Interaction Of Monomers Research Articles

Hydride and halide abstraction reactions behind the enhanced basicity of Be and Mg clusters with nitrogen bases.

In this study, we investigate the protonation effects on the structure, relative stability and basicity of complexes formed by the interaction of monomers and dimers of BeX2 and MgX2 (X = H, F) with NH3, CH2NH, HCN, and NC5H5 bases. Calculations were performed using the M06-2X/aug-cc-pVTZ formalism, along with QTAIM, ELF and NCI methods for electron density analysis and MBIE and LMO-EDA energy decomposition analyses for interaction enthalpies. The protonation of the MH2- and M2H4-Base complexes occurs at the negatively charged hydrogen atoms of the MH2 and M2H4 moieties through typical hydride abstraction reactions, while protonation at the N atom of the base is systematically less exothermic. The preference for the hydride transfer mechanism is directly associated with the significant exothermicity of H2 formation through the interaction between H- and H+, and the high hydride donor ability of these complexes. The basicity of both, MH2 and M2H4 compounds increases enormously upon association with the corresponding bases, with the increase exceeding 40 orders of magnitude in terms of ionization constants. Due to the smaller exothermicity of HF formation, the basicity of fluorides is lower than that of hydrides. In Be complexes, the protonation at the N atom of the base dominates over the fluoride abstraction mechanism. However, for the Mg complexes the fluoride abstraction mechanism is energetically the most favorable process, reflecting the greater facility of Mg complexes to lose F-.

Competitive Interaction of the SGFRKMAF Peptide with 3CLpro Dimerization Intermediates: A Brownian Dynamics Investigation.

The SGFRKMAF peptide is known to inhibit the dimerization of 3CLpro monomers, which is essential for SARS-CoV-2 replication. The mechanism behind this, however, is largely unknown. In this work, we used Brownian dynamics simulations to compare and contrast 3CLpro monomer-monomer interactions and 3CLpro monomer-SGFRKMAF peptide interactions. We found that formation of the 3CLpro wild-type dimer could potentially involve formation of three intermediates that are primarily stabilized by G11-G124, S1-S301, and T118-G278 interactions. Analysis of 3CLpro monomer interaction with the SGFRKMAF peptide, however, revealed the presence of eight basins of interactions where the peptide assumes the highest local densities at the 3CLPro monomer surface. The second highest-density basin was found to coincide with the interface region of the wild-type 3CLpro dimer, thereby directly blocking the 3CLpro dimer-dimer interactions. The other basins, however, were found to lie far from the interface region. Notably, we found that only 6% of the BD trajectories end up directly into the basin at the interface region and ∼39% of the trajectories end up into those basins lying away from the interface region, indicating a greater role for peptide binding at sites away from the dimer interface region. Importantly, the locations of the basins lying away from the interface were found to coincide with the 3CLpro residues involved in stabilization of the 3CLpro monomer-monomer intermediates. Given that the rate constant of the peptide reaching the monomer surface was found to be almost an order of magnitude higher than the rate constant of monomer-monomer association, the SGFRKMAF peptide has the potential to inhibit dimerization of 3CLpro monomers not only through blocking the interface region but also through blocking the formation of the intermediates involved in the dimerization process. This could potentially open new avenues for 3CLpro dimerization inhibitors that transcend traditional X-ray-based discovery approaches.

Gramicidin A as a mechanical sensor for mixed nonideal lipid membranes.

Gramicidin A (gA) is a short hydrophobic β-helical peptide that forms cation-selective channels in lipid membranes in the course of transbilayer dimerization. The length of the gA helix is smaller than the thickness of a typical lipid monolayer. Consequently, elastic deformations of the membrane arise in the configurations of gA monomers, conducting dimer, and the intermediate state of coaxial pair, where gA monomers from opposing membrane monolayers are located one on top of the other. The gA channel is characterized by the average lifetime of the conducting state. The elastic properties of the membrane influence the average lifetime, thus making gA a convenient sensor of membrane elasticity. However, the utilization of gA to investigate the elastic properties of mixed membranes comprising two or more components frequently relies on the assumption of ideality, namely that the elastic parameters of mixed-lipid bilayers depend linearly on the concentrations of the components. Here, we developed a general approach that does not rely on the aforementioned assumption. Instead, we explicitly accounted for the possibility of inhomogeneous lateral distribution of all lipid components, as well as for membrane-mediated lateral interactions of gA monomers, dimer, coaxial pair, and minor lipid components. This approach enabled us to derive unknown elastic parameters of lipid monolayer from experimentally determined lifetimes of gA channel in mixed-lipid bilayers. A general algorithm was formulated that allows the unknown elastic parameters of a lipid monolayer to be obtained using gA as a mechanical sensor.

