Titration Calorimetry Research Articles

Cu(II) binding to the λ6aJL2-R24G antibody light chain protein associated with light chain amyloidosis disease: The role of histidines

Light chain amyloidosis is a conformational disease caused by the abnormal proliferation and deposition of antibody light chains as amyloid fibers in organs and tissues. The effect of Cu(II) binding to the model recombinant protein 6aJL2-R24G was previously characterized in our group, and we found an acceleration of the aggregation kinetics of the protein. In this study, in order to confirm the Cu(II) binding sites, histidine variants of 6aJL2-R24G were prepared and the effects of their interaction with Cu(II) were analyzed by circular dichroism, fluorescence spectroscopy, isothermal calorimetry titrations, and molecular dynamics simulations. Confirming our earlier work, we found that His8 and His99 are the highest affinity Cu(II) binding sites, and that Cu(II) binding to both sites is a cooperative event.

Two Squares in a Barrel: An Axially Disubstituted Conformationally Rigid Aliphatic Binding Motif for Cucurbit[6]uril.

Novel binding motifs suitable for the construction of multitopic guest-based molecular devices (e.g., switches, sensors, data storage, and catalysts) are needed in supramolecular chemistry. No rigid, aliphatic binding motif that allows for axial disubstitution has been described for cucurbit[6]uril (CB6) so far. We prepared three model guests combining spiro[3.3]heptane and bicyclo[1.1.1]pentane centerpieces with imidazolium and ammonium termini. We described their binding properties toward CB6/7 and α-/β-CD using NMR, titration calorimetry, mass spectrometry, and single-crystal X-ray diffraction. We found that a bisimidazolio spiro[3.3]heptane guest forms inclusion complexes with CB6, CB7, and β-CD with respective association constants of 4.0 × 104, 1.2 × 1012, and 1.4 × 102. Due to less hindering terminal groups, the diammonio analogue forms more stable complexes with CB6 (K = 1.4 × 106) and CB7 (K = 3.8 × 1012). The bisimidazolio bicyclo[1.1.1]pentane guest forms a highly stable complex only with CB7 with a K value of 1.1 × 1011. The high selectivity of the new binding motifs implies promising potential in the construction of multitopic supramolecular components.

Anion Complexation by an Azocalix[4]arene Derivative and the Scope of Its Fluoride Complex Salt to Capture CO2 from the Air.

A newly synthesized upper rim azocalix[4]arene, namely 5,11,17,23-tetra[(4-ethylacetoxyphenyl) (azo)]calix[4]arene, CA-AZ has been fully characterized, and its chromogenic and selective properties for anions are reported. Among univalent anions, the receptor is selective for the fluoride anion, and its mode of interaction in solution is discussed. The kinetics of the complexation process were found to be very fast as reflected in the immediate colour change observed with a naked eye resulting from the receptor-anion interaction. An emphasis is made about the relevance in selecting a solvent in which the formulation of the process is representative of the events taking place in the solution. The composition of the fluoride complex investigated using UV/VIS spectrophotometry, conductance measurements and titration calorimetry was 1:1, and the thermodynamics of complexation of anions and CA-AZ in DMSO were determined. The fluoride complex salt was isolated, and a detailed investigation was carried out to assess its ability to remove CO2 from the air. The recycling of the complex was easily achieved. Final conclusions are given.

The Anthocyanidin Peonidin Interferes with an Early Step in the Fibrillation Pathway of α-Synuclein and Modulates It toward Amorphous Aggregates.

