Recombinant multiepitope proteins expressed in Escherichia coli cells and their potential for immunodiagnosis.

Recombinant multiepitope proteins (RMPs) are a promising alternative for application in diagnostic tests and, given their wide application in the most diverse diseases, this review article aims to survey the use of these antigens for diagnosis, as well as discuss the main points surrounding these antigens. RMPs usually consisting of linear, immunodominant, and phylogenetically conserved epitopes, has been applied in the experimental diagnosis of various human and animal diseases, such as leishmaniasis, brucellosis, cysticercosis, Chagas disease, hepatitis, leptospirosis, leprosy, filariasis, schistosomiasis, dengue, and COVID-19. The synthetic genes for these epitopes are joined to code a single RMP, either with spacers or fused, with different biochemical properties. The epitopes' high density within the RMPs contributes to a high degree of sensitivity and specificity. The RMPs can also sidestep the need for multiple peptide synthesis or multiple recombinant proteins, reducing costs and enhancing the standardization conditions for immunoassays. Methods such as bioinformatics and circular dichroism have been widely applied in the development of new RMPs, helping to guide their construction and better understand their structure. Several RMPs have been expressed, mainly using the Escherichia coli expression system, highlighting the importance of these cells in the biotechnological field. In fact, technological advances in this area, offering a wide range of different strains to be used, make these cells the most widely used expression platform. RMPs have been experimentally used to diagnose a broad range of illnesses in the laboratory, suggesting they could also be useful for accurate diagnoses commercially. On this point, the RMP method offers a tempting substitute for the production of promising antigens used to assemble commercial diagnostic kits.

Standard operation procedure for switchSENSE DRX systems.

There is currently a large panel of technologies available to address molecular interactions in vitro. Each technology presents individual advantages and drawbacks, and it becomes challenging to choose which technology will be best suited for a molecular interaction of interest. Approaches can be broadly categorized as either microfluidic surface-bound methods (such as Surface Plasmon Resonance (SPR) or switchSENSE) or in-solution methods (such as Isothermal Titration Calorimetry (ITC) or MicroScale Thermophoresis (MST)). In-solution methods are advantageous in terms of sample preparation and ease of use as none of the binding partners are subjected to immobilization. On the other hand, surface-based techniques require only small amounts of immobilized interaction partner and provide off-rate characterization as unbound analytes can be removed from the surface to observe analyte dissociation. Here, a standard operating procedure (SOP) for the switchSENSE method is presented, which aims to guide new users through the process of a switchSENSE measurement, covering sample preparation, instrument and biochip handling as well as data acquisition and analysis. This guide will help researchers decide whether switchSENSE is the right method for their application as well as supporting novice users to get the most information out of a switchSENSE measurement. switchSENSE technology offers the unique advantage of a controlled DNA-based ligand surface within a microfluidic channel which allows the user to distribute specifically up to two different ligand molecules on the surface at a customized density and ratio. The technology offers multi-parameter characterization of binding kinetics, affinity, enzymatic activity, and changes in protein conformation.

Insight into the Molecular Interaction of Cloxyquin (5-chloro-8-hydroxyquinoline) with Bovine Serum Albumin: Biophysical Analysis and Computational Simulation.

Cloxyquin is a potential therapeutic compound possessing various bioactivities, especially antibacterial, antifungal, cardioprotective, and pain relief activities. Herein, the interaction mechanism between cloxyquin and bovine serum albumin (BSA) has been elucidated in order to fulfill its pharmacokinetic and pharmacodynamic gaps essential for further development as a therapeutic drug. Multi-spectroscopic and biophysical model analysis suggested that cloxyquin interacts with BSA via a static process by ground-state complex formation. Its binding behavior emerged as a biphasic fashion with a moderate binding constant at the level of 104 M-1. Thermodynamic analysis and molecular docking simulation concurrently revealed that hydrophobic interaction is a major driving force for BSA-cloxyquin complexation. Binding of cloxyquin tends to slightly enlarge the monomeric size of BSA without a significant increase of aggregate fraction. Cloxyquin preferentially binds into the fatty acid binding site 5 (FA5) of the BSA via hydrophobic interaction amongst its quinoline scaffold and Phe550, Leu531, and Leu574 residues of BSA. The quinoline ring and hydroxyl moiety of cloxyquin also form the π-π interaction and the hydrogen bond with Phe506. Our data indicate a potential function of serum albumin as a carrier of cloxyquin in blood circulation.