Investigation of Polyvinyl Alcohol (PVA)-Sakacin P Interaction by Molecular Docking Method

Aim: Group IIA bacteriocins, which contain unmodified amino acids and have antimicrobial activity, are a very broad group. Sakacins in this group are bacteriocins produced by Lactobacillus sakei. The most well-known sakacins are Sakacin A, G, K, P, and Q. In particular, Sakacin A and P are well characterized. This study aims to examine the interaction between a single monomer of PVA polymer and Sakacin P bacteriocin. Method: In t this study, the interaction of a single monomer of PVA polymer, which is among the water-soluble, biocompatible synthetic polymers, and Sakacin P bacteriocin, which has a protein structure, was investigated by molecular docking method. Results: As a result of our molecular docking study, the presence of binding affinity between the C2H4O monomer of PVA selected as the ligand and the Sakacin P protein selected as the receptor was determined. Conclusion: According to the results of the analysis, the presence of a strong inhibitor was detected between the ligand and the target. Therefore, this study can serve as a template for polymer-bacteriocin materials to be produced in the laboratory.

Revealing the Regulation Effect of Surface Charge at Aromatic Interface to Dynamic Conformational Changes of α‐Synuclein at Early Aggregation Stage

Comprehensive SummaryThe aggregation of α‐synuclein (α‐syn) is strongly influenced by membrane interfaces, but the mechanism of transition from monomers to oligomers at early aggregation stage is not clear. Here, we investigate the adsorption and structure changes of α‐syn on oppositely charged aromatic interfaces through in‐situ surface‐enhanced infrared absorption (SEIRA) spectroscopy and nano‐IR technique. The results show that the synergy of electrostatic and hydrophobic interactions leads to a “fast‐slow” two‐step aggregation pathway on negatively charged interface. Surface adsorption induces the formation of an extended helix structure and subsequently partial helix unwinding in NAC region, which enables the hydrophobic stacking between nearby NAC regions. Stable antiparallel β‐sheet rich aggregates are gradually emerging as further interactions of monomers with the fast formed “first layer”. Monomers electrostatically adsorb on positively charged interface by C‐terminus with NAC region and N‐terminus stretched in solvent, which serve as an aggregation core and induce further adsorption and gradual formation of aggregates with C‐terminus exposure. Our results demonstrate the modulation of surface charge and synergy of electrostatic and hydrophobic interactions on the interaction modes and aggregation pathways, which provide insights into dynamic conformation changes of α‐syn at early aggregation stage and imply the important role of spatial‐temporal heterogeneity of membranes in α‐synucleinopathies.

The role of organic solvent and nano-additives loading in preparing and characterizing graphene oxide based polystyrene nanocomposites

ABSTRACT In this work, two types of organic solvents: namely, dimethylformamide (DMF) and tetrahydrofuran (THF); were investigated individually to prepare graphene oxide (GO) based polystyrene (PS) nanocomposites. Both were effective exfoliation agents for GO nano-sheets using sonication process. However, DMF showed poor solubility for PS beads compared to THF due to its high boiling point, poor volatility, and the prevailing monomer–monomer interaction rather than monomer–solvent interaction. Hot pressing was not an efficient preparatory approach for those samples prepared by DMF. Hence, solution casting, and THF were adopted to prepare PS and its GO-based nanocomposites. (0.5 and 1.0) wt. % of GO were dispersed homogeneously in PS using different dispersive techniques. Range of properties were tested for the pure polymer and its GO-based nanocomposites prepared by THF. A good dispersion of GO nano-sheets in PS was observed by different microscopic techniques. A significant improvement of mechanical and thermo-mechanical (storage modulus) performances for the polymer nanocomposites (PNCs) were achieved compared to the pure polymer. Young’s modulus for PS/GO nanocomposites was improved by 122% and 143% for GO loadings of 0.5 and 1.0 wt. %; respectively, and storage modulus for the nanocomposites recorded a considerable promotion compared to the pure polymer.