Parkinson's disease (PD) is characterized by progressive degeneration of the dopaminergic neurons in the brain, accompanied by the accumulation of proteinaceous inclusions, Lewy bodies (LB), mainly comprised of alpha synuclein (α-syn) aggregates. The heterogeneity and the transient nature of the intermediate species formed in the α-syn fibrillation pathway have made it difficult to develop an effective therapeutic intervention. Therefore, any therapeutic molecule that could prevent as well as treat PD would be of great interest. Anthocyanidins are natural flavonoid compounds that have been shown to have neuroprotective properties and to modulate factors that cause neuronal death. Herein, we have explored the modulation and inhibition of α-syn fibrillation by the anthocyanidins cyanidin, delphinidin, and peonidin using a number of biophysical and structural tools. α-Syn fibrillation monitored using thioflavin T (ThT) fluorescence and light scattering suggested concentration dependent inhibition of α-syn fibrillation by all the three anthocyanidins. While cyanidin and delphinidin induced the formation of oligomers and small fibrillar structures of α-syn, respectively, peonidin led to the formation of amorphous aggregates, as observed by Atomic Force Microscopy (AFM). Peonidin proved to be most effective of the three anthocyanidins toward alleviating cell toxicity of SH-SY5Y neuroblastoma cells at concentrations where α-synuclein fibrillation was completely suppressed. Hence, the inhibition mechanism of peonidin was further explored by studying its interaction with α-syn using titration calorimetry and molecular docking. The results show its weak binding (in mM range) to the NAC region of α-syn through hydrogen bonding interactions. Also, circular dichroism and Raman spectroscopy revealed the structural aspects of peonidin-induced α-syn amorphous aggregates showing alpha helical structures with exposed Phe and Tyr regions. Due to the neuroprotective nature of peonidin, the findings reported here are significant and can be further explored toward developing a modifying therapy that could address both disease onset as well as the progression of PD.

Assembling a supramolecular 3D network with tuneable mechanical properties using adamantylated cross-linking agents and β-cyclodextrin-modified hyaluronan

Hydrogels based on the supramolecular host–guest concept can be prepared if at least one constituent is a polymer chain modified with supramolecular host or guest (or both) units. Low-molecular-weight multitopic counterparts can also be used, however, guest molecules in the role of cross-linking agents are seldom reported, although such an approach offers wide-ranging possibilities for tuning the system properties via easily achievable structural modifications. In this paper, a series of adamantane-based star-like guest molecules was used for cross-linking of two types of β-cyclodextrin-modified hyaluronan (CD-HA). The prepared 3D supramolecular networks were characterised using nuclear magnetic resonance, titration calorimetry and rheological measurements to confirm the formation of the host–guest complexes between adamantane moieties and β-cyclodextrin units, including their typical properties such as self-healing and dynamic nature. The results indicate that the nature of the cross-linker (amides versus esters) has a greater impact on mechanical properties than the length of the guest's arms. In addition, the results show that the length of the HA polymer chain is more important than the degree of modification with supramolecular units. In conclusion, it was proven that the modular concept employing low-molecular-weight cross-linking guests is valuable for the formulation of supramolecular networks, including hydrogels.

Thermodynamics Evaluation of Selective Hydride Reduction for α,β-Unsaturated Carbonyl Compounds

The selective reduction of α,β-unsaturated carbonyl compounds is one of the core reactions and also a difficult task for organic synthesis. We have been attempting to study the thermodynamic data of these compounds to create a theoretical basis for organic synthesis and computational chemistry. By electrochemical measurement method and titration calorimetry, in acetonitrile at 298 K, the hydride affinity of two types of unsaturated bonds in α,β-unsaturated carbonyl compounds, their single-electron reduction potential, and the single-electron reduction potential of the corresponding radical intermediate are determined. Their hydrogen atom affinity, along with the hydrogen atom affinity and proton affinity of the corresponding radical anion, is also derived separately based on thermodynamic cycles. The above data are used to establish the corresponding "Molecule ID Card" (Molecule identity card) and analyze the reduction mechanism of unsaturated carbonyl compounds. Primarily, the mixture of any carbonyl hydride ions and Ac-tempo+ will stimulate hydride transfer process and create corresponding α,β-unsaturated carbonyl compounds and Ac-tempoH from a thermodynamic point of view.

Small Molecule Screening Discovers Compounds that Reduce FMRpolyG Protein Aggregates and Splicing Defect Toxicity in Fragile X-Associated Tremor/Ataxia Syndrome.

