Formulation development of a stable influenza recombinant neuraminidase vaccine candidate.

Current influenza vaccines could be augmented by including recombinant neuraminidase (rNA) protein antigen to broaden protective immunity and improve efficacy. Toward this goal, we investigated formulation conditions to optimize rNA physicochemical stability. When rNA in sodium phosphate saline buffer (NaPBS) was frozen and thawed (F/T), the tetrameric structure transitioned from a "closed" to an "open" conformation, negatively impacting functional activity. Hydrogen deuterium exchange experiments identified differences in anchorage binding sites at the base of the open tetramer, offering a structural mechanistic explanation for the change in conformation and decreased functional activity. Change to the open configuration was triggered by the combined stresses of acidic pH and F/T. The desired closed conformation was preserved in a potassium phosphate buffer (KP), minimizing pH drop upon freezing and including 10% sucrose to control F/T stress. Stability was further evaluated in thermal stress studies where changes in conformation were readily detected by ELISA and size exclusion chromatography (SEC). Both tests were suitable indicators of stability and antigenicity and considered potential critical quality attributes (pCQAs). To understand longer-term stability, the pCQA profiles from thermally stressed rNA at 6 months were modeled to predict stability of at least 24-months at 5°C storage. In summary, a desired rNA closed tetramer was maintained by formulation selection and monitoring of pCQAs to produce a stable rNA vaccine candidate. The study highlights the importance of understanding and controlling vaccine protein structural and functional integrity.

Structural and biochemical rationale for Beta variant protein booster vaccine broad cross-neutralization of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, uses a surface expressed trimeric spike glycoprotein for cell entry. This trimer is the primary target for neutralizing antibodies making it a key candidate for vaccine development. During the global pandemic circulating variants of concern (VOC) caused several waves of infection, severe disease, and death. The reduced efficacy of the ancestral trimer-based vaccines against emerging VOC led to the need for booster vaccines. Here we present a detailed characterization of the Sanofi Beta trimer, utilizing cryo-EM for structural elucidation. We investigate the conformational dynamics and stabilizing features using orthogonal SPR, SEC, nanoDSF, and HDX-MS techniques to better understand how this antigen elicits superior broad neutralizing antibodies as a variant booster vaccine. This structural analysis confirms the Beta trimer preference for canonical quaternary structure with two RBD in the up position and the reversible equilibrium between the canonical spike and open trimer conformations. Moreover, this report provides a better understanding of structural differences between spike antigens contributing to differential vaccine efficacy.

Epitope screening using Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS): An accelerated workflow for evaluation of lead monoclonal antibodies.

BACKGROUND: Epitope mapping is an increasingly important aspect of biotherapeutic and vaccine development. Recent advances in therapeutic antibody design and production have enabled candidate mAbs to be identified at a rapidly increasing rate, resulting in a significant bottleneck in the characterization of "structural" epitopes, that are challenging to determine using existing high throughput epitope mapping tools. Here, a Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) epitope screening workflow was introduced that is well suited for accelerated characterization of epitopes with a common antigen. MAIN METHODS AND MAJOR RESULTS: The method is demonstrated on set of six candidate mAbs targeting Pertactin (PRN). Using this approach, five of the six epitopes were unambiguously determined using two HDX mixing timepoints in 24 h total run time, which is equivalent to the instrument time required to map a single epitope using the conventional workflow. CONCLUSION: An accelerated HDX-MS epitope screening workflow was developed. The "screening" workflow successfully characterized five (out of six attempted) novel epitopes on the PRN antigen; information that can be used to support vaccine antigenicity assays.

Genetically detoxified pertussis toxin displays near identical structure to its wild-type and exhibits robust immunogenicity.

The mutant gdPT R9K/E129G is a genetically detoxified variant of the pertussis toxin (PTx) and represents an attractive candidate for the development of improved pertussis vaccines. The impact of the mutations on the overall protein structure and its immunogenicity has remained elusive. Here we present the crystal structure of gdPT and show that it is nearly identical to that of PTx. Hydrogen-deuterium exchange mass spectrometry revealed dynamic changes in the catalytic domain that directly impacted NAD+ binding which was confirmed by biolayer interferometry. Distal changes in dynamics were also detected in S2-S5 subunit interactions resulting in tighter packing of B-oligomer corresponding to increased thermal stability. Finally, antigen stimulation of human whole blood, analyzed by a previously unreported mass cytometry assay, indicated broader immunogenicity of gdPT compared to pertussis toxoid. These findings establish a direct link between the conserved structure of gdPT and its ability to generate a robust immune response.