Development and Fabrication of a Molecularly Imprinted Polymer-Based Electroanalytical Sensor for the Determination of Acyclovir.

Acyclovir (ACV), a synthetic nucleoside derivative of purine, is one of the most potent antiviral medications recommended in the specific management of varicella-zoster and herpes simplex viruses. The molecularly imprinted polymer (MIP) was utilized to create an effective and specific electrochemical sensor using a straightforward photopolymerization process to determine ACV. The polymeric thin coating was developed using the template molecule ACV, a functional monomer acrylamide, a basic monomer 2-hydroxyethyl methacrylate, a cross-linker ethylene glycol dimethacrylate, and a photoinitiator 2-hydroxy-2-methyl propiophenone on the exterior of the glassy carbon electrode (GCE). Scanning electron microscopy, attenuated total reflectance-Fourier transform infrared spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetry were employed for the purpose of characterizing the constructed sensor (AM-ACV@MIP/GCE). Differential pulse voltammetry and a 5 mM ferrocyanide/ferricyanide ([Fe(CN)6]3-/4-) redox reagent were used to detect the ACV binding to the specific cavities on MIP. The study involves density functional theory (DFT) calculations, which were conducted to investigate template-functional monomer interactions thoroughly, calculate template-functional monomer interaction energies, and determine the optimal template/functional monomer ratio. DFT calculations were performed using Becke's three-parameter hybrid functional with the Lee-Yang-Parr correlation functional (B3LYP) method and 6-31G(d,p) basis set. The sensor exhibits linear performance throughout the concentration region 1 × 10-11 to 1 × 10-10 M, and the limit of detection and limit of quantification were 7.15 × 10-13 M and 2.38 × 10-12 M, respectively. For the electrochemical study of ACV, the sensor demonstrated high accuracy, precision, robustness, and a short detection time. Furthermore, the developed electrochemical sensor exhibited exceptional recovery in tablet dosage form and commercial human blood samples, with recoveries of 99.40 and 100.44%, respectively. The findings showed that the AM-ACV@MIP/GCE sensor would effectively be used to directly assess pharmaceuticals from actual specimens and would particularly detect ACV compared to structurally similar pharmaceutical compounds.

SYNTHESIS OF TETRACYCLINE IMPRINTED POLYMERS WITH METHACRYLIC ACID AS FUNCTIONAL MONOMER IN METHANOL-CHLOROFORM MIXTURE USING BULK AND PRECIPITATION POLYMERIZATION METHOD

The content of tetracycline residues in poultry meat products can cause antibiotic resistance in humans who consume these products, so it is necessary to develop sensitive analytical techniques to determine the levels of tetracycline residues to assess the safety of these products for consumption. The molecular imprinting technique is a method to produce sorbents with molecular recognition capability of target compounds that can be used to increase the selectivity of solid phase extraction to extract tetracycline residues for further analysis. This study aimed to obtain a sorbent synthesized by molecular imprinting technique to analyze tetracycline in poultry meat products. The stages of the research began with the study of the interaction of functional monomers with template molecules, determining the association constants of functional monomers with template molecules, synthesis of imprinted polymer molecules using bulk and precipitation methods, evaluating the ability and adsorption capacity of the synthesized polymers, assess the selectivity of polymers for analog structures and physical characteristics with FTIR. The research showed that methacrylic acid was the best functional monomer with a binding energy value of -27.3776 kcal/mol. The higher adsorption capacity was achieved by Molecularly Imprinted Polymer (MIP) that was synthesized by precipitation method (MIP2) than the other MIP synthesized by bulk polymerization (MIP1) with a value of 0.8748 mg/g and 0.4077 mg/g, respectively. The analogous compounds' imprinting factor values for each MIP were 1.197 and 1.1272. The polymer synthesized by molecular imprinting technique is selective for extracting and analyzing tetracycline from poultry meat matrix.