Expansion of CGG trinucleotide repeats in 5' untranslated region of the FMR1 gene is the causative mutation of neurological diseases such as fragile X syndrome (FXS), fragile X-associated tremor/ataxia syndrome (FXTAS), and ovarian disorder such as fragile X-associated primary ovarian insufficiency (FXPOI). CGG repeats containing FMR1 transcripts form the toxic ribonuclear aggregates, abrupt pre-mRNA splicing, and cause repeat-associated non-AUG translation, leading to the disease symptoms. Here, we utilized a small molecule library of ~ 250,000 members obtained from the National Cancer Institute (NCI) and implemented a shape-based screening approach to identify the candidate small molecules that mitigate toxic CGG RNA-mediated pathogenesis. The compounds obtained from screening were further assessed for their affinity and selectivity towards toxic CGG repeat RNA by employing fluorescence-binding experiment and isothermal calorimetry titration assay. Three candidate molecules B1, B4, and B11 showed high affinity and selectivity for expanded CGG repeats RNA. Further, NMR spectroscopy, gel mobility shift assay, CD spectroscopy, UV-thermal denaturation assay, and molecular docking affirmed their high affinity and selectivity for toxic CGG RNAs. Next, these lead compounds selectively improved the pre-mRNA alternative splicing defects with no perturbation in global splicing efficacy and simultaneouslyreduced the FMR1polyG protein aggregate formation without affecting the downstream expression of the gene. Taken together these findings, we addressed compound B1, B4, and B11 as potential lead molecules for developing promising therapeutics against FXTAS. Herein, this study, we have utilized shape similarity approach to screen the NCI library and found out the potential candidate which improves the pre-mRNA splicing defects and reduces FMR1polyG aggregations.

Phosphorescence Enables Identification of Electronic State for Acridinium Salt in Solutions.

Acridinium derivatives are an important class of photocatalysts, where the interaction between the catalyst and the environment is under-reported. Here we show that the Lewis acidic acridinium salt exhibits various degrees of interactions with different Lewis bases, including water (HOH), methanol (CH3OH), tetrahydrofuran (THF, ROR), amines (R3N), and tert-butoxide (RO-) due to distinct physical properties stemming from different resonance forms. Interactions with water and methanol produce almost identical 1H NMR spectra but lead to drastically different UV absorption and luminescence emission, particularly phosphorescence; interactions with CH3OH/methanol and THF, which are differentiated by heat calorimetry titration, share the same luminescence spectra but show two different sets of 1H NMR peaks. These distinct physical properties could only be revealed by a combination of NMR and molecular fluorescence/phosphorescence spectroscopic methods. The current report serves as an example of using phosphorescence spectroscopy as a complementary tool for identifying interactions between organic molecules.

Citrate Stabilizes Hydroxylapatite Precursors: Implications for Bone Mineralization.

Mineralization of hydroxylapatite (HAp), the main inorganic phase in bone, follows nonclassical crystallization routes involving metastable precursors and is strongly influenced by organic macromolecules. However, the effect of small organic molecules such as citrate on the formation of HAp is not well constrained. Using potentiometric titration experiments and titration calorimetry, in combination with a multianalytical approach, we show that citrate stabilizes prenucleation species as well as a liquid-like calcium phosphate precursor formed before any solid phase nucleates in the system. The stabilization of a liquid-like precursor phase could facilitate infiltration into the cavities of the collagen fibrils during bone mineralization, explaining the enhancement of collagen-mediated mineralization by citrate reported in previous studies. Hence, citrate can influence bone mineralization way before any solid phase (amorphous or crystalline) is formed. We also show that HAp formation after amorphous calcium phosphate (ACP) in the absence and presence of citrate results in nanoplates of about 5–12 nm thick, elongated along the c axis. Such nanoplates are made up of HAp nanocrystallites with a preferred c axis orientation and with interspersed ACP. The nanoplatelet morphology, size, and preferred crystallographic orientation, remarkably similar to those of bone HAp nanocrystals, appear to be an intrinsic feature of HAp formed from an amorphous precursor. Our results challenge current models for HAp mineralization in bone and the role of citrate, offering new clues to help answer the long-standing question as to why natural evolution favored HAp as the mineral phase in bone.

