Saquinavir-Piperine Eutectic Mixture: Preparation, Characterization, and Dissolution Profile.

The dissolution rate of the anti-HIV drug saquinavir base (SQV), a poorly water-soluble and extremely low absolute bioavailability drug, was improved through a eutectic mixture formation approach. A screening based on a liquid-assisted grinding technique was performed using a 1:1 molar ratio of the drug and the coformers sodium saccharinate, theobromine, nicotinic acid, nicotinamide, vanillin, vanillic acid, and piperine (PIP), followed by differential scanning calorimetry (DSC). Given that SQV-PIP was the only resulting eutectic system from the screening, both the binary phase and the Tammann diagrams were adapted to this system using DSC data of mixtures prepared from 0.1 to 1.0 molar ratios in order to determine the exact eutectic composition. The SQV-PIP system formed a eutectic at a composition of 0.6 and 0.40, respectively. Then, a solid-state characterization through DSC, powder X-ray diffraction (PXRD), including small-angle X-ray scattering (SAXS) measurements to explore the small-angle region in detail, Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and a powder dissolution test were performed. The conventional PXRD analyses suggested that the eutectic mixture did not exhibit structural changes; however, the small-angle region explored through the SAXS instrument revealed a change in the crystal structure of one of their components. FT-IR spectra showed no molecular interaction in the solid state. Finally, the dissolution profile of SQV in the eutectic mixture was different from the dissolution of pure SQV. After 45 min, approximately 55% of the drug in the eutectic mixture was dissolved, while, for pure SQV, 42% dissolved within this time. Hence, this study concludes that the dissolution rate of SQV can be effectively improved through the approach of using PIP as a coformer.

Unveiling protein-protein interaction potential through Monte Carlo simulation combined with small-angle X-ray scattering.

Protein interactions are investigated under different conditions of lysozyme concentration, temperature and ionic strength by means of in-solution small angle X-Ray scattering (SAXS) experiments and Monte Carlo (MC) simulations. Initially, experimental data were analysed through a Hard-Sphere Double Yukawa (HSDY) model combined with Random Phase Approximation (RPA), a closure relationship commonly used in the literature for monodisperse systems. We realized by means of MC that the HSDY/RPA modelling fails to describe the protein-protein pair potential for moderated and dense systems at low ionic strength, mainly due to inherent distortions of the RPA approximation. Our SAXS/MC results thus show that lysozyme concentrations between 2 (diluted) and 20 mg/mL (not crowded) present similar protein-protein pair potential preserving the values of surface net charge around 7 e, protein diameter of 28 Å, decay range of attractive well potential of 3 Å and a depth of the well potential varying from 1 to 5 kBT depending on temperature and salt addition. Noteworthy, we here propose a novel method to analyse the SAXS data from interacting proteins through MC simulations, which overcomes the deficiencies presented by the use of a closure relationship. Furthermore, this new methodology of combining SAXS with MC simulations gives a step forward to investigate more complex systems as those composed of a mixture of proteins of distinct species presenting different molecular weights (and hence sizes) and surface net charges at low, moderate and very dense systems.

A Fijivirus Major Viroplasm Protein Shows RNA-Stimulated ATPase Activity by Adopting Pentameric and Hexameric Assemblies of Dimers.

Fijiviruses replicate and package their genomes within viroplasms in a process involving RNA-RNA and RNA-protein interactions. Here, we demonstrate that the 24 C-terminal residues (C-arm) of the P9-1 major viroplasm protein of the mal de Río Cuarto virus (MRCV) are required for its multimerization and the formation of viroplasm-like structures. Using an integrative structural approach, the C-arm was found to be dispensable for P9-1 dimer assembly but essential for the formation of pentamers and hexamers of dimers (decamers and dodecamers), which favored RNA binding. Although both P9-1 and P9-1ΔC-arm catalyzed ATP with similar activities, an RNA-stimulated ATPase activity was only detected in the full-length protein, indicating a C-arm-mediated interaction between the ATP catalytic site and the allosteric RNA binding sites in the (do)decameric assemblies. A stronger preference to bind phosphate moieties in the decamer was predicted, suggesting that the allosteric modulation of ATPase activity by RNA is favored in this structural conformation. Our work reveals the structural versatility of a fijivirus major viroplasm protein and provides clues to its mechanism of action. IMPORTANCE The mal de Río Cuarto virus (MRCV) causes an important maize disease in Argentina. MRCV replicates in several species of Gramineae plants and planthopper vectors. The viral factories, also called viroplasms, have been studied in detail in animal reovirids. This work reveals that a major viroplasm protein of MRCV forms previously unidentified structural arrangements and provides evidence that it may simultaneously adopt two distinct quaternary assemblies. Furthermore, our work uncovers an allosteric communication between the ATP and RNA binding sites that is favored in the multimeric arrangements. Our results contribute to the understanding of plant reovirids viroplasm structure and function and pave the way for the design of antiviral strategies for disease control.

