Exploring the Higher Order Structure and Conformational Transitions in Insulin Microcrystalline Biopharmaceuticals by Proton-Detected Solid-State Nuclear Magnetic Resonance at Natural Abundance.

The integrity of a higher order structure (HOS) is an essential requirement to ensure the efficacy, stability, and safety of protein therapeutics. Solution-state nuclear magnetic resonance (NMR) occupies a unique niche as one of the most promising methods to access atomic-level structural information on soluble biopharmaceutical formulations. Another major class of drugs is poorly soluble, such as microcrystalline suspensions, which poses significant challenges for the characterization of the active ingredient in its native state. Here, we have demonstrated a solid-state NMR method for HOS characterization of biopharmaceutical suspensions employing a selective excitation scheme under fast magic angle spinning (MAS). The applicability of the method is shown on commercial insulin suspensions at natural isotopic abundance. Selective excitation aided with proton detection and non-uniform sampling (NUS) provides improved sensitivity and resolution. The enhanced resolution enabled us to demonstrate the first experimental evidence of a phenol-escaping pathway in insulin, leading to conformational transitions to different hexameric states. This approach has the potential to serve as a valuable means for meticulously examining microcrystalline biopharmaceutical suspensions, which was previously not attainable in their native formulation states and can be seamlessly extended to other classes of biopharmaceuticals such as mAbs and other microcrystalline proteins.

Combined Liquid-State and Solid-State Nuclear Magnetic Resonance at Natural Abundance for Comparative Higher Order Structure Assessment in the Formulated-State of Biphasic Biopharmaceutics.

A higher-order structure (HOS) is critical to a biopharmaceutical drug as the three-dimensional structure governs its function. Even the partial perturbation in the HOS of the drug can alter the biological efficiency and efficacy. Due to current limitations in analytical technologies, it is imperative to develop a protocol to characterize the HOS of biopharmaceuticals in the native formulated state. This becomes even more challenging for the suspension formulations where solution and solid phases co-exist. Here, we have used a combinatorial approach using liquid (1D 1H) and solid-state (13C CP MAS) NMR methodology to demonstrate the HOS in the biphasic microcrystalline suspension drug in its formulated state. The data were further assessed by principal component analysis and Mahalanobis distance (DM) calculation for quantitative assessment. This approach is sufficient to provide information regarding the protein HOS and the local dynamics of the molecule when combined with orthogonal techniques such as X-ray scattering. Our method can be an elegant tool to investigate batch-to-batch variation in the process of manufacture and storage as well as a biosimilarity comparison study for biphasic/microcrystalline suspension.

Conjugates of ibuprofen inhibit CHIKV infection and inflammation.

Chikungunya virus infection has become a global health concern because of its high rates of morbidity and mortality in patients with preexisting conditions. Inflammation and arthritis are the major symptoms of CHIKV that persist even after clearance of CHIKV. To develop an antiviral that can reduce infection and manage inflammation independent of the CHIKV infection, ibuprofen (IBU) conjugates with sulfonamide and thiosemicarbazide were synthesized. The conjugates, IBU-SULFA, IBU-ISS and IBU-IBT significantly inhibited CHIKV infection in vitro with a selectivity index (CC50/IC50) of > 11.9, > 25.1 and > 21, respectively. The reduction in infection was attributed to the interference of the conjugates in the early stages of CHIKV life cycle. With no acute oral toxicity, these compounds significantly reduced inflammation and arthritis in rats. Unlike IBU, the conjugates were not ulcerogenic. In conclusion, the conjugation imparted anti-CHIKV properties while retaining the anti-inflammatory properties of IBU. These findings can encourage further validation and research to develop an antiviral for CHIKV to manage both infection and arthritis.

Methodologies for Operando ATR-IR Spectroscopy of Magnesium Battery Electrolytes.