Combined spectroscopic and theoretical analysis of the binding of a water-soluble perylene diimide to DNA/RNA polynucleotides and G-quadruplexes

We present here a combined spectroscopic and theoretical analysis of the binding of N,N’-bis(2-(1-piperazino)ethyl)-3,4,9,10-perylenetetracarboxylic acid diimide dichloride (PZPERY) to different biosubstrates. Absorbance titrations and circular dichroism experiments, melting studies and isothermal calorimetry (ITC) titrations reveal a picture where the binding to natural double-stranded DNA is very different from that to double and triple-stranded RNAs (poly(A)∙poly(U) and poly(U)∙poly(A)⁎poly(U)). As confirmed also by the structural and energetic details clarified by density functional theory (DFT) calculations, intercalation occurs for DNA, with a process driven by the combination of aggregates disruption and monomers intercalation. Oppositely, for RNAs, no intercalation but groove binding with the formation of supramolecular aggregates is observed. Among all the tested biosubstrates, the affinity of PZPERY towards DNA G-quadruplexes (G4) is the greatest one with a preference for human telomeric G4s. Focusing on hybrid G4 forms, either sitting-atop (“tetrad-parallel”) or lateral (“groove-parallel”) binding modes were considered in the discussion of the experimental results and molecular dynamics (MD) simulations. Both turned out to be possible concurrently, in agreement also with the experimental binding stoichiometries higher than 2:1.

Supramolecular Gold Stripping from Activated Carbon Using α-Cyclodextrin.

We report the molecular recognition of the Au(CN)2- anion, a crucial intermediate in today's gold mining industry, by α-cyclodextrin. Three X-ray single-crystal superstructures-KAu(CN)2⊂α-cyclodextrin, KAu(CN)2⊂(α-cyclodextrin)2, and KAg(CN)2⊂(α-cyclodextrin)2-demonstrate that the binding cavity of α-cyclodextrin is a good fit for metal-coordination complexes, such as Au(CN)2- and Ag(CN)2- with linear geometries, while the K+ ions fulfill the role of linking α-cyclodextrin tori together as a result of [K+···O] ion-dipole interactions. A 1:1 binding stoichiometry between Au(CN)2- and α-cyclodextrin in aqueous solution, revealed by 1H NMR titrations, has produced binding constants in the order of 104 M-1. Isothermal calorimetry titrations indicate that this molecular recognition is driven by a favorable enthalpy change overcoming a small entropic penalty. The adduct formation of KAu(CN)2⊂α-cyclodextrin in aqueous solution is sustained by multiple [C-H···π] and [C-H···anion] interactions in addition to hydrophobic effects. The molecular recognition has also been investigated by DFT calculations, which suggest that the 2:1 binding stoichiometry between α-cyclodextrin and Au(CN)2- is favored in the presence of ethanol. We have demonstrated that this molecular recognition process between α-cyclodextrin and KAu(CN)2 can be applied to the stripping of gold from the surface of activated carbon at room temperature. Moreover, this stripping process is selective for Au(CN)2- in the presence of Ag(CN)2-, which has a lower binding affinity toward α-cyclodextrin. This molecular recognition process could, in principle, be integrated into commercial gold-mining protocols and lead to significantly reduced costs, energy consumption, and environmental impact.

Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers

Reliable measurement of the binding kinetics of low molecular weight analytes to their targets is still a challenging task. Often, the introduction of labels is simply impossible in such measurements, and the application of label-free methods is the only reliable choice. By measuring the binding kinetics of Ni(II) ions to genetically modified flagellin layers, we demonstrate that: (1) Grating-Coupled Interferometry (GCI) is well suited to resolve the binding of ions, even at very low protein immobilization levels; (2) it supplies high quality kinetic data from which the number and strength of available binding sites can be determined, and (3) the rate constants of the binding events can also be obtained with high accuracy. Experiments were performed using a flagellin variant incorporating the C-terminal domain of the nickel-responsive transcription factor NikR. GCI results were compared to affinity data from titration calorimetry. We found that besides the low-affinity binding sites characterized by a micromolar dissociation constant (Kd), tetrameric FliC-NikRC molecules possess high-affinity binding sites with Kd values in the nanomolar range. GCI enabled us to obtain real-time kinetic data for the specific binding of an analyte with molar mass as low as 59 Da, even at signals lower than 1 pg/mm2.

Measuring boundaries in phase diagrams of ternary systems using titration calorimetry

This paper demonstrates how isothermal titration microcalorimetry (ITC) can be used for the global construction of several kinds of phase diagrams for ternary systems at 298.15 K and atmospheric pressure. The solid-solid-liquid system of KCl-KNO3-H2O has not yet been studied using ITC, and thus, it is the main system discussed. It is clear that thermal effects associated with transformations in a system (appearance or disappearance of a phase) change with the composition of the system in ampoule. During titration, the solubility curves (including the liquid-liquid and solid-liquid equilibrium lines) in ternary systems can be detected as changes in the slope of the heat evolved vs time plot or in the slope of the observed heat vs solvent concentration plot. ITC is more precise and repeatable than the visual-cloud-point method. This isothermal and isobaric titration method can also provide additional information regarding the dissolution heat and the dilution heat. On the basis of the three experimental solubilities of KCl, KNO3, and KCl + KNO3 (co-saturated solution) in H2O, the other intermediate points on the solubility curve can be obtained based on the lever rule and the assumption of ideal solution, and these results agree well with the experimental values.

Solid-liquid equilibria and excess enthalpies in binary mixtures of propiophenone with some aliphatic amides

The solid-liquid equilibria and excess enthalpies of the binary systems of propiophenone and {N-methylformamide, or N,N-dimethylformamide, or N,N-dimethylacetamide, or N-methyl-2-pyrrolidone} were measured. The SLE data were determined by the cloud-point method, partly supplemented by the DSC technique. The excess enthalpies were measured by the titration calorimetry at 298.15 K and 308.15 K for all the systems and additionally at 293.15 K and 318.15 K for the system with N-methylformamide, and at 293.15 K for the system with N,N-dimethylformamide. The activity coefficients of both components as a function of concentration and temperature were calculated by the correlation which included both types of data. The results of the prediction by the modified UNIFAC model were compared with the experimental data. The observed trends and differences between the systems were discussed.

Selective Protein Separation Based on Charge Anisotropy by Spherical Polyelectrolyte Brushes.

Protein purification is of vital importance in the food industry, drug discovery, and other related fields. Among many separation methods, polyelectrolyte (PE)-based phase separation was developed and recognized as a low-cost purification technique. In this work, spherical polyelectrolyte brushes (SPBs) with a high specific surface area were utilized to study the protein accessibility and selective protein binding on highly charged nanoparticles (NPs) as well as the selective phase separation of proteins. The correlation between charge anisotropy, protein binding, and phase separation was investigated on various protein systems including those proteins with similar isoelectric points (pI) such as bovine serum albumin (BSA) and β-lactoglobulin (BLG), proteins with similar molecular weights such as BSA and hemoglobin (HB), and even protein variants (BLG-A and -B) with a tiny difference of amino acids. The nonspecific electrostatic interaction studied by turbidimetric titrations and isothermal calorimetry titration (ITC) indicates a specific binding between proteins and SPBs arising from the charge anisotropy of proteins. An optimized output based on selective protein binding on SPBs could be correlated for efficient protein separation through tuning external conditions including pH and ionic strength. These findings, therefore, proved that phase separation based on selective protein adsorption by SPBs was an efficient alternative for protein separation compared with the traditional practice.

