Aqueous solution and solid-state behaviour of l-homophenylalanine: experiment, modelling, and DFT calculations.

In the present study, the solid-state and aqueous solubility behaviour of l-homophenylalanine (l-Hpa) is explored. Different characterization techniques such as TG, DSC, temperature-resolved PXRD, and hot-stage microscopy were used to investigate basic thermal solid-state characteristics. Solubilities of l-Hpa in water were determined as a function of temperature and pH. Moreover, a thermodynamic model based on perturbation theory (PC-SAFT) is applied to represent the data. In addition, aqueous density data of l-Hpa were measured in a broader temperature range. To model the solubility data as a function of pH, pKa values are needed, which were accessed by employing density functional theory (DFT) calculations. The solid-state investigation did not show a simple melting process of l-Hpa, but a complete decomposition of the prevalent initial solid phase at elevated temperatures approximately above 520 K. This system exhibited extraordinarily low solubilities for an amino acid at all investigated temperatures. While the solubility does not differ from its isoelectric-point value over a wide pH range, it dramatically increases as the pH falls below 2.5 and rises above 9.5. The PC-SAFT model was able to calculate the solubilities as a function of pH and predict the density values.

Pharmacological effects of methysticin and L-sulforaphane through the Nrf2/ARE signaling pathway in MLO-Y4 osteocytes: in vitro study.

BACKGROUND: Oxidative stress plays a crucial role in the pathogenesis of many skeletal diseases by inducing osteocyte death. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of various antioxidant gene expressions through antioxidant response element (ARE) against cellular oxidative stress and can be induced by various stimulants, including the phytochemicals methysticin (MET) and L-sulforaphane (SFN). This study aimed to establish an osteocyte in vitro model to investigate the pharmacological effects of MET and SFN on the Nrf2/ARE pathway. METHODS: MLO-Y4 murine osteocytes and the stably transduced MLO-Y4-SIN-lenti-ARE reporter gene cell line were used. MET and SFN were used as Nrf2 inducers. The cytotoxicity of MET, SFN, and hydrogen peroxide (H2O2) was evaluated using the CytoTox-Glo™ Assay. Time- and dose-dependent ARE induction was examined by Monoluciferase Assay. The mRNA and protein expressions of Nrf2 target markers, such as heme-oxygenase 1 (Ho-1), NADPH quinone dehydrogenase 1 (Nqo1), and thioredoxin reductase 1 (Txnrd1), were detected by RT-qPCR, Western Blot, and immunofluorescence staining, respectively. Osteogenesis markers, osteopontin, and osteocalcin were compared with and without treatment by immunofluorescence staining. RESULTS: The experimental data showed that MET and SFN induced ARE activity in a time- and dose-dependent manner and increased the mRNA and protein expression of antioxidant markers compared to vehicle-treated controls. The protein expression of osteopontin and osteocalcin in the samples treated with SFN were significantly higher than without treatment, and the number of cell death treated with SFN was significantly lower than without treatment under H2O2-induced stress conditions. CONCLUSIONS: Nrf2 inducers MET and SFN increased the mRNA expression of antioxidant genes through the Nrf2/ARE pathway in osteocytes. Notably, SFN increased the protein expression of osteocyte-associated osteogenic markers and suppressed cell death under H2O2-induced stress condition. Thus, Nrf2 stimulators can exert stress-relieving and osteogenic effects on osteocytes.

Heteroelements in polyoxometalates: a study on the influence of different group 15 elements on polyoxometalate formation.

In the field of polyoxometalate (POM) chemistry, different heteroelements are integrated into the cage-like structures, to obtain different structural types of so-called heteropolyanions (HPAs). While it is generally accepted, that some elements favor certain types of structure, a systematic study is still missing. In this article, we present a systematic investigation of the influence of the group 15 elements nitrogen, phosphorous, arsenic, and antimony on the formation of different POM structure types. Our study is comprised of DFT calculations and corresponding experimental structural analysis. In this context, the DFT study establishes the thermodynamics of formation of different coordination geometries with various heteroelements on two POM structure types, the Keggin and the Anderson-Evans structures. Our POM synthesis experiments were performed at two different pH values (1 and 5) and resulted in a variety of heteropolytungstates, which were identified and characterized by elemental analysis as well as single crystal X-ray diffraction and vibrational spectroscopy. With these methods, we were able to establish a clear trend, showing that heavier elements lead to formation of different structure types than lighter elements. These results signify a large step towards a better understanding of POM formation specifically with respect to the choice of heteroelement.

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications-Updated and completely revised edition.