We explore the suitability of operando attenuated total reflection infrared (ATR-IR) spectroscopy methodologies for the study of organoaluminate electrolytes for Mg battery applications. The "all-phenyl complex" in tetrahydrofuran (THF), with the molecular structure [Mg2Cl3·6THF]+[AlPh4]-, is used as an exemplar electrolyte to compare two different spectroelectrochemical cell configurations. In one case, a Pt gauze is used as a working electrode, while in the second case, a thin (∼10 nm) Pt film working electrode is deposited directly on the surface of the ATR crystal. Spectroscopic measurements indicate substantial differences in the ATR-IR response for the two configurations, reflecting the different spatial arrangements of the working electrode with respect to the ATR sampling volume. The relative merits and potential pitfalls associated with the two approaches are discussed.

Atom-efficient synthesis of a benchmark electrolyte for magnesium battery applications.

The benchmark magnesium electrolyte, [Mg2Cl3]+ [AlPh4]-, can be prepared in a 100% atom-economic fashion by a ligand exchange reaction between AlCl3 and two molar equivalents of MgPh2. NMR and vibrational spectroscopy indicate that the reported approach results in a simpler ionic composition than the more widely adopted synthesis route of combining PhMgCl with AlCl3. Electrochemical performance has been validated by polarisation tests and cyclic voltammetry, which demonstrate excellent stability of electrolytes produced via this atom-efficient approach.

Identification of suitable reference genes for normalization of quantitative real-time PCR-based gene expression in chicken (Gallus gallus).

The recent availability of genome information greatly facilitates the fundamental research on chicken. In different organs, gene expression patterns can provide clues to understanding the biological functions. For rapid and accurate quantification of gene expression, quantitative real-time PCR (qPCR) has become one of the most widely used methods. However, the success of qPCR data normalization depends on the use of a suitable reference gene and a single reference gene is not universally suitable for all the experiments. Therefore, reference gene validation is a crucial step for different organ tissues of chicken where suitable reference genes for qPCR analysis in varieties of tissues have not been investigated exhaustively so far. In this study, we have selected 30 Gallus gallus candidate reference genes from NCBI, amplified and studied their expression profiles by qPCR in different organ tissues (breast muscle, thigh muscle, heart, liver, spleen, gizzard, and bursa) of chicken. The result showed that, for breast muscle HSP10 and RPL23, thigh muscle RPL14 and RPL13, liver ALB and HSP70, spleen ALB and GAPDH, heart CYCS and TUBA8B, gizzard RPL5 and 18S rRNA, and bursa EEF1A1 and PGK2 are most stable genes respectively. The results also showed that for different organ tissues, individual or a combination of reference genes should be selected for data normalization. In this study, we have identified and validated 30 reference genes in seven different organ tissues to provide accurate transcript normalization and quantification, which can be useful for gene expression studies in other avian species.

Probing Watson-Crick and Hoogsteen base pairing in duplex DNA using dynamic nuclear polarization solid-state NMR spectroscopy.

The majority of base pairs in double-stranded DNA exist in the canonical Watson-Crick geometry. However, they can also adopt alternate Hoogsteen conformations in various complexes of DNA with proteins and small molecules, which are key for biological function and mechanism. While detection of Hoogsteen base pairs in large DNA complexes and assemblies poses considerable challenges for traditional structural biology techniques, we show here that multidimensional dynamic nuclear polarization-enhanced solid-state NMR can serve as a unique spectroscopic tool for observing and distinguishing Watson-Crick and Hoogsteen base pairs in a broad range of DNA systems based on characteristic NMR chemical shifts and internuclear dipolar couplings. We illustrate this approach using a model 12-mer DNA duplex, free and in complex with the antibiotic echinomycin, which features two central adenine-thymine base pairs with Watson-Crick and Hoogsteen geometry, respectively, and subsequently extend it to the ∼200 kDa Widom 601 DNA nucleosome core particle.

Transition Metal Phthalocyanines as Redox Mediators in Li-O2 Batteries: A Combined Experimental and Theoretical Study of the Influence of 3d Electrons in Redox Mediation.