Ligand stabilization and effect on unfolding by polymorphism in human flavin-containing monooxygenase 3

Pharmacogenomics is a powerful tool to prevent adverse reactions caused by different response of individuals to drug administration. Single nucleotide polymorphisms (SNPs) represent up to 90% of genetic variations among individuals. Drug metabolizing enzymes are highly polymorphic therefore the kinetic parameters of their catalytic reactions can be significantly influenced. This work reports on the unfolding process of a phase I drug metabolizing enzyme, human flavin-containing monooxygenase 3 (hFMO3) and its single nucleotide polymorphic variants (SNPs) V257M, E158K and E308G. Differential scanning calorimetry (DSC) indicates that the thermal denaturation of the enzyme is irreversible. The melting temperature (Tm) for the (Wild Type) WT and its polymorphic variants is found to be in a range from 46 °C to 50 °C. Also the activation energies of unfolding (Ea) show no significant differences among all proteins investigated (290–328 KJ/mol), except for the E308G variant that showed a significantly higher Ea of 412 KJ/mol. The presence of the bound NADP+ cofactor is found to stabilize all the variants by shifting the main Tm by 4–5 °C for all the proteins, exception made for E308G where no changes are observed.Isothermal titration calorimetry (ITC) was used to characterize the interaction of the protein with NADP+ in terms of dissociation constant (Kd), enthalpy (ΔH) and entropy (ΔS). Kd values of 1.6 and 0.7 μM, ΔH of −13.9 Kcal/mol and −16.8 Kcal/mol, ΔS of −20.5 cal/mol/deg, and −28.5 cal/mol/deg were found for V257M and E158K respectively. E308G was found to be unable to bind the NADP+ cofactor, a result that is in line with the Tm results. Circular dichroism also confirmed an overall lower stability of E308G, while NADP+ was found to give a strong positive shift of the Tm stabilizing the structure of E158K (46.2 to 50.6 °C).Previous data highlighted significant differences in terms of activity among the SNPs of hFMO3. In this work a minor impact of the SNPs was found on the stability of the enzyme in the ligand free form, except for E308G, whereas the binding of NADP+ reveals major differences among WT and polymorphic variants that are all measurable in terms of heat capacity, enthalpy and secondary structure content. These data provide the first direct evidence of ligand stabilization effects on hFMO3 that can explain the differences observed in catalytic efficiencies and serve as the starting point for the development of inhibitors of this enzyme.

Targeting nucleic acid with a bioactive fluorophore: Insights from spectroscopic and calorimetric studies

The Schiff base (H2SALNN) (N,N′-bis(4-methoxy-salicylaldehyde)azine) has been designed to develop a DNA targeted fluorescent probe. The H2SALNN was synthesized and geometry optimized by DFT/B3LYP. The interaction of H2SALNN with Calf thymus DNA (CT-DNA) was studied by spectroscopic and calorimetric techniques. The compound was found to bind with CT-DNA through groove binding mode. From isothermal calorimetry (ITC) titration experiment, the binding constant between the compound and DNA was estimated to be (1.52 ± 0.03) × 105 M−1. The negative ΔG0 and positive ΔS0 values obtained from the calorimetric study confirmed the spontaneity of the binding of H2SALNN with DNA. Analysis of thermodynamic parameters indicated that the process of interaction of H2SALNN with DNA is entropy driven.

Lumefantrine, an antimalarial drug, reverses radiation and temozolomide resistance in glioblastoma