Small-angle X-ray scattering to quantify the incorporation and analyze the disposition of magnetic nanoparticles inside cells.

Access to detailed information on cells loaded with nanoparticles with nanoscale precision is of a long-standing interest in many areas of nanomedicine. In this context, designing a single experiment able to provide statistical mean data from a large number of living unsectioned cells concerning information on the nanoparticle size and aggregation inside cell endosomes and accurate nanoparticle cell up-take is of paramount importance. Small-angle X-ray scattering (SAXS) is presented here as a tool to achieve such relevant data. Experiments were carried out in cultures of B16F0 murine melanoma and A549 human lung adenocarcinoma cell lines loaded with various iron oxide nanostructures displaying distinctive structural characteristics. Five systems of water-dispersible magnetic nanoparticles (MNP) of different size, polydispersity and morphology were analyzed, namely, nearly monodisperse MNP with 11 and 13 nm mean size coated with meso-2,3-dimercaptosuccinic acid, more polydisperse 6 nm colloids coated with citric acid and two nanoflowers (NF) systems of 24 and 27 nm in size resulting from the aggregation of 8 nm MNP. Up-take was determined for each system using B16F0 cells. Here we show that SAXS pattern provides high resolution information on nanoparticles disposition inside endosomes of the cytoplasm through the structure factor analysis, on nanoparticles size and dispersity after their incorporation by the cell and on up-take quantification from the extrapolation of the intensity in absolute scale to null scattering vector. We also report on the cell culture preparation to reach sensitivity for the observation of MNP inside cell endosomes using high brightness SAXS synchrotron source. Our results show that SAXS can become a valuable tool for analyzing MNP in cells and tissues.

Cubosomal lipid nanoassemblies with pH-sensitive shells created by biopolymer complexes: A synchrotron SAXS study.

We report a strategy for sustainable development of pH-responsive cubic liquid crystalline nanoparticles (cubosomes), in which the structure-defining lyotropic nonlamellar lipid and the eventually encapsulated guest molecules can be protected by pH-sensitive polyelectrolyte shells with mucoadhesive properties. Bulk non-lamellar phases as well as pH-responsive polyelectrolyte-modified nanocarriers were formed by spontaneous assembly of the nonlamellar lipid monoolein and two biopolymers tailored in nanocomplexes with pH-dependent net charge. The mesophase particles involved positively charged N-arginine-modified chitosan (CHarg) and negatively charged alginate (ALG) chains assembled at different biopolymer concentrations and charge ratios into a series of pH-responsive complexes. The roles of Pluronic F127 as a dispersing agent and a stabilizer of the nanoscale dispersions were examined. Synchrotron small-angle X-ray scattering (SAXS) investigations were performed at several N-arginine-modified chitosan/alginate ratios (CHarg/ALG with 10, 15 and 20 wt% ALG relative to CHarg) and varying pH values mimicking the pH conditions of the gastrointestinal route. The structural parameters characterizing the inner cubic liquid crystalline organizations of the nanocarriers were determined as well as the particle sizes and stability on storage. The surface charge variations, influencing the measured zeta-potentials, evidenced the inclusion of the CHarg/ALG biopolymer complexes into the lipid nanoassemblies. The polyelectrolyte shells rendered the hybrid cubosome nanocarriers pH-sensitive and influenced the swelling of their lipid-phase core as revealed by the acquired SAXS patterns. The pH-responsiveness and the mucoadhesive features of the cubosomal lipid/polyelectrolyte nanocomplexes may be of interest for in vivo drug delivery applications.

X-ray Characterization of Conformational Changes of Human Apo- and Holo-Transferrin.