Exploring the interactions of iron and zinc with the microtubule binding repeats R1 and R4.

The dyshomeostasis of copper, iron and zinc ions in pathological conditions, which are critically involved in many brain activities, may result in an accumulation of them in the brain that has been reported for the patients with Alzheimer's disease. Conformational change is one of the consequences of metal-peptide interaction as we observed for the interaction of the Cu2+ with microtubule binding repeats of tau protein, which ultimately cause peptide aggregation. Herein, we show that interaction of Zn2+, Fe2+, and Fe3+ with full-length tau peptide R1 (tau244-274) and R4 (tau337-368), the first and fourth microtubule binding repeats of tau protein, lead to the conformational changes. And while the Electrospray ionization-mass spectrometry (ESI-MS) confirmed the complexation of Zn2+ and Fe2+ with both R1 and R4, there is no evidence for metalation of R1 or R4 with Fe3+.

Identification of Soybean Seed Varieties Based on Hyperspectral Imaging Technology.

Hyperspectral imaging is a nondestructive testing technology that integrates spectroscopy and iconology technologies, which enables us to quickly obtain both internal and external information of objects and identify crop seed varieties. First, the hyperspectral images of ten soybean seed varieties were collected and the reflectance was obtained. Savitzky-Golay smoothing (SG), first derivative (FD), standard normal variate (SNV), fast Fourier transform (FFT), Hilbert transform (HT), and multiplicative scatter correction (MSC) spectral reflectance pretreatment methods were used. Then, the feature wavelengths and feature information of the pretreated spectral reflectance data were extracted using competitive adaptive reweighted sampling (CARS), the successive projections algorithm (SPA), and principal component analysis (PCA). Finally, 5 classifiers, Bayes, support vector machine (SVM), k-nearest neighbor (KNN), ensemble learning (EL), and artificial neural network (ANN), were used to identify seed varieties. The results showed that MSC-CARS-EL had the highest accuracy among the 90 combinations, with training set, test set, and 5-fold cross-validation accuracies of 100%, 100%, and 99.8%, respectively. Moreover, the contribution of spectral pretreatment to discrimination accuracy was higher than those of feature extraction and classifier selection. Pretreatment methods determined the range of the identification accuracy, feature-selective methods and classifiers only changed within this range. The experimental results provide a good reference for the identification of other crop seed varieties.

A highly conductive conjugated coordination polymer for fast-charge sodium-ion batteries: reconsidering its structures.

Conjugated coordination polymers (CCPs), showing high conductivity, can be expected to be possibly applied in batteries, which, however, have not been well studied. Furthermore, CCPs are so unique that most of their chemical structures have not been well determined. Herein, a highly conductive CCP is reported for sodium-ion batteries, which shows high rate performance and high capacity retention of 84% even at 30C. What's more, the chemical structure is successfully revealed based on the structure variation during the electrochemical redox process.

Aggregation of Microtubule Binding Repeats of Tau Protein is Promoted by Cu2.

Understanding the factors that give rise to tau aggregation and reactive oxygen species (ROS) is the key aspect in Alzheimer's disease pathogenesis. Microtubule (MT) binding repeats of tau protein were suggested to play a critical role in tau aggregation. Here, we show that the interaction of Cu2+ with full-length MT binding repeats R1-R4 leads to the aggregation, and a Cys-based redox chemistry is critically involved in tau aggregation leading to disulfide-bridge dimerization of R2 and R3 and further aggregation into a fibrillar structure. Notably, ascorbate and glutathione, the most abundant antioxidants in neurons, cannot prevent the effect of Cu2+ on R2 and R3 aggregation. Detailed ESI-MS and NMR experiments demonstrate the interaction of Cu2+ with MT binding repeats. We show that redox activity of copper increases when bound to the MT repeats leading to ROS formation, which significantly contribute to cellular damage and neuron death. Results presented here provide new insights into the molecular mechanism of tau aggregation and ROS formation and suggest a new target domain for tau aggregation inhibitors.

Interaction of metal ions with tau protein. The case for a metal-mediated tau aggregation.