Redox mediation is an innovative strategy for ensuring efficient energy harvesting from metal-oxygen systems. This work presents a systematic exploratory analysis of first-row transition-metal phthalocyanines as solution-state redox mediators for lithium-oxygen batteries. Our findings, based on experiment and theory, convincingly demonstrate that d5 (Mn), d7 (Co), and d8 (Ni) configurations function better compared to d6 (Fe) and d9 (Cu) in redox mediation of the discharge step. The d10 configuration (Zn) and non-d analogues (Mg) do not show any redox mediation because of the inability of binding with oxygen. The solution-state discharge product, transition-metal bound Li2O2, undergoes dissociation and oxidation in the charging step of the battery, thus confirming a bifunctional redox mediation. Apart from the reaction pathways predicted based on thermodynamic considerations, density functional theory calculations also reveal interesting effects of electrochemical perturbation on the redox mediation mechanisms and the role of the transition-metal center.

Generation of specialized blood vessels via lymphatic transdifferentiation.

The lineage and developmental trajectory of a cell are key determinants of cellular identity. In the vascular system, endothelial cells (ECs) of blood and lymphatic vessels differentiate and specialize to cater to the unique physiological demands of each organ1,2. Although lymphatic vessels were shown to derive from multiple cellular origins, lymphatic ECs (LECs) are not known to generate other cell types3,4. Here we use recurrent imaging and lineage-tracing of ECs in zebrafish anal fins, from early development to adulthood, to uncover a mechanism of specialized blood vessel formation through the transdifferentiation of LECs. Moreover, we demonstrate that deriving anal-fin vessels from lymphatic versus blood ECs results in functional differences in the adult organism, uncovering a link between cell ontogeny and functionality. We further use single-cell RNA-sequencing analysis to characterize the different cellular populations and transition states involved in the transdifferentiation process. Finally, we show that, similar to normal development, the vasculature is rederived from lymphatics during anal-fin regeneration, demonstrating that LECs in adult fish retain both potency and plasticity for generating blood ECs. Overall, our research highlights an innate mechanism of blood vessel formation through LEC transdifferentiation, and provides in vivo evidence for a link between cell ontogeny and functionality in ECs.

Conformational Dynamics of Histone H3 Tails in Chromatin.

Chromatin is a supramolecular DNA-protein complex that compacts eukaryotic genomes and regulates their accessibility and functions. Dynamically disordered histone H3 N-terminal tails are among key chromatin regulatory components. Here, we used high-resolution-magic-angle-spinning NMR measurements of backbone amide 15N spin relaxation rates to investigate, with residue-specific detail, the dynamics and interactions of H3 tails in recombinant 13C,15N-enriched nucleosome arrays containing 15, 30, or 60 bp linker DNA between the nucleosome repeats. These measurements were compared to analogous data available for mononucleosomes devoid of linker DNA or containing two 20 bp DNA overhangs. The H3 tail dynamics in nucleosome arrays were found to be considerably attenuated compared with nucleosomes with or without linker DNA due to transient electrostatic interactions with the linker DNA segments and the structured chromatin environment. Remarkably, however, the H3 tail dynamics were not modulated by the specific linker DNA length within the 15-60 bp range investigated here.

Chirp pulse sequences for broadband π rotation.

In this paper, we present various chirp pulse sequences for implementing a broadband π rotation, which can serve as an ideal refocusing pulse element in a spin echo pulse sequence. These sequences are composed of three pulse elements each of which do adiabatic passage at rate a or 2a in either backward or forward direction. Various possible combinations are considered and different variants are presented. They all implement a broadband π rotation. We present the theory of such composite chirp sequences along with simulations and experiments.

Structure-Redox Response Correlation in a Few Select Heme Systems Using X-ray Absorption Spectroelectrochemistry.

Heme based biomolecules control some of the most crucial life processes, such as oxygen and electron transport during respiration and energy metabolism, respectively. The active site of the heme, viz., the metal center, plays a key role and attributes functionality to these biomolecules. During the oxygen binding and debinding processes, it is important to note that the oxidation state of iron in hemoglobin (+II in the native form) does not undergo any change. However, the spin states of the metal center change. We present here a comprehensive study of the redox response of such molecules, based on the electronic structure of the active site. The local electronic structure of heme in a few selective molecular systems is studied in operando via synchrotron X-ray absorption spectroscopy (Fe K-edge) and cyclic voltammetry. Our objective is to identify the electronic structural parameters that can effectively be correlated with the redox reversibility. Evolution in these parameters can be followed to trace the overall changes in redox state of the system. Our data indicate that axial coordination and spin state of the iron center are two such parameters that are intimately connected with the redox response.