Noninvasive analysis and minimally invasive in vivo experimental challenges of the skin barrier.

In this review, we aim to give a concise and selective overview of noninvasive biophysical analysis techniques for skin barrier analysis (transepidermal water loss, electrical methods, confocal Raman microspectroscopy, sebumeter, reflectance spectrophotometry, tristimulus colorimetry, diffuse reflectance spectroscopy and reflectance confocal microscopy), including advantages and limitations. Rather than giving an exhaustive description of the many techniques currently available, we show the usefulness of a representative selection of techniques in the functional and morphological evaluation of the skin barrier. Furthermore, we introduce human minimally invasive skin challenging models as a means to study the mechanisms regulating skin homoeostasis and disease and subsequently show how biophysical analysis techniques can be combined with these in vivo skin challenging models in the functional and morphological evaluation of the skin barrier in healthy human skin. We are convinced that the widespread application of biophysical analysis techniques in dermatological practice and in cosmetic sciences will prove invaluable in offering personalized and noninvasive skin treatment solutions. Furthermore, combining the human in vivo challenging models with these novel noninvasive techniques will provide valuable methodology and tools for detailed characterization of the skin barrier in health and disease.

Mixed up minor groove binders: Convincing A·T specific compounds to recognize a G·C base pair.

DNA minor-groove-binding compounds have limited biological applications, in part due to problems with sequence specificity that cause off-target effects. A model to enhance specificity has been developed with the goal of preparing compounds that bind to two AT sites separated by G·C base pairs. Compounds of interest were probed using thermal melting, circular dichroism, mass spectrometry, biosensor-SPR, and molecular modeling methods. A new minor groove binder that can strongly and specifically recognize a single G·C base pair with flanking AT sequences has been prepared. This multi-site DNA recognition mode offers novel design principles to recognize entirely new DNA motifs.

Fundamental analysis of recombinant human epidermal growth factor in solution with biophysical methods.

Correlation of thermodynamic and secondary structural stability of proteins at various buffer pHs was investigated using differential scanning calorimetry (DSC), dynamic light scattering (DLS) and attenuated total reflection Fourier-transform infrared spectroscopy (ATR FT-IR). Recombinant human epithelial growth factor (rhEGF) was selected as a model protein at various pHs and in different buffers, including phosphate, histidine, citrate, HEPES and Tris. Particle size and zeta potential of rhEGF at each selected pH of buffer were observed by DLS. Four factors were used to characterize the biophysical stability of rhEGF in solution: temperature at maximum heat flux (Tm), intermolecular ß-sheet contents, zeta size and zeta potential. It was possible to predict the apparent isoelectric point (pI) of rhEGF as 4.43 by plotting pH against zeta potential. When the pH of the rhEGF solution increased or decreased from pI, the absolute zeta potential increased indicating a reduced possibility of protein aggregation, since Tm increased and ß-sheet contents decreased. The contents of induced intermolecular ß-sheet in Tris and HEPES buffers were the lowest. Thermodynamic stability of rhEGF markedly increased when pH is higher than 6.2 in histidine buffer where Tm of first transition was all above 70 °C. Moreover, rhEGF in Tris buffer was more thermodynamically stable than in HEPES with higher zeta potential. Tris buffer at pH 7.2 was concluded to be the most favorable.

Complex biophysical changes and reduced neuronal firing in an SCN8A variant associated with developmental delay and epilepsy.

Mutations in the SCN8A gene, encoding the voltage-gated sodium channel NaV1.6, are associated with a range of neurodevelopmental syndromes. The p.(Gly1625Arg) (G1625R) mutation was identified in a patient diagnosed with developmental epileptic encephalopathy (DEE). While most of the characterized DEE-associated SCN8A mutations were shown to cause a gain-of-channel function, we show that the G1625R variant, positioned within the S4 segment of domain IV, results in complex effects. Voltage-clamp analyses of NaV1.6G1625R demonstrated a mixture of gain- and loss-of-function properties, including reduced current amplitudes, increased time constant of fast voltage-dependent inactivation, a depolarizing shift in the voltage dependence of activation and inactivation, and increased channel availability with high-frequency repeated depolarization. Current-clamp analyses in transfected cultured neurons revealed that these biophysical properties caused a marked reduction in the number of action potentials when firing was driven by the transfected mutant NaV1.6. Accordingly, computational modeling of mature cortical neurons demonstrated a mild decrease in neuronal firing when mimicking the patients' heterozygous SCN8A expression. Structural modeling of NaV1.6G1625R suggested the formation of a cation-π interaction between R1625 and F1588 within domain IV. Double-mutant cycle analysis revealed that this interaction affects the voltage dependence of inactivation in NaV1.6G1625R. Together, our studies demonstrate that the G1625R variant leads to a complex combination of gain and loss of function biophysical changes that result in an overall mild reduction in neuronal firing, related to the perturbed interaction network within the voltage sensor domain, necessitating personalized multi-tiered analysis for SCN8A mutations for optimal treatment selection.