This review is in support of the development of selective, precise, fast, and validated capillary electrophoresis (CE) methods. It follows up a similar article from 1998, Wätzig H, Degenhardt M, Kunkel A. "Strategies for capillary electrophoresis: method development and validation for pharmaceutical and biological applications," pointing out which fundamentals are still valid and at the same time showing the enormous achievements in the last 25 years. The structures of both reviews are widely similar, in order to facilitate their simultaneous use. Focusing on pharmaceutical and biological applications, the successful use of CE is now demonstrated by more than 600 carefully selected references. Many of those are recent reviews; therefore, a significant overview about the field is provided. There are extra sections about sample pretreatment related to CE and microchip CE, and a completely revised section about method development for protein analytes and biomolecules in general. The general strategies for method development are summed up with regard to selectivity, efficiency, precision, analysis time, limit of detection, sample pretreatment requirements, and validation.

Experimentally Observed Conformational Changes in Antibodies Due to Binding and Paratope-epitope Asymmetries.

Understanding binding related changes in antibody conformations is important for epitope prediction and antibody refinement. The increase of available data in the PDB allowed a more detailed investigation of the conformational landscape for free and bound antibodies. A dataset containing a total of 835 unique PDB entries of antibodies that were crystallized in complex with their antigen and in a free state was constructed. It was examined for binding related conformation changes. We present further evidence supporting the theory of a pre-existing-equilibrium in experimental data. Multiple sequence alignments did not show binding induced tendencies in the solvent accessibility of residues in any specific position. Evaluating the changes in solvent accessibility per residue revealed a certain binding induced increase for several amino acids. Antibody-antigen interaction statistics were established and quantify a significant directional asymmetry between many interacting antibody and antigen residue pairs, especially a richness in tyrosine in the antibody epitope compared to its paratope. This asymmetry could potentially facilitate an increase in the success rate of computationally guided antibody refinement.

Bilayer lipid membrane formation on surface assemblies with sparsely distributed tethers.

A combined computational and experimental study of small unilamellar vesicle (SUV) fusion on mixed self-assembled monolayers (SAMs) terminated with different deuterated tether moieties (-(CD2)7CD3 or -(CD2)15CD3) is reported. Tethered bilayer lipid membrane (tBLM) formation of synthetic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine was initially probed on SAMs with controlled tether (d-alkyl tail) surface densities and lateral molecular packing using quartz crystal microbalance with dissipation monitoring (QCM-D). Long time-scale coarse-grained molecular dynamics (MD) simulations were then employed to elucidate the mechanisms behind the interaction between the SUVs and the different phases formed by the -(CD2)7CD3 and -(CD2)15CD3 tethers. Furthermore, a series of real time kinetics was recorded under different osmotic conditions using QCM-D to determine the accumulated lipid mass and for probing the fusion process. It is shown that the key factors driving the SUV fusion and tBLM formation on this type of surfaces involve tether insertion into the SUVs along with vesicle deformation. It is also evident that surface densities of the tethers as small as a few mol% are sufficient to obtain stable tBLMs with a high reproducibility. The described "sparsely tethered" tBLM system can be advantageous in studying different biophysical phenomena, such as membrane protein insertion, effects of receptor clustering, and raft formation.

The Fully Oxidized State of the Glutamate Coordinated O2-Tolerant [NiFe]-Hydrogenase Shows a Ni(III)/Fe(III) Open-Shell Singlet Ground State.

The oxygen tolerance of the [NiFe]-hydrogenase from H. thermoluteolus was recently assigned to originate from an unusual coordination sphere of the active site nickel atom (Shomura et al. Science 2017, 357, 928-932, 10.1126/science.aan4497). In the oxidized state, a terminal cysteine residue is displaced by a bidentate coordinating nearby Glu32 and thus moves to occupy a third µ-cysteine bridging position. Spectral features of the oxidized state were assigned to originate from a closed-shell Ni(IV)/Fe(II) state (Kulka-Peschke et al. J. Am. Chem. Soc. 2022, 144, 17022-17032, 10.1021/jacs.2c06400). Such a high-valent nickel oxidation state is unprecedented in biological systems. The spectral properties and the coordination sphere of that [NiFe]-hydrogenase can, however, also be rationalized by an energetically lower broken-symmetry Ni(III)/Fe(III) state of the active site which was not considered. In this open-shell singlet, the ligand-mediated antiferromagnetic spin-coupling leads to an overall S = 0 spin state with evenly distributed spin densities over the metal atoms. Experiments are suggested that may clarify the final assignment of redox states.