Histone H4 Tails in Nucleosomes: a Fuzzy Interaction with DNA.

The interaction of positively charged N-terminal histone tails with nucleosomal DNA plays an important role in chromatin assembly and regulation, modulating their susceptibility to post-translational modifications and recognition by chromatin-binding proteins. Here, we report residue-specific 15 N NMR relaxation rates for histone H4 tails in reconstituted nucleosomes. These data indicate that H4 tails are strongly dynamically disordered, albeit with reduced conformational flexibility compared to a free peptide with the same sequence. Remarkably, the NMR observables were successfully reproduced in a 2-µs MD trajectory of the nucleosome. This is an important step toward resolving an apparent inconsistency where prior simulations were generally at odds with experimental evidence on conformational dynamics of histone tails. Our findings indicate that histone H4 tails engage in a fuzzy interaction with nucleosomal DNA, underpinned by a variable pattern of short-lived salt bridges and hydrogen bonds, which persists at low ionic strength (0-100 mM NaCl).

Hemoglobin Dynamics in Solution vis-à-vis Under Confinement: An Electrochemical Perspective.

Confining heme protein in silico often leads to beneficial functionalities such as an enhanced electrochemical response from the heme center. This can be harnessed to design effective biosensors for medical diagnostics. Proteins under confinement, surface confinement on the electrode to be precise, have more ordered and monodisperse structure compared to the protein in bulk solution. As the electrochemical response of a protein comes from those protein molecules that are confined within the electrical double layer across the electrode-electrolyte interface, it is expected that restriction of conformational fluctuations of the polymeric protein will help in enhancement of the electrochemical response. This is probably the prima facie reason for electrochemical response enhancement under confinement. We examine the dynamic features of hemoglobin under confinement vis-à-vis that in bulk solution. We use a variety of spectroscopic techniques across a wide time-space window to establish the following facts: (a) hardening of the protein polypeptide backbone, (b) slowing down of protein diffusion, (c) increase in relaxation times in NMR, and (d) slowing down of dielectric relaxation times under confinement. This indicates an overall quenching of protein dynamics when the protein is confined inside silica matrix. Thus, we hypothesize that along with retention of secondary structure, this quenching of dynamics contributes to the enhancement of electrochemical response observed.

Studying Hemoglobin and a Bare Metal-Porphyrin Complex Immobilized on Functionalized Silicon Surfaces Using Synchrotron X-ray Reflectivity.

We evaluate here, using synchrotron X-ray reflectivity, hemoglobin adsorption characteristics on silicon substrates with varying chemical functionalities. Hemoglobin at isoelectronic point and at negative charge is immobilized on functionalized hydrophilic (hydroxyl, carboxylic, amine) and hydrophobic (alkylated) silicon surfaces for the study. As a control, the bare cofactor hemin (containing only the metal and porphyrin with no amino acid residues) is also studied under similar conditions. Ordered layers (grown using the Langmuir-Blodgett technique) are observed to be less affected by the surface chemistry compared to the multilayers formed by physical absorption. Surface chemistry and charge of the proteins are critical in controlling the protein adsorption characteristics on silicon, such as thickness (correlated to molecule size) and roughness. In this study, this is very well realized by varying both the hydrophobicity and hydrophilicity of the substrate. The fundamental studies discussed here provide us with a set of important guidelines as to how electrode surface functionalization can control molecular conformation/orientation, especially protein adsorption on the substrate. This in turn is expected to have a significant impact on the protein electrochemical function and response of biomolecular devices.

Using porphyrin-amino acid pairs to model the electrochemistry of heme proteins: experimental and theoretical investigations.