Exciton interactions of chlorophyll tetramer in water-soluble chlorophyll-binding protein BoWSCP

The exciton interaction of four chlorophyll a (Chl a) molecules in a symmetrical tetrameric complex of the water-soluble chlorophyll-binding protein BoWSCP was analyzed in the pH range of 3–11. Exciton splitting ΔE = 232 ± 2 cm−1 of the Qy band of Chl a into two subcomponents with relative intensities of 78.1 ± 0.7 % and 21.9 ± 0.7 % was determined by a joint decomposition of the absorption and circular dichroism spectra into Gaussian functions. The exciton coupling parameters were calculated based on the BoWSCP atomic structure in three approximations: the point dipole model, the distributed atomic monopoles, and direct ab initio calculations in the TDDFT/PCM approximation. The Coulomb interactions of monomers were calculated within the continuum model using three values of optical permittivity. The models based on the properties of free Chl a in solution suffer from significant errors both in estimating the absolute value of the exciton interaction and in the relative intensity of exciton transitions. Calculations within the TDDFT/PCM approximation reproduce the experimentally determined parameters of the exciton splitting and the relative intensities of the exciton bands. The following factors of pigment-protein and pigment–pigment interactions were examined: deviation of the macrocycle geometry from the planar conformation of free Chl; the formation of hydrogen bonds between the macrocycle and water molecules; the overlap of wave functions of monomers at close distances. The most significant factor is the geometrical deformation of the porphyrin macrocycle, which leads to an increase in the dipole moment of Chl monomer from 5.5 to 6.9 D and to a rotation of the dipole moment by 15° towards the cyclopentane ring. The contributions of resonant charge-transfer states to the wave functions of the Chl dimer were determined and the transition dipole moments of the symmetric and antisymmetric charge-transfer states were estimated.

Jamming crossovers in a confined driven polymer in solution.

We use lattice-Boltzmann molecular dynamics (LBMD) simulations to study the compression of a confined polymer immersed in a fluid and pushed by a large spherical colloid with a diameter comparable to the channel width. We examined the chain's deformation with both purely repulsive and weakly attractive Lennard-Jones (LJ) potentials applied between the monomers. The sphere's velocity was varied over 3 orders of magnitude. The chain is in a non-dense state at low pushing velocities for both repulsive and attractive monomer interactions. When the velocity of the spherical colloid exceeds a threshold v*, the back end of the chain transitions to a high density state with low mean square monomer displacement (MSD) values. The front end, however, remains in a non-dense state with high MSD indicating a pseudo two-state coexistence. This crossover is also revealed through volume per monomer and MSD as a function of the sphere's velocity. We also studied polymer dynamics by investigating folding events at different times.

Cyclization of Peptides Enhances the Inhibitory Activity against Ganglioside-Induced Aβ Fibril Formation.

Alzheimer's disease is a progressive neurodegenerative disease and is the most common cause of dementia. It has been reported that the assembly of amyloid β-protein (Aβ) on the cell membrane is induced by the interaction of the Aβ monomer with gangliosides such as GM1. The ganglioside-bound Aβ (GAβ) complex acts as a seed to promote the toxic assembly of the Aβ fibrils. In a previous study, we found that a GM1 cluster-binding peptide (GCBP) specifically recognizes Aβ-sensitive ganglioside nanoclusters and inhibits the assembly of Aβ on a GM1-containing lipid membrane. In this study, cysteine-substituted double mutants of GCBP were designed and cyclized by intramolecular disulfide bond formation. Affinity assays indicated that one of the cyclic peptides had a higher affinity to a GM1-containing membrane compared to that of GCBP. Furthermore, surface topography analysis indicated that this peptide recognizes GM1 nanoclusters on the lipid membrane. An evaluation of the inhibitory kinetics indicated that the cyclic peptide could inhibit the formation of Aβ fibrils with an IC50 value of 1.2 fM, which is 10,000-fold higher than that of GCBP. The cyclic peptide was also shown to have a clearance effect on Aβ fibrils deposited on the lipid membrane and suppressed the formation of toxic Aβ assemblies. Our results indicate that the cyclic peptide that binds to the Aβ-sensitive ganglioside nanocluster is a potential novel inhibitor of ganglioside-induced Aβ assembly.