Human serum transferrin (Tf) is a bilobed glycoprotein whose function is to transport iron through receptor-mediated endocytosis. The mechanism for iron release is pH-dependent and involves conformational changes in the protein, thus making it an attractive system for possible biomedical applications. In this contribution, two powerful X-ray techniques, namely Macromolecular X-ray Crystallography (MX) and Small Angle X-ray Scattering (SAXS), were used to study the conformational changes of iron-free (apo) and iron-loaded (holo) transferrin in crystal and solution states, respectively, at three different pH values of physiological relevance. A crystallographic model of glycosylated apo-Tf was obtained at 3.0 Å resolution, which did not resolve further despite many efforts to improve crystal quality. In the solution, apo-Tf remained mostly globular in all the pH conditions tested; however, the co-existence of closed, partially open, and open conformations was observed for holo-Tf, which showed a more elongated and flexible shape overall.

Identification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus covalently closed circular DNA.

Approximately 250 million people worldwide are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing cirrhosis and hepatocellular carcinoma. The HBV genome persists as covalently closed circular DNA (cccDNA), which serves as the template for all HBV mRNA transcripts. Current nucleos(t)ide analogs used to treat HBV do not directly target the HBV cccDNA genome and thus cannot eradicate HBV infection. Here, we report the discovery of a unique G-quadruplex structure in the pre-core promoter region of the HBV genome that is conserved among nearly all genotypes. This region is central to critical steps in the viral life cycle, including the generation of pregenomic RNA, synthesis of core and polymerase proteins, and genome encapsidation; thus, an increased understanding of the HBV pre-core region may lead to the identification of novel anti-HBV cccDNA targets. We utilized biophysical methods (circular dichroism and small-angle X-ray scattering) to characterize the HBV G-quadruplex and the effect of three distinct G to A mutants. We also used microscale thermophoresis to quantify the binding affinity of G-quadruplex and its mutants with a known quadruplex-binding protein (DHX36). To investigate the physiological relevance of HBV G-quadruplex, we employed assays using DHX36 to pull-down cccDNA and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids by monitoring the levels of genomic DNA, pregenomic RNA, and antigens. Further evaluation of this critical host-protein interaction site in the HBV cccDNA genome may facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template.

Nanostructured lipid carriers as a novel tool to deliver sclareol: physicochemical characterisation and evaluation in human cancer cell lines

Sclareol (SC) is arousing great interest due to its cytostatic and cytotoxic activities in several cancer cell lines. However, its hydrophobicity is a limiting factor for its in vivo administration. One way to solve this problem is through nanoencapsulation. Therefore, solid lipid nanoparticles (SLN-SC) and nanostructured lipid carriers (NLC-SC) loaded with SC were produced and compared regarding their physicochemical properties. NLC-SC showed better SC encapsulation than SLN-SC and was chosen to be compared with free SC in human cancer cell lines (MDA-MB-231 and HCT-116). Free SC had slightly higher cytotoxicity than NLC-SC and produced subdiploid DNA content in both cell lines. On the other hand, NLC-SC led to subdiploid content in MDA-MB-231 cells and G2/M checkpoint arrest in HCT-116 cells. These findings suggest that SC encapsulation in NLC is a way to allow the in vivo administration of SC and might alter its biological properties

High-intensity ultrasound-assisted extraction of phenolic compounds from cowpea pods and its encapsulation in hydrogels.

Currently and according to the growing worldwide interest in the revaluation of agricultural by-products, the use of legumes waste presents great potential to obtain bioactive compounds. In this context, an extract rich in phenolic compounds was obtained from Vigna unguiculata (cowpea) pods by optimizing the high-intensity ultrasound conditions (10 min and 36% of amplitude) using response surface methodology. Then, the extract was encapsulated in Ca(II)-alginate beads with the addition of arabic or guar gums or cowpea isolated proteins. A complete morphological study by image analysis and microstructural evaluation by SAXS has been carried out. Results showed that beads containing alginate and alginate-guar gum have the highest loading efficiency of total phenolic compounds (47 ± 5%) and antioxidant activity (44 ± 3%). However, the coupled effect of the cowpea extract and the isolated proteins (at it higher concentration) increased the antioxidant capacity of the beads due to the contribution of the phenolic compounds and the amino acids with anti-radical activity, reaching a value of 67 ± 3 % of inhibition of ABTS.+. Finally, the microstructural analyses revealed that cowpea pod extract increased the interconnectivity of the rods due to the presence of trivalent cations, conferring versatility, and larger coordination to the network. Also, it was observed that the addition of cowpea proteins produced more interconnected bigger and fewer compacts rods than beads containing only alginate, increasing 12 and 49 % the interconnection and the size, respectively, and decreasing 10 % their compactness. This research demonstrated the use of cowpea sub-products as a source of bioactive compounds that further modulate the microstructure of the hydrogel network, and the outstanding potential for being incorporated in techno-functional foods by using Ca(II)-alginate as a carrier.