Structural Dynamics of Lys11-Selective Deubiquitinylase Cezanne-1 during the Catalytic Cycle.

Deubiquitinylating enzymes (DUBs) regulate the deubiquitinylation process of post-translationally modified proteins and thus control protein signaling in various cellular processes. The DUB Cezanne-1 catalyzes the cleavage of the iso-peptide bond of Lys11-linked polyubiquitin chains with high selectivity. Crystal structures of Cezanne-1 in different states provide important insight regarding the complex formation and global changes during the catalytic cycle but are lacking details of dynamics and control of activation. Activity-based probes are used to isolate intermediate states upon forming covalent bonds with the DUB active site. Those, however, may lead to structures that are non-native. Conformational changes of Cezanne-1, during its process of activation and proteolytic activity, are investigated using all-atom molecular dynamics (MD) simulations of the ubiquitin-free, diubiquitin-bound, and monoubiquitin-bound Cezanne-1 DUB for a total of ∼18 µs. Our results show that ubiquitin-free Cezanne-1 dynamically shuttles between catalytically competent and incompetent states which suggests that its activation is independent of substrate binding. The catalytically competent substrate-free Cezanne-1 promotes distal ubiquitin substrate access to the catalytic center. The subsequent binding of the proximal ubiquitin shifts the equilibrium toward the catalytically competent state of the dyad, thereby promoting proteolysis of the iso-peptide bond. After cleavage of the scissile bond, sequential dissociation of first the proximal ubiquitin induces the inactivation of Cezanne-1. The subsequent release of the distal ubiquitin fully reconstitutes the inactive substrate-free state of Cezanne-1. The process of activation and catalytic turnover of DUB Cezanne-1 is a multistage cycle with several critical dynamic transitions that cannot be characterized based on protein structures alone. Activity-based probes of cysteine proteases lead to non-native protein-protein contacts, which need to be resolved in order to be able to issue statements about physiological states and substrate binding.

Conformational Control of Fast Asparagine Deamidation in a Norovirus Capsid Protein.

Accelerated spontaneous deamidation of asparagine 373 and subsequent conversion into an isoaspartate has been shown to attenuate the binding of histo blood group antigens (HBGAs) to the protruding domain (P-domain) of the capsid protein of a prevalent norovirus strain (GII.4). Here, we link an unusual backbone conformation of asparagine 373 to its fast site-specific deamidation. NMR spectroscopy and ion exchange chromatography have been used to monitor the deamidation reaction of P-domains of two closely related GII.4 norovirus strains, specific point mutants, and control peptides. MD simulations over several microseconds have been instrumental to rationalize the experimental findings. While conventional descriptors such as available surface area, root-mean-square fluctuations, or nucleophilic attack distance fail as explanations, the population of a rare syn-backbone conformation distinguishes asparagine 373 from all other asparagine residues. We suggest that stabilization of this unusual conformation enhances the nucleophilicity of the backbone nitrogen of aspartate 374, in turn accelerating the deamidation of asparagine 373. This finding should be relevant to the development of reliable prediction algorithms for sites of rapid asparagine deamidation in proteins.

Impact of N-heteroaromatic N-termini in Cu(II) ATCUN metallopeptides on their biorelevant redox activity.

Cu(II) complexes with ATCUN peptide ligands have been investigated for their ROS (reactive oxygen species) generation and oxidative DNA degradation abilities. The biological activity of most ATCUN complexes such as Cu-GGH (Gly-Gly-His) is, however, low. Tuning the redox chemistry by incorporation of N-heteroaromatics reinstates ROS production which leads to efficient DNA cleavage.

A benchmark for non-covalent interactions in organometallic crystals.

Organometallic complexes are the basis for homogeneous catalysis, have applications in materials science and are also active pharmaceutical ingredients. The interaction between transition metal complexes in the solid state is determining their thermodynamics and bio-availability. Non-covalent interactions such as hydrogen bonding and van der Waals are stabilizing crystals of transition metal complexes. The variation of ligand field, central metal atoms and their oxidation and spin states are determinants of the magnitude of their inter-molecular interactions. A comparison of a set of 43 manually curated experimental heats of sublimation (the new XTMC43 set) and results from periodic DFT calculations shows that an agreement to within 9% can be achieved using GGA or mGGA functionals with atom-centred Gaussian-type basis functions. The need for careful assessments of consistency, calibration and reproducibility of experimental and computational data is discussed. Results regarding the new XTMC43 benchmark set are suggested to serve as a starting point for further method development, systematic screening and crystal engineering.