Glioblastoma multiforme (GBM) is an aggressive cancer without currently effective therapies. Radiation and temozolomide (radio/TMZ) resistance are major contributors to cancer recurrence and failed GBM therapy. Heat shock proteins (HSPs), through regulation of extracellular matrix (ECM) remodeling and epithelial mesenchymal transition (EMT), provide mechanistic pathways contributing to the development of GBM and radio/TMZ-resistant GBM. The Friend leukemia integration 1 (Fli-1) signaling network has been implicated in oncogenesis in GBM, making it an appealing target for advancing novel therapeutics. Fli-1 is linked to oncogenic transformation with up-regulation in radio/TMZ-resistant GBM, transcriptionally regulating HSPB1. This link led us to search for targeted molecules that inhibit Fli-1. Expression screening for Fli-1 inhibitors identified lumefantrine, an antimalarial drug, as a probable Fli-1 inhibitor. Docking and isothermal calorimetry titration confirmed interaction between lumefantrine and Fli-1. Lumefantrine promoted growth suppression and apoptosis in vitro in parental and radio/TMZ-resistant GBM and inhibited tumor growth without toxicity in vivo in U87MG GBM and radio/TMZ-resistant GBM orthotopic tumor models. These data reveal that lumefantrine, an FDA-approved drug, represents a potential GBM therapeutic that functions through inhibition of the Fli-1/HSPB1/EMT/ECM remodeling protein networks.

Small Molecule Inhibits Enterovirus 71 Replication by Targeting the IRES Domain

Human Enterovirus 71 (EV71), one of the major etiological agents of the hand, foot, and mouth disease (HFMD), represents a serious global health threat against which there is no FDA‐approved treatment. Enteroviruses utilize a highly structured RNA domain, known as a type I Internal Ribosome Entry Site (IRES), to facilitate cap‐independent translation and genome replication by recruiting multiple host RNA‐binding proteins (ITAFs) that fine‐tune translation. Of particular interest, the stem‐loop II (SLII) IRES domain has been shown to specifically interact with several ITAFs such as hnRNP A1 and AUF1, having the SLII‐bulge as the binding surface to effectively modulate viral translation. Further studies showed that deletions and mutations to the bulge motif inhibit EV71 replication by offsetting IRES‐driven translation. Altogether, the results provide strong basis to classify the EV71 SLII domain as a promising target to develop novel antiviral agents. Here, we screened an RNA focused small molecule (rfsm) library against the EV71 SLII domain which yielded in the identification of six rfms that specifically bind to the bulge motif when detected by NMR spectroscopy and titration calorimetry. One of the aforementioned rfms impairs IRES‐dependent translation and displays dose‐dependent antiviral properties in cell culture. Furthermore, the NMR‐derived 3D structure of the SLII‐rfsm complex provides evidence that the small molecule binds to the bulge to induce a conformational change that exposes key nucleotides on the binding site for AUF1. This was further validated both in an in vitro biochemical assay and in vivo. Collectively, these results suggest an allosteric mechanism of inhibition in which the rfsm stabilizes the SLII‐AUF1 complex to repress translation. Therefore, this study provides structural and functional insight to develop novel RNA‐focused small molecule inhibitors of the Enterovirus genus.Support or Funding InformationNIH R01GM126833

Site-Specific Interactions with Copper Promote Amyloid Fibril Formation for λ6aJL2-R24G.

Light-chain amyloidosis (AL) is one of the most common systemic amyloidoses, and it is characterized by the deposition of immunoglobulin light chain (LC) variable domains as insoluble amyloid fibers in vital organs and tissues. The recombinant protein 6aJL2-R24G contains λ6a and JL2 germline genes and also contains the Arg24 by Gly substitution. This mutation is present in 25% of all amyloid-associated λ6 LC cases, reduces protein stability, and increases the propensity to form amyloid fibers. In this study, it was found that the interaction of 6aJL2-R24G with Cu(II) decreases the thermal stability of the protein and accelerates the amyloid fibril formation, as observed by fluorescence spectroscopy. Isothermal calorimetry titration showed that Cu(II) binds to the protein with micromolar affinity. His99 may be one of the main Cu(II) interaction sites, as observed by nuclear magnetic resonance spectroscopy. The binding of Cu(II) to His99 induces larger fluctuations of the CDR1 and loop C″, as shown by molecular dynamics simulations. Thus, Cu(II) binding may be inducing the loss of interactions between CDR3 and CDR1, making the protein less stable and more prone to form amyloid fibers. This study provides insights into the mechanism of metal-induced aggregation of the 6aJL2-R24G protein and sheds light on the bio-inorganic understanding of AL disease.