Masoprocol: a promising candidate for targeting insulin resistance by inhibiting resistin with optimal druglikeness Potentials: an in silico approach

Resistin is a cysteine-rich secretory hormone that induces resistance to insulin, and its elevated expression is correlated with the onset of diabetes and several related metabolic disorders. Resistin performs its inhibitory role by connecting three identical subunits through Cys22-based disulfide linkages. The necessity to inhibit the formation of resistin trimer is one of the essential means to prevent the aggravation of diabetes mellitus type 2, obesity, and atherosclerosis. This study was conducted to screen the clinically approved drugs to find the most potent one to inhibit resistin with the best pharmacokinetics and drug-likeness properties. A total of 4654 clinically approved drugs were docked against the Cys22 residue of resistin. The top ten drugs with the highest high-precision (XP) docking scores were selected. Ioversol and masoprocol showed the highest XP docking and Molecular Mechanics-Generalized Born Surface Area (MMGBSA) scores, respectively, with double hydrogen bonding with the targeted Cys22. Molecular dynamics (MD) simulations showed that the masoprocol-resistin complex exhibited lower root mean square deviation (RMSD), radius of gyration, and root mean square fluctuation (RMSF) values than those observed in the ioversol-resistin complex. Both drugs induced drastic conformational changes in resistin monomer interactions. However, ioversol did not prove satisfying drug-likeness properties, while masoprocol showed the most favourable pharmacokinetic and drug-likeness properties. This study has demonstrated that masoprocol offers a novel inhibitory effect on resistin with the highest ligand affinity, making it a promising drug for combating insulin resistance. Communicated by Ramaswamy H. Sarma

A mechanistic study on homo- and copolymerization of L-Lactide and ε-caprolactone catalyzed by an aluminum complex bearing a Bis(phenoxy)amine ligand: A DFT study

Polymerization of biodegradable lactide and lactone has been the subject of intense research during the past decade. We used density functional theory (DFT) calculations to investigate the initiation and first propagation for ring-opening polymerization (ROP) mechanisms of L-lactide (LA) and ε-caprolactone (CL) on aluminum complexes bearing the bis(phenoxy)-amine ligand with pyridine (Catalyst I) and diethylamine (Catalyst II) sidearm. Here, the ROP mechanisms of LA and CL on both catalysts were classified into 3 parts: (1) LA homopolymerization, (2) CL homopolymerization, and (3) LA and CL copolymerization. For homopolymerization, Catalyst I shows a greater performance than Catalyst II on both LA and CL due to its less bulky sidearm. However, CL homopolymerization on Catalyst I is more favorable than that of LA, which is caused by the stronger CL coordination on the catalyst. For LA and CL copolymerization, the amine sidearm variation and the vdW interactions of monomers on Catalysts I and II provide different copolymer products, the tapered and random copolymers, respectively. These calculated results are in good agreement with our previous experimental reports. The understanding gained in the current study might be helpful in the development of high-performance catalysts for the ROP reaction of biodegradable lactide and lactone.

PAP(248-286) Conformational Changes during the Lag Phase of Amyloid Fibril Formation.

The initial stage of fibril formation of C-terminal region PAP(248-286) of human seminal plasma protein prostatic acid phosphatase was considered. Amyloid fibrils from the peptide PAP(248-286) are termed as a semen-derived enhancer of viral infection (SEVI) found in abundant quantities in semen. The kinetics of the amyloid fibril formation process consists of two characteristic phases (lag phase/nucleation phase and growth phase/elongation phase). The lag phase can be caused by the presence of mature amyloid fibrils (seeds) in protein solution, so-called secondary nucleation. The secondary nucleation includes interaction of protein monomers with the mature fibril surface that leads to protein spatial structural changes for further amyloid fibril formation. In this work, changes of the PAP(248-286) spatial structure were obtained during the secondary nucleation phase. Pulsed-field gradient (PFG) NMR was used to characterize the behavior of monomeric PAP(248-286) in water solution after PAP(248-286) seed addition. The self-diffusion coefficient showed compactization of the peptide monomer due to fibril-monomer interactions. PAP(248-286) spatial structural changes were detected with the help of high-resolution NMR spectroscopy and molecular dynamics (MD) simulation. The folding of PAP(248-286) occurs due to backbone chain bending in the region of H270 and T275 amino acid residues. Obtained folded conformation of PAP(248-286) emerging in the secondary nucleation process is energetically favorable and retains after monomer-amyloid interaction. The structural changes are associated with localization of PAP(248-286) hydrophobic surface regions, which are probably responsible for peptide monomer-amyloid interactions.

