = 75.8%, specificity = 90.7%). Additionally, the method allowed for a neat separation between spectral profiles of individuals with intermediate and advanced pathologic staging, as well as for the discrimination of samples based on tumor localization. NMR spectroscopy analysis of urinary metabolic profiles proved successful in identifying a complex molecular signature of PDAC. Furthermore, results of a descriptive-level analysis show the possibility to follow disease evolution and to carry out tumor site mapping. Given the high reproducibility and the noninvasive nature of the analytical procedure, the described method bears potential to impact large-scale screening programs.

Structural requirements for cooperativity in ileal bile acid-binding proteins.

Ileal bile acid-binding proteins (I-BABP), belonging to the family of intracellular lipid-binding proteins, control bile acid trafficking in enterocytes and participate in regulating the homeostasis of these cholesterol-derived metabolites. I-BABP orthologues share the same structural fold and are able to host up to two ligands in their large internal cavities. However variations in the primary sequences determine differences in binding properties such as the degree of binding cooperativity. To investigate the molecular requirements for cooperativity we adopted a gain-of-function approach, exploring the possibility to turn the noncooperative chicken I-BABP (cI-BABP) into a cooperative mutant protein. To this aim we first solved the solution structure of cI-BABP in complex with two molecules of the physiological ligand glycochenodeoxycholate. A comparative structural analysis with closely related members of the same protein family provided the basis to design a double mutant (H99Q/A101S cI-BABP) capable of establishing a cooperative binding mechanism. Molecular dynamics simulation studies of the wild type and mutant complexes and essential dynamics analysis of the trajectories supported the role of the identified amino acid residues as hot spot mediators of communication between binding sites. The emerging picture is consistent with a binding mechanism that can be described as an extended conformational selection model.

Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy.

The use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications. In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxation rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products.