Low-resolution molecular shape, biochemical characterization and emulsification properties of a halotolerant esterase from Bacillus licheniformis.

Bacterial esterases are highly versatile enzymes, currently widely used in detergents, biosurfactants, bioemulsifiers and as biocatalysts in paper and food industries. Present work describes heterologous expression, purification, and biophysical and biochemical characterization of a halotolerant esterase from Bacillus licheniformis (BlEstA). BlEstA preferentially cleaves pNP-octanoate and both activity and stability of the enzyme increased in the presence of 2 M NaCl, and also with several organic solvents (ethanol, methanol and DMSO). Furthermore, BlEstA has considerable emulsifying properties, particularly with olive oil as substrate. Our studies also show that the enzyme is monomeric in solution and its small-angle X-ray scattering low-resolution molecular envelope fits well its high-resolution homology model.

Cell wall structure and composition is affected by light quality in tomato seedlings.

Light affects many aspects of cell development. Tomato seedlings growing at different light qualities (white, blue, green, red, far-red) and in the dark displayed alterations in cell wall structure and composition. A strong and negative correlation was found between cell wall thickness and hypocotyl growth. Cell walls was thicker under blue and white lights and thinner under far-red light and in the dark, while intermediate values was observed for red or green lights. Additionally, the inside layer surface of cell wall presented random deposited microfibrillae angles under far-red light and in the dark. However, longitudinal transmission electron microscopy indicates a high frequency of microfibrils close to parallels related to the elongation axis in the outer layer. This was confirmed by ultra-high resolution small angle X-ray scattering. These data suggest that cellulose microfibrils would be passively reoriented in the longitudinal direction. As the cell expands, the most recently deposited layers (inside) behave differentially oriented compared to older (outer) layers in the dark or under FR lights, agreeing with the multinet growth hypothesis. High Ca and pectin levels were found in the cell wall of seedlings growing under blue and white light, also contributing to the low extensibility of the cell wall. Low Ca and pectin contents were found in the dark and under far-red light. Auxins marginally stimulated growth in thin cell wall circumstances. Hypocotyl growth was stimulated by gibberellins under blue light.

Biochemical characterization and low-resolution SAXS shape of a novel GH11 exo-1,4-ß-xylanase identified in a microbial consortium.

Biotechnologies that aim to produce renewable fuels, chemicals, and bioproducts from residual ligno(hemi)cellulosic biomass mostly rely on enzymatic depolymerization of plant cell walls (PCW). This process requires an arsenal of diverse enzymes, including xylanases, which synergistically act on the hemicellulose, reducing the long and complex xylan chains to oligomers and simple sugars. Thus, xylanases play a crucial role in PCW depolymerization. Until recently, the largest xylanase family, glycoside hydrolase family 11 (GH11) has been exclusively represented by endo-catalytic ß-1,4- and ß-1,3-xylanases. Analysis of a metatranscriptome library from a microbial lignocellulose community resulted in the identification of an unusual exo-acting GH11 ß-1,4-xylanase (MetXyn11). Detailed characterization has been performed on recombinant MetXyn11 including determination of its low-resolution small-angle X-ray scattering (SAXS) molecular envelope in solution. Our results reveal that MetXyn11 is a monomeric globular enzyme that liberates xylobiose from heteroxylans as the only product. MetXyn11 has an optimal activity in a pH range from 6 to 9 and an optimal temperature of 50 °C. The enzyme maintained above 65% of its original activity in the pH range 5 to 6 after being incubated for 72 h at 50 °C. Addition of the enzyme to a commercial enzymatic cocktail (CelicCtec3) promoted a significant increase of enzymatic hydrolysis yields of hydrothermally pretreated sugarcane bagasse (16% after 24 h of hydrolysis).

Dispersion of reduced graphene oxide within thermoplastic starch/poly(lactic acid) blends investigated by small-angle X-ray scattering.