Tau protein aggregation and its hyperphosphorylation play an important role in the pathogenesis of Alzheimer's disease. There is also considerable evidence for the accumulation of Fe2/3+, Cu2+, and Zn2+ in the brain of Alzheimer's patients, although their involvement in the etiology of the disease remains unknown. Here, interactions of the 3d metal ions Fe2/3+, Cu2+, and Zn2+ with the longest isoform of the human tau protein (htau40) are studied in detail. Electrospray mass spectrometry and ion mobility mass spectrometry analyses confirm the interactions of metal species with tau and that these interactions cause structural changes. Phosphorylation of the full-length htau40 with glycogen synthase kinase 3ß (GSK3ß), a protein kinase, causes a reduction in metal interactions. Transmission electron microscopy studies of the tau aggregates formed in the presence of metal ions suggest that the presence of metal ions influences the aggregation process. Fluorescence studies of full-length htau40 in the presence of Cu2+ indicate the formation of reactive oxygen species, which may contribute further to oxidative stress and neuronal death.

A Rapid and Highly Efficient Method for the Identification of Soybean Seed Varieties: Hyperspectral Images Combined with Transfer Learning.

Convolutional neural network (CNN) can be used to quickly identify crop seed varieties. 1200 seeds of ten soybean varieties were selected, hyperspectral images of both the front and the back of the seeds were collected, and the reflectance of soybean was derived from the hyperspectral images. A total of 9600 images were obtained after data augmentation, and the images were divided into a training set, validation set, and test set with a 3:1:1 ratio. Pretrained models (AlexNet, ResNet18, Xception, InceptionV3, DenseNet201, and NASNetLarge) after fine-tuning were used for transfer training. The optimal CNN model for soybean seed variety identification was selected. Furthermore, the traditional machine learning models for soybean seed variety identification were established by using reflectance as input. The results show that the six models all achieved 91% accuracy in the validation set and achieved accuracy values of 90.6%, 94.5%, 95.4%, 95.6%, 96.8%, and 97.2%, respectively, in the test set. This method is better than the identification of soybean seed varieties based on hyperspectral reflectance. The experimental results support a novel method for identifying soybean seeds rapidly and accurately, and this method also provides a good reference for the identification of other crop seeds.

Hydrogen-Deuterium Exchange Epitope Mapping Reveals Distinct Neutralizing Mechanisms for Two Monoclonal Antibodies against Diphtheria Toxin.

The diphtheria toxoid (DT) antigen is one of the major components in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. However, a structurally resolved analysis of diphtheria toxin (DTx) epitopes with underlying molecular mechanisms of antibody neutralization has not yet been reported. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Biolayer Interferometry (BLI) assays, we have characterized two neutralizing anti-DTx monoclonal antibodies (mAbs), 2-25 and 2-18, by identifying the specific epitopes on the diphtheria toxin responsible for antibody binding. Our results show that both epitopes are conformational, and mechanistically distinct. Monoclonal antibody 2-25 binds selectively to the B-subunit (translocation and receptor domain) of DTx, blocking the heparin-binding EGF-like growth factor (HBEGF) binding site. In contrast, mAb 2-18 binds to the A-subunit (catalytic domain), partially covering the catalytic loop region that shuttles NAD during catalysis. The results are discussed in the context of antigen neutralization mechanisms and can ultimately help to reveal the underlying factors that contribute to Diptheria vaccine efficacy.

Constructing Universal Ionic Sieves via Alignment of Two-Dimensional Covalent Organic Frameworks (COFs).

The shuttle effect of electrode materials always leads to capacity loss and poor cycle life of batteries. Two-dimensional (2D) covalent organic frameworks (COFs) with uniform and controllable nanopores provide a promising strategy for fabricating ionic sieves to inhibit the shuttle effect. However, the insoluble nature of COFs made it difficult to fabricate compact and ordered membranes of COFs. Herein, we report a novel method for facilely anisotropic ordering of 2D COFs via depositing COFs onto graphene. The resulted double-layer membranes acting as ionic sieves impressively inhibit the shuttle effect and exhibit versatility to both organic sodium-ion batteries and Li-S batteries, leading to high cyclability.

Large π-Conjugated Porous Frameworks as Cathodes for Sodium-Ion Batteries.