Effect of β-amyloid on blood-brain barrier properties and function.

The deposition of beta-amyloid (Aβ) aggregates in the brain, accompanied by impaired cognitive function, is a characteristic feature of Alzheimer's disease (AD). An important role in this process is played by vascular disorders, in particular, a disturbance of the blood-brain barrier (BBB). The BBB controls the entry of Aβ from plasma to the brain via the receptor for advanced glycation end products (RAGE) and the removal of brain-derived Aβ via the low-density lipoprotein receptor-related protein (LRP1). The balance between the input of Aβ to the brain from the periphery and its output is disturbed during AD. Aβ changes the redox-status of BBB cells, which in turn changes the functioning of mitochondria and disrupts the barrier function of endothelial cells by affecting tight junction proteins. Aβ oligomers have the greatest toxic effect on BBB cells, and oligomers are most rapidly transferred by transcytosis from the brain side of the BBB to the blood side. Both the cytotoxic effect of Aβ and the impairment of barrier function are partly due to the interaction of Aβ monomers and oligomers with membrane-bound RAGE. AD therapies based on the disruption of this interaction or the creation of decoys for Aβ are being developed. The question of the transfer of various Aβ isoforms through the BBB is important, since it can influence the development of AD. It is shown that the rate of input of Aβ40 and Aβ42 from the blood into the brain is different. The actual question of the transfer of pathogenic Aβ isoforms with post-translational modifications or mutations through the BBB still remains open.

The interaction of triglycidyl phosphate with europium nitrate and properties of obtained metal-containing polymer

The interaction of phosphorus-containing epoxy trifunctional monomer – triglycidyl phosphate (TGPhT), with europium (III) nitrate hexahydrate is studied. A dissolution of europium salt added into TGPhT is occurred due to the interaction of the cation with phosphoryl groups and oxygen atoms of the epoxy ring, that is accompanied by the opening reaction and led to the formation of a polymer. The interaction of triepoxide with salt is investigated by FTIR-spectroscopy and differential scanning calorimetry (DSC). The thermal stability of the samples is determined by combined method of thermogravimetry and differential scanning calorimetry (TG/DSC), coupled with a quadrupole mass spectrometry to identify the gaseous products evolved during the thermal analysis of the samples. The kinetic parameters of the curing reaction are computed by using Thermokinetics software. The interaction of TGPhT with europium nitrate is suggested to occur through opening of epoxy rings in the presence of europium ions via the mechanism of cationic polymerization. The cured epoxy polymer exhibits luminescent properties.

Boron Nitride Nanotubes: Force Field Parameterization, Epoxy Interactions, and Comparison with Carbon Nanotubes for High-Performance Composite Materials

Boron nitride nanotubes (BNNTs) are a very promising reinforcement for future high-performance composites because of their excellent thermo-mechanical properties. To take full advantage of BNNTs in composite materials, it is necessary to have a comprehensive understanding of the wetting characteristics of various high-performance resins. Molecular dynamics (MD) simulations provide an accurate and efficient approach to establish the contact angle values of engineering polymers on reinforcement surfaces, which offers a measure for the interaction between the polymer and reinforcement. In this research, MD simulations and experiments are used to determine the wettability of various epoxy systems on BNNT surfaces. The reactive interface force field (IFF-R) is parameterized and utilized in the simulations to accurately describe the interaction of the epoxy monomers with the BNNT surface. The effect of the epoxy monomer type, hardener type, local atomic charges, and temperature on the contact angle is established. The results show that contact angles decrease with increases in temperature for all the epoxy/hardener systems. The bisphenol-A-based epoxy system demonstrates better wettability with the BNNT surface than the bisphenol-F based epoxy system. Furthermore, the MD predictions demonstrate that these observations are validated with experimental results, wherein the same contact angle trends are observed for macroscopic epoxy drops on nonwoven nanotube papers. As wetting properties drive the resin infusion in the reinforcement materials, these results are important for the future manufacturing of high-quality BNNT/epoxy nanocomposites for high-performance applications such as aerospace and aeronautical vehicles.

Biochemical and Bioinformatic Studies of Mutations of Residues at the Monomer-Monomer Interface of Human Ornithine Aminotransferase Leading to Gyrate Atrophy of Choroid and Retina.