Reduced graphene oxide (rGO) was incorporated into plasticized cornstarch (TPS), poly(lactic acid) (PLA) and their blends. Small-angle X-ray scattering (SAXS) was used to investigate rGO dispersion within the materials. For the TPS/PLA blend at 70:30 composition, the incorporation of rGO led to a larger fraction of small-sized rGO sheets, which at 5.0 mass% content developed stable fractal structures and domains of correlated sheets. The formation of fractal structures resulted in substantial enhancements in macroscopic properties. For these hybrids, the electrical conductivity, melt viscosity, storage moduli and biodegradation rates presented enhancements in relation to the neat blend. For the TPS/PLA blend at 30:70 composition, SAXS results indicated the formation of smaller fractions of well-dispersed rGO sheets and of segregated larger rGO sheets. With rGO at 5 mass% content, less expressive increases in electrical conductivity, melt viscosity, storage moduli and biodegradation rates were observed.

Biochemical characterization and low-resolution SAXS structure of two-domain endoglucanase BlCel9 from Bacillus licheniformis.

Lignocellulose feedstock constitutes the most abundant carbon source in the biosphere; however, its recalcitrance remains a challenge for microbial conversion into biofuel and bioproducts. Bacillus licheniformis is a microbial mesophilic bacterium capable of secreting a large number of glycoside hydrolase (GH) enzymes, including a glycoside hydrolase from GH family 9 (BlCel9). Here, we conducted biochemical and biophysical studies of recombinant BlCel9, and its low-resolution molecular shape was retrieved from small angle X-ray scattering (SAXS) data. BlCel9 is an endoglucanase exhibiting maximum catalytic efficiency at pH 7.0 and 60 °C. Furthermore, it retains 80% of catalytic activity within a broad range of pH values (5.5-8.5) and temperatures (up to 50 °C) for extended periods of time (over 48 h). It exhibits the highest hydrolytic activity against phosphoric acid swollen cellulose (PASC), followed by bacterial cellulose (BC), filter paper (FP), and to a lesser extent carboxymethylcellulose (CMC). The HPAEC-PAD analysis of the hydrolytic products demonstrated that the end product of the enzymatic hydrolysis is primarily cellobiose, and also small amounts of glucose, cellotriose, and cellotetraose are produced. SAXS data analysis revealed that the enzyme adopts a monomeric state in solution and has a molecular mass of 65.8 kDa as estimated from SAXS data. The BlCel9 has an elongated shape composed of an N-terminal family 3 carbohydrate-binding module (CBM3c) and a C-terminal GH9 catalytic domain joined together by 20 amino acid residue long linker peptides. The domains are closely juxtaposed in an extended conformation and form a relatively rigid structure in solution, indicating that the interactions between the CBM3c and GH9 catalytic domains might play a key role in cooperative cellulose biomass recognition and hydrolysis.

The Machado-Joseph disease-associated expanded form of ataxin-3: Overexpression, purification, and preliminary biophysical and structural characterization.

An expansion of the polyglutamine (polyQ) tract within the deubiquitinase ataxin-3 protein is believed to play a role in a neurodegenerative disorder. Ataxin-3 contains a Josephin catalytic domain and a polyQ tract that renders it intrinsically prone to aggregate, and thus full-length protein is difficult to characterize structurally by high-resolution methods. We established a robust protocol for expression and purification of wild-type and expanded ataxin-3, presenting 19Q and 74Q, respectively. Both proteins are monodisperse as assessed by analytical size exclusion chromatography. Initial biophysical characterization was performed, with apparent transition melting temperature of expanded ataxin-3 lower than the wild-type counterpart. We further characterize the molecular envelope of wild-type and expanded polyQ tract in ataxin-3 using small angle X-ray scattering (SAXS). Characterization of protein-protein interactions between ataxin-3 and newly identified binding partners will benefit from our protocol.

Structure and stability of the Human respiratory syncytial virus M2-1 RNA-binding core domain reveals a compact and cooperative folding unit.

Human syncytial respiratory virus is a nonsegmented negative-strand RNA virus with serious implications for respiratory disease in infants, and has recently been reclassified into a new family, Pneumoviridae. One of the main reasons for this classification is the unique presence of a transcriptional antiterminator, called M2-1. The puzzling mechanism of action of M2-1, which is a rarity among antiterminators in viruses and is part of the RNA polymerase complex, relies on dissecting the structure and function of this multidomain tetramer. The RNA-binding activity is located in a monomeric globular `core' domain, a high-resolution crystal structure of which is now presented. The structure reveals a compact domain which is superimposable on the full-length M2-1 tetramer, with additional electron density for the C-terminal tail that was not observed in the previous models. Moreover, its folding stability was determined through chemical denaturation, which shows that the secondary and tertiary structure unfold concomitantly, which is indicative of a two-state equilibrium. These results constitute a further step in the understanding of this unique RNA-binding domain, for which there is no sequence or structural counterpart outside this virus family, in addition to its implications in transcription regulation and its likeliness as an antiviral target.