Biochemical and structural analysis of N-terminal acetyltransferases.

N-terminal acetylation is a co- and post-translational modification catalyzed by the conserved N-terminal acetyltransferase (NAT) family of enzymes. A majority of the human proteome is modified by the human NATs (NatA-F and H), which are minimally composed of a catalytic subunit and as many as two auxiliary subunits. Together, NATs specifically regulate many cellular functions by influencing protein activities such as their degradation, membrane targeting, and protein-protein interactions. This chapter will describe methods developed for their preparation, and their biochemical and structural characterization. This will include methodologies for expression and purification of recombinant NAT protein, kinetic assays, biochemical and biophysical assays, and strategies for structural studies.

[Biophysical Analysis of the Protein-Small Molecule Interactions to Develop Small Molecule Drug Discovery].

In small molecule drug discovery, researchers must find specific binders that interact with a target protein and inhibit its function in connection with human diseases. It is of critical importance to know the binding mode of compounds interacting with a target protein to assure hit validation and optimization. Biophysical analysis is a powerful quantitative approach to evaluate the binding modes of such candidates. Since the level of sensitivity of biophysical analysis is suitable to quantitatively detect the binding of fragment compounds, and because of the remarkable success of compound libraries of small molecules, the development and adaptation of biophysical analysis for these applications is in great demand. Herein, we describe the technical developments of biophysical methods, especially thermodynamic and kinetic analysis, for the purpose of screenings which employ small molecules. In addition, we discuss the interaction mechanisms of small molecules to find hit compounds based on these biophysical analyses.

Is perturbation in the quaternary structure of bacterial CysE, another regulatory mechanism for cysteine synthesis?

Drug resistance to almost all antibiotics of Shigella flexneri, a major cause of shigellosis in developing countries, necessitates continuous discovery of novel therapeutics. This study reports a structure-function analysis of a potential drug target serine acetyltransferase (CysE), an enzyme of de novo cysteine biosynthesis pathway that is absent in humans. Analysis of CysE sequences of S. flexneri species and serotypes displayed only two variants that differed by a single amino acid substitution at position 241. Structural inspection of the available crystal structure disclosed this site to be distinct from the substrate/cofactor binding pockets or dimer/trimer interfaces. This study discovers that V241 variant of S. flexneri CysE has nearly null enzymatic activity. The observation is explained by molecular dynamic studies which reveal that the disorder generated by A241V substitution is the basis of dissociation of the quaternary assembly of S. flexneri CysE leading to loss of enzymatic activity and stability. The study provides the first evidence that position 241 of CysE, affects the catalytic efficiency of enzyme and suggests this locus as a 'hot spot' for the propagation of conformational changes. It may be postulated that transient quaternary structure of CysE maybe another mechanism for regulating the intracellular level of cysteine.

Potent α-Synuclein Aggregation Inhibitors, Identified by High-Throughput Screening, Mainly Target the Monomeric State.

α-Synuclein (αSN) aggregation is central to the etiology of Parkinson's disease (PD). Large-scale screening of compounds to identify aggregation inhibitors is challenged by stochastic αSN aggregation and difficulties in detecting early-stage oligomers (αSOs). We developed a high-throughput screening assay combining SDS-stimulated αSN aggregation with FRET to reproducibly detect initial stages in αSN aggregation. We screened 746,000 compounds, leading to 58 hits that markedly inhibit αSN aggregation and reduce αSOs' membrane permeabilization activity. The most effective aggregation inhibitors were derivatives of (4-hydroxynaphthalen-1-yl)sulfonamide. They interacted strongly with the N-terminal part of monomeric αSN and reduced αSO-membrane interactions, possibly by affecting electrostatic interactions. Several compounds reduced αSO toxicity toward neuronal cell lines. The inhibitors introduced chemical modifications of αSN that were, however, not a prerequisite for inhibitory activity. We also identified several phenyl-benzoxazol compounds that promoted αSN aggregation (proaggregators). These compounds may be useful tools to modulate αSN aggregation in cellula.