Deficit of human ornithine aminotransferase (hOAT), a mitochondrial tetrameric pyridoxal-5'-phosphate (PLP) enzyme, leads to gyrate atrophy of the choroid and retina (GA). Although 70 pathogenic mutations have been identified, only few enzymatic phenotypes are known. Here, we report biochemical and bioinformatic analyses of the G51D, G121D, R154L, Y158S, T181M, and P199Q pathogenic variants involving residues located at the monomer-monomer interface. All mutations cause a shift toward a dimeric structure, and changes in tertiary structure, thermal stability, and PLP microenvironment. The impact on these features is less pronounced for the mutations of Gly51 and Gly121 mapping to the N-terminal segment of the enzyme than those of Arg154, Tyr158, Thr181, and Pro199 belonging to the large domain. These data, together with the predicted ΔΔG values of monomer-monomer binding for the variants, suggest that the proper monomer-monomer interactions seem to be correlated with the thermal stability, the PLP binding site and the tetrameric structure of hOAT. The different impact of these mutations on the catalytic activity was also reported and discussed on the basis of the computational information. Together, these results allow the identification of the molecular defects of these variants, thus extending the knowledge of enzymatic phenotypes of GA patients.

Stimuli responsive hierarchical assembly of supramolecular conductive fibers from coiled-coil building blocks.

Proteins and peptides provide inspiration for synthetic stimuli-responsive nanomaterials. In this work, the assembly mechanism of a de novo designed peptide that forms α-helical coiled-coil hexamer fibers was probed through point mutations of the amino acid sequence and varying assembly solution pH. Buffer pH affects multi-scale aspects of supramolecular assembly, including peptide monomer secondary structure and interactions between α-helical coiled-coil hexamer building blocks. These higher order assembly interfaces are formed by electrostatic networks between solvent-exposed polarizable side chains, as observed by cryo-electron microscopy. One of the resulting hierarchical structures was a novel assembly of α-helical cross coiled-coils that exhibits electrical conductivity. Here, we show that pH-gated assembly can be achieved with only one amino acid mutation. This work opens materials design for more stimuli-responsive and functional materials with simple pH-gating structural motifs.

New protocol for kinetic assay seeding ability recovery "KASAR" from formalin-fixed paraffin-embedded tissues.

The real-time quaking-induced conversion (RT-QuIC) alpha-synuclein (aSyn) protein kinetic seeding assay has been very useful for detecting pathological aggregates in various synucleinopathies including Parkinson's disease (PD). This biomarker assay relies on fresh frozen tissue to effectively seed and amplify aSyn aggregating protein. With vast repositories of formalin-fixed paraffin-embedded (FFPE) tissues, it is paramount to harness the power of kinetic assays to unlock the diagnostic potential of archived FFPE biospecimens. However, the major challenge posed by significantly reduced amplification of formalin-fixed tissues in the assay suggests that formalin fixation deterred monomer interaction with the sample seed and depressed subsequent protein aggregation. To overcome this challenge, we developed a kinetic assay seeding ability recovery (KASAR) protocol to maintain the integrity of the tissue and seeding protein. For this, we implemented a series of heating steps with the brain tissue suspended in a buffer composed of 500mM tris-HCl (pH 7.5) and 0.02% SDS after the standard deparaffinization of the tissue sections. Initially, samples from seven human brain samples, including four samples from patients diagnosed with dementia with Lewy bodies (DLB) and three samples from healthy controls without DLB, were compared to fresh frozen samples under three different, but clinically common sample storage conditions: formalin-fixed, FFPE, and FFPE slices cut 5µm thick. The KASAR protocol was able to recover seeding activity for all positive samples in all storage conditions. Next, 28 FFPE samples from the submandibular gland (SMG) of patients diagnosed with PD, incidental Lewy body disease (ILBD), or healthy controls were tested with 93% of results replicating when blinded. With samples of only a few milligrams, this protocol recovered the same quality of seeding in formalin-fixed tissue as fresh frozen tissue. Moving forward, protein aggregate kinetic assays, in conjunction with the KASAR protocol, can be used to understand and diagnose neurodegenerative diseases more comprehensively. Overall, our KASAR protocol unlocks and restores the seeding ability of formalin-fixed paraffin-embedded tissues for the amplification of biomarker protein aggregates in kinetic assays.