The Critical Role of Thioacetamide Concentration in the Formation of ZnO/ZnS Heterostructures by Sol-Gel Process.

ZnO/ZnS heterostructures have emerged as an attractive approach for tailoring the properties of particles comprising these semiconductors. They can be synthesized using low temperature sol-gel routes. The present work yields insight into the mechanisms involved in the formation of ZnO/ZnS nanostructures. ZnO colloidal suspensions, prepared by hydrolysis and condensation of a Zn acetate precursor solution, were allowed to react with an ethanolic thioacetamide solution (TAA) as sulfur source. The reactions were monitored in situ by Small Angle X-ray Scattering (SAXS) and UV-vis spectroscopy, and the final colloidal suspensions were characterized by High Resolution Transmission Electron Microscopy (HRTEM). The powders extracted at the end of the reactions were analyzed by X-ray Absorption spectroscopy (XAS) and X-ray diffraction (XRD). Depending on TAA concentration, different nanostructures were revealed. ZnO and ZnS phases were mainly obtained at low and high TAA concentrations, respectively. At intermediate TAA concentrations, we evidenced the formation of ZnO/ZnS heterostructures. ZnS formation could take place via direct crystal growth involving Zn ions remaining in solution and S ions provided by TAA and/or chemical conversion of ZnO to ZnS. The combination of all the characterization techniques was crucial to elucidate the reaction steps and the nature of the final products.

Spectroscopic and calorimetric assays reveal dependence on dCTP and two metals (Zn2++Mg2+) for enzymatic activity of Schistosoma mansoni deoxycytidylate (dCMP) deaminase.

The parasite Schistosoma mansoni possess all pathways for pyrimidine biosynthesis, whereby deaminases play an essential role in the thymidylate cycle, a crucial step to controlling the ratio between cytidine and uridine nucleotides. In this study, we heterologously expressed and purified the deoxycytidylate (dCMP) deaminase from S. mansoni to obtain structural, biochemical and kinetic information. Small-angle X-ray scattering of this enzyme showed that it is organized as a hexamer in solution. Isothermal titration calorimetry was used to determine the kinetic constants for dCMP-dUMP conversion and the role of dCTP and dTTP in enzymatic regulation. We evaluated the metals involved in activating the enzyme and show for the first time the dependence of correct folding on the interaction of two metals. This study provides information that may be useful for understanding the regulatory mechanisms involved in the metabolic pathways of S. mansoni. Thus, improving our understanding of the function of these essential pathways for parasite metabolism and showing for the first time the hitherto unknown deaminase function in this parasite.

Allosteric Transmission along a Loosely Structured Backbone Allows a Cardiac Troponin C Mutant to Function with Only One Ca2+ Ion.

Hypertrophic cardiomyopathy (HCM) is one of the most common cardiomyopathies and a major cause of sudden death in young athletes. The Ca2+ sensor of the sarcomere, cardiac troponin C (cTnC), plays an important role in regulating muscle contraction. Although several cardiomyopathy-causing mutations have been identified in cTnC, the limited information about their structural defects has been mapped to the HCM phenotype. Here, we used high-resolution electron-spray ionization mass spectrometry (ESI-MS), Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG-RD), and affinity measurements of cTnC for the thin filament in reconstituted papillary muscles to provide evidence of an allosteric mechanism in mutant cTnC that may play a role to the HCM phenotype. We showed that the D145E mutation leads to altered dynamics on a µs-ms time scale and deactivates both of the divalent cation-binding sites of the cTnC C-domain. CPMG-RD captured a low populated protein-folding conformation triggered by the Glu-145 replacement of Asp. Paradoxically, although D145E C-domain was unable to bind Ca2+, these changes along its backbone allowed it to attach more firmly to thin filaments than the wild-type isoform, providing evidence for an allosteric response of the Ca2+-binding site II in the N-domain. Our findings explain how the effects of an HCM mutation in the C-domain reflect up into the N-domain to cause an increase of Ca2+ affinity in site II, thus opening up new insights into the HCM phenotype.