Mononuclear manganese complexes as hydrogen evolving catalysts.

Molecular hydrogen (H2) is one of the pillars of future non-fossil energy supply. In the quest for alternative, non-precious metal catalysts for hydrogen generation to replace platinum, biological systems such as the enzyme hydrogenase serve as a blueprint. By taking inspiration from the bio-system, mostly nickel- or iron-based catalysts were explored so far. Manganese is a known oxygen-reducing catalyst but has received much less attention for its ability to reduce protons in acidic media. Here, the synthesis, characterization, and reaction mechanisms of a series of four mono-nuclear Mn(I) complexes in terms of their catalytic performance are reported. The effect of the variation of equatorial and axial ligands in their first and second coordination spheres was assessed pertaining to their control of the turnover frequencies and overpotentials. All four complexes show reactivity and reduce protons in acidic media to release molecular hydrogen H2. Quantum chemical studies were able to assign and interpret spectral characterizations from UV-Vis and electrochemistry and rationalize the reaction mechanism. Two feasible reaction mechanisms of electrochemical (E) and protonation (C) steps were compared. Quantum chemical studies can assign peaks in the cyclic voltammetry to structural changes of the complex during the reaction. The first one-electron reduction is essential to generate an open ligand-based site for protonation. The distorted octahedral Mn complexes possess an inverted second one-electron redox potential which is a pre-requisite for a swift and facile release of molecular hydrogen. This series on manganese catalysts extends the range of elements of the periodic table which are able to catalyze the hydrogen evolution reaction and will be explored further.

Comparison of mobility shift affinity capillary electrophoresis and capillary electrophoresis frontal analysis for binding constant determination between human serum albumin and small drugs.

In this study, two capillary electrophoresis-based ligand binding assays, namely, mobility shift affinity capillary electrophoresis (ms-ACE) and capillary electrophoresis-frontal analysis (CE-FA), were applied to determine binding parameters of human serum albumin toward small drugs under similar experimental conditions. The substances S-amlodipine (S-AML), lidocaine (LDC), l-tryptophan (l-TRP), carbamazepine (CBZ), ibuprofen (IBU), and R-verapamil (R-VPM) were used as the main binding partners. The scope of this comparative study was to estimate and compare both the assays in terms of their primary measure's precision and the reproducibility of the derived binding parameters. The effective mobility could be measured with pooled CV values between 0.55% and 7.6%. The precision of the r values was found in the range between 1.5% and 10%. Both assays were not universally applicable. The CE-FA assay could successfully be applied to measure the drugs IBU, CBZ, and LDC, and the interaction toward CBZ, S-AML, l-TRP, and R-VPM could be determined using ms-ACE. The average variabilities of the estimated binding constants were 64% and 67% for CE-FA and ms-ACE, respectively.

Quality by Design Assisted Optimization of a Chiral Capillary Electrokinetic Chromatographic Method for the Separation of Amlodipine Enantiomers Using Maltodextrin as Chiral Selector.

Analytical-method development based on design of experiment has been applied for optimizing the enantioseparation of amlodipine by chiral capillary electrokinetic chromatography using maltodextrin as the chiral selector. The effect of different factors on the enantioresolution quality was screened. Three separation factors, namely maltodextrin concentration, pH of the background electrolyte and applied voltage were selected as independent variables. The number of experiments was reduced while maximizing the information content using design of experiment. Based on a full-quadratic design that included three variables on three levels, the total design space could be reduced to fifteen factor combinations using a D-optimal algorithm. The aim of the experiment was to find the optimal factor combinations with respect to resolution. The maltodextrin concentration (7.5-10% w/v) demonstrated the strongest effect on the resolution followed by pH (2-4) of the background electrolyte and the applied voltage (15-20 kV). An increase in the maltodextrin concentration was found to result in a greater stereoselectivity, represented by the higher resolution values (Rs ≥ 1.5). The separation conditions in the proposed method were feasible to be adjusted within the applied range with an acceptable resolution.

Mechanism of Diiron Hydrogenase Complexes Controlled by Nature of Bridging Dithiolate Ligand.