Organic sodium-ion batteries (OSIBs) are promising alternatives of inorganic lithium-ion batteries. The cathodes of OSIBs still suffer from low capacity, poor rate performance, and low cyclability. For the first time, we demonstrate the large π-conjugated porous frameworks (CPFs) as cathodes for OSIBs, motivated by the speculation that the CPFs are capable of enhancing charge transport, facilitating ionic diffusion, inhibiting dissolution, as well as improving stability. The batteries based on the obtained CPFs indeed delivered much better electrochemical performance than the small molecular construction units without any complex post-treatments. The moderate BET surface area of CPFs and the detailed analyses suggested that the micropores and the lamellar structure should be responsible for the fast ionic diffusion. We believe that this work will provoke growing interest of CPFs for OSIBs with functional molecular design toward high performance and pave a venue to achieve OSIBs in large-scale applications.

Suppressing allostery in epitope mapping experiments using millisecond hydrogen / deuterium exchange mass spectrometry.

Localization of the interface between the candidate antibody and its antigen target, commonly known as epitope mapping, is a critical component of the development of therapeutic monoclonal antibodies. With the recent availability of commercial automated systems, hydrogen / deuterium eXchange (HDX) is rapidly becoming the tool for mapping epitopes preferred by researchers in both industry and academia. However, this approach has a significant drawback in that it can be confounded by 'allosteric' structural and dynamic changes that result from the interaction, but occur far from the point(s) of contact. Here, we introduce a 'kinetic' millisecond HDX workflow that suppresses allosteric effects in epitope mapping experiments. The approach employs a previously introduced microfluidic apparatus that enables millisecond HDX labeling times with on-chip pepsin digestion and electrospray ionization. The 'kinetic' workflow also differs from conventional HDX-based epitope mapping in that the antibody is introduced to the antigen at the onset of HDX labeling. Using myoglobin / anti-myoglobin as a model system, we demonstrate that at short 'kinetic' workflow labeling times (i.e., 200 ms), the HDX signal is already fully developed at the 'true' epitope, but is still largely below the significance threshold at allosteric sites. Identification of the 'true' epitope is supported by computational docking predictions and allostery modeling using the rigidity transmission allostery algorithm.

Enhanced Binding Affinity via Destabilization of the Unbound State: A Millisecond Hydrogen-Deuterium Exchange Study of the Interaction between p53 and a Pleckstrin Homology Domain.

The incorporation of intrinsically disordered domains enables proteins to engage a wide variety of targets, with phosphorylation often modulating target specificity and affinity. Although phosphorylation can clearly act as a chemical driver of complexation in structured proteins, e.g., by abrogating or permitting new charge-charge interactions, the basis for enhancement of the hydrophobically driven interactions that are typical of disordered protein-target complexation is less clear. To determine how phosphorylation can positively impact target recruitment in disordered domains, we have examined the interaction between the disordered N-terminal transactivation domain (TAD) of p53 and the pleckstrin homology (PH) domain of p62. Using time-resolved electrospray ionization with hydrogen-deuterium exchange, we demonstrate that phosphorylation has little effect on the conformation of the p53 TAD when it is bound to the PH domain but instead increases the degree of conformational disorder in the unbound state. We propose that this increase in the degree of disorder creates a wider free energy gap between the free and bound states, providing a target-independent mechanism for enhanced binding when the phosphorylated and unphosphorylated p53-target complexes have similar free energies.

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Differential Mobility Spectrometry-Hydrogen Deuterium Exchange (DMS-HDX) as a Probe of Protein Conformation in Solution.

Differential mobility spectrometry (DMS) is an ion mobility technique that has been adopted chiefly as a pre-filter for small- to medium-sized analytes (<1 000 Da). With the exception of a handful of studies that employ an analogue of DMS-field asymmetric waveform ion mobility spectroscopy (FAIMS)-the application of DMS to intact biomacromolecules remains largely unexplored. In this work, we employ DMS combined with gas-phase hydrogen deuterium exchange (DMS-HDX) to probe the gas-phase conformations generated from proteins that were initially folded, partially-folded, and unfolded in solution. Our findings indicate that proteins with distinct structural features in solution exhibit unique deuterium uptake profiles as function of their optimal transmission through the DMS. Ultimately we propose that DMS-HDX can, if properly implemented, provide rapid measurements of liquid-phase protein structural stability that could be of use in biopharmaceuticals development. Graphical Abstract ᅟ.