Quasi reversibility in electrochemical cycling between different oxidation states of iron is an often seen characteristic of iron containing heme proteins that bind dioxygen. Surprisingly, the system becomes fully reversible in the bare iron-porphyrin complex: hemin. This leads to the speculation that the polypeptide bulk (globin) around the iron-porphyrin active site in these heme proteins is probably responsible for the electrochemical quasi reversibility. To understand the effect of such polypeptide bulk on iron-porphyrin, we study the interaction of specific amino acids with the hemin center in solution. We choose three representative amino acids-histidine (a well-known iron coordinator in bio-inorganic systems), tryptophan (a well-known fluoroprobe for proteins), and cysteine (a redox-active organic molecule). The interactions of these amino acids with hemin are studied using electrochemistry, spectroscopy, and density functional theory. The results indicate that among these three, the interaction of histidine with the iron center is strongest. Further, histidine maintains the electrochemical reversibility of iron. On the other hand, tryptophan and cysteine interact weakly with the iron center but disturb the electrochemical reversibility by contributing their own redox active processes to the system. Put together, this study attempts to understand the molecular interactions that can control electrochemical reversibility in heme proteins. The results obtained here from the three representative amino acids can be scaled up to build a heme-amino acid interaction database that may predict the electrochemical properties of any protein with a defined polypeptide sequence.

Unique Features of Metformin: A Combined Experimental, Theoretical, and Simulation Study of Its Structure, Dynamics, and Interaction Energetics with DNA Grooves.

There are certain small molecules that exhibit extraordinarily diverse biological activities. Metformin is one of them. It is widely used as an antidiabetic drug for type-two diabetes. Recent lines of evidence of its role in antitumor activities and increasing the survival rates of cancer patients (namely, colorectal, breast, pancreas, and prostate cancer) are emerging. However, theoretical studies of the structure and dynamics of metformin have not yet been fully explored. In this work, we investigate the characteristic structural and dynamical features of three monoprotonated forms of metformin hydrochloride with the help of experiments, quantum chemical calculations, and atomistic molecular dynamics simulations. We validate our force field by comparing simulation results to those of the experimental findings. Energetics of proton transfer between two planar monoprotonated forms reveals a low energy barrier, which leads us to speculate a possible coexistence of them. Nevertheless, among the protonation states, we find that the nonplanar tautomeric form is the most stable. Our calculated values of the self-diffusion coefficient agree quantitatively with NMR results. Metformin forms strong hydrogen bonds with surrounding water molecules, and its solvation dynamics shows unique features. Because of an extended positive charge distribution, metformin possesses features of being a permanent cationic partner toward several targets. We study its interaction and binding ability with DNA using UV spectroscopy, circular dichroism, fluorimetry, and metadynamics simulation. We find a nonintercalative mode of interaction. Metformin feasibly forms a minor/major groove-bound state within a few tens of nanoseconds, preferably with AT-rich domains. A significant decrease in the free energy of binding is observed when it binds to a minor groove of DNA.

Determination of Redox Sensitivity in Structurally Similar Biological Redox Sensors.

Redox stimuli govern a variety of biological processes. The relative sensitivity of redox sensors plays an important role in providing a calibrated response to environmental stimuli and cellular homeostasis. This cellular machinery plays a crucial role in the human pathogen Mycobacterium tuberculosis as it encounters diverse microenvironments in the host. The redox sensory mechanism in M. tuberculosis is governed by two component and one-component systems, alongside a class of transcription factors called the extra cytoplasmic function (ECF) σ factors. ECF σ factors that govern the cellular response to redox stimuli are negatively regulated by forming a complex with proteins called zinc associated anti-σ factors (ZAS). ZAS proteins release their cognate σ factor in response to oxidative stress. The relative sensitivity of the ZAS sensors to redox processes dictate the concentration of free ECF σ factors in the cell. However, factors governing the redox threshold of these sensors remain unclear. We describe here, the molecular characterization of three σ factor/ZAS pairs-σL/RslA, σE/RseA, and σH/RshA-using a combination of biophysical and electrochemical techniques. This study reveals, conclusively, the differences in redox sensitivity in these proteins despite apparent structural similarity and rationalizes the hierarchy in the activation of the cognate ECF σ factors. Put together, the study provides a basis for examining sequence and conformational features that modulate redox sensitivity within the confines of a conserved structural scaffold. The findings provide the guiding principles for the design of intracellular redox sensors with tailored sensitivity and predictable redox thresholds, providing a much needed biochemical tool for understanding host-pathogen interaction.