Bio-inorganic complexes inspired by hydrogenase enzymes are designed to catalyze the hydrogen evolution reaction (HER). A series of new diiron hydrogenase mimic complexes with one or two terminal tris(4-methoxyphenyl)phosphine and different µ-bridging dithiolate ligands and show catalytic activity towards electrochemical proton reduction in the presence of weak and strong acids. A series of propane- and benzene-dithiolato-bridged complexes was synthesized, crystallized, and characterized by various spectroscopic techniques and quantum chemical calculations. Their electrochemical properties as well as the detailed reaction mechanisms of the HER are elucidated by density functional theory (DFT) methods. The nature of the µ-bridging dithiolate is critically controlling the reaction and performance of the HER of the complexes. In contrast, terminal phosphine ligands have no significant effects on redox activities and mechanism. Mono- or di-substituted propane-dithiolate complexes afford a sequential reduction (electrochemical; E) and protonation (chemical; C) mechanism (ECEC), while the µ-benzene dithiolate complexes follow a different reaction mechanism and are more efficient HER catalysts.

Ovarian tumor domain proteases in pathogen infection.

With the aim of overcoming host immune responses, and to permit persistence, numerous bacterial and viral pathogens have evolved effective strategies to control the activity of ovarian tumor domain proteases (OTUs), a group of deubiquitinylases crucial for regulating ubiquitin-modified proteins. Due to the important role of eukaryotic OTUs in cellular physiology, it is not surprising that pathogens have evolutionarily developed effector proteins which mimic host OTUs. Here, we focus on recent findings that illustrate how pathogen-encoded OTUs modulate eukaryotic host proteins and how they are implicated in cellular dysregulation. Further, we discuss the biological effects of OTUs in the context of structural features and pharmacological targeting. We point out the potentiality of selective OTU inhibitors, which shield ubiquitin-binding sites, as pharmacologic targets to treat harmful infections.

Substrate-assisted activation and selectivity of the bacterial RavD effector deubiquitinylase.

Deubiquitinylases (DUBs) catalyze the peptide bond cleavage of specific ubiquitin linkages at distinct protein substrates. Pathogens from viruses and bacteria independently developed effector proteins with DUB activity to mimic host DUB functions and circumvent immune responses. The effector protein RavD from Legionella pneumophila cleaves linear ubiquitin chains with an exclusive methionine-1 selectivity. It thus performs as a functional analogue of the human DUB OTULIN, which achieves its selectivity only via a specialized proximal ubiquitin S1' binding site as well as a substrate-assisted activation of the catalytic triad. An analysis of the crystal structures of bacterial RavD in its free and di-ubiquitin-bound forms, in order to rationalize the structural basis for its selectivity and activation mechanism, is not fully conclusive. As these ambiguities might arise from the introduced double mutation of the di-ubiquitin substrate in the RavD-di-ubiquitin complex crystal structure, biomolecular modeling, and molecular dynamics sampling (1-2 µs for each system of RavD and OTULIN) were employed to reconstitute the physiological RavD-di-ubiquitin complex. The simulations show that the distal S1 ubiquitin binding sites of RavD and OTULIN are similar in terms of interface area, composition, and ubiquitin binding affinity. The proximal S1' site of RavD, in contrast, is significantly smaller and ubiquitin binding is weaker and more flexible than in OTULIN. Upon substrate access, the residues of the catalytic triad of RavD show a reduction of flexibility and a conformational transition toward a catalytically active state. Thus, the enzymatic activation of RavD is presumably also substrate-assisted and a clear rationale for the common M1-substrate selectivity.

Incorporation of ß-Alanine in Cu(II) ATCUN Peptide Complexes Increases ROS Levels, DNA Cleavage and Antiproliferative Activity.

Redox-active Cu(II) complexes are able to form reactive oxygen species (ROS) in the presence of oxygen and reducing agents. Recently, Faller et al. reported that ROS generation by Cu(II) ATCUN complexes is not as high as assumed for decades. High complex stability results in silencing of the Cu(II)/Cu(I) redox cycle and therefore leads to low ROS generation. In this work, we demonstrate that an exchange of the α-amino acid Gly with the ß-amino acid ß-Ala at position 2 (Gly2→ß-Ala2) of the ATCUN motif reinstates ROS production (• OH and H2 O2 ). Potentiometry, cyclic voltammetry, EPR spectroscopy and DFT simulations were utilized to explain the increased ROS generation of these ß-Ala2-containing ATCUN complexes. We also observed enhanced oxidative cleavage activity towards plasmid DNA for ß-Ala2 compared to the Gly2 complexes. Modifications with positively charged Lys residues increased the DNA affinity through electrostatic interactions as determined by UV/VIS, fluorescence, and CD spectroscopy, and consequently led to a further increase in nuclease activity. A similar trend was observed regarding the cytotoxic activity of the complexes against several human cancer cell lines where ß-Ala2 peptide complexes had lower IC50 values compared to Gly2. The higher cytotoxicity could be attributed to an increased cellular uptake as determined by ICP-MS measurements.