Conformational Dynamics and Allostery in Pyruvate Kinase.

Pyruvate kinase catalyzes the final step in glycolysis and is allosterically regulated to control flux through the pathway. Two models are proposed to explain how Escherichia coli pyruvate kinase type 1 is allosterically regulated: the "domain rotation model" suggests that both the domains within the monomer and the monomers within the tetramer reorient with respect to one another; the "rigid body reorientation model" proposes only a reorientation of the monomers within the tetramer causing rigidification of the active site. To test these hypotheses and elucidate the conformational and dynamic changes that drive allostery, we performed time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange studies followed by mutagenic analysis to test the activation mechanism. Global exchange experiments, supported by thermostability studies, demonstrate that fructose 1,6-bisphosphate binding to the allosteric domain causes a shift toward a globally more dynamic ensemble of conformations. Mapping deuterium exchange to peptides within the enzyme highlight site-specific regions with altered conformational dynamics, many of which increase in conformational flexibility. Based upon these and mutagenic studies, we propose an allosteric mechanism whereby the binding of fructose 1,6-bisphosphate destabilizes an α-helix that bridges the allosteric and active site domains within the monomeric unit. This destabilizes the ß-strands within the (ß/α)8-barrel domain and the linked active site loops that are responsible for substrate binding. Our data are consistent with the domain rotation model but inconsistent with the rigid body reorientation model given the increased flexibility at the interdomain interface, and we can for the first time explain how fructose 1,6-bisphosphate affects the active site.

Hyperphosphorylation of intrinsically disordered tau protein induces an amyloidogenic shift in its conformational ensemble.

Tau is an intrinsically disordered protein (IDP) whose primary physiological role is to stabilize microtubules in neuronal axons at all stages of development. In Alzheimer's and other tauopathies, tau forms intracellular insoluble amyloid aggregates known as neurofibrillary tangles, a process that appears in many cases to be preceded by hyperphosphorylation of tau monomers. Understanding the shift in conformational bias induced by hyperphosphorylation is key to elucidating the structural factors that drive tau pathology, however, as an IDP, tau is not amenable to conventional structural characterization. In this work, we employ a straightforward technique based on Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) and Hydrogen/Deuterium Exchange (HDX) to provide a detailed picture of residual structure in tau, and the shifts in conformational bias induced by hyperphosphorylation. By comparing the native and hyperphosphorylated ensembles, we are able to define specific conformational biases that can easily be rationalized as enhancing amyloidogenic propensity. Representative structures for the native and hyperphosphorylated tau ensembles were generated by refinement of a broad sample of conformations generated by low-computational complexity modeling, based on agreement with the TRESI-HDX profiles.

Oxidative protein labeling with analysis by mass spectrometry for the study of structure, folding, and dynamics.

SIGNIFICANCE: Analytical approaches that can provide insights into the mechanistic processes underlying protein folding and dynamics are few since the target analytes-high-energy structural intermediates-are short lived and often difficult to distinguish from coexisting structures. Folding "intermediates" can be populated at equilibrium using weakly denaturing solvents, but it is not clear that these species are identical to those that are transiently populated during folding under "native" conditions. Oxidative labeling with mass spectrometric analysis is a powerful alternative for structural characterization of proteins and transient protein species based on solvent exposure at specific sites. RECENT ADVANCES: Oxidative labeling is increasingly used with exceedingly short (µs) labeling pulses, both to minimize the occurrence of artifactual structural changes due to the incorporation of label and to detect short-lived species. The recent introduction of facile photolytic approaches for producing reactive oxygen species is an important technological advance that will enable more widespread adoption of the technique. CRITICAL ISSUES: The most common critique of oxidative labeling data is that even with brief labeling pulses, covalent modification of the protein may cause significant artifactual structural changes. FUTURE DIRECTIONS: While the oxidative labeling with the analysis by mass spectrometry is mature enough that most basic methodological issues have been addressed, a complete systematic understanding of side chain reactivity in the context of intact proteins is an avenue for future work. Specifically, there remain issues around the impact of primary sequence and side chain interactions on the reactivity of "solvent-exposed" residues. Due to its analytical power, wide range of applications, and relative ease of implementation, oxidative labeling is an increasingly important technique in the bioanalytical toolbox.