Design and fabrication of 3D-printed in situ crystallization plates for probing microcrystals in an external electric field.

X-ray crystallography is an established tool to probe the structure of macromolecules with atomic resolution. Compared with alternative techniques such as single-particle cryo-electron microscopy and micro-electron diffraction, X-ray crystallography is uniquely suited to room-temperature studies and for obtaining a detailed picture of macromolecules subjected to an external electric field (EEF). The impact of an EEF on proteins has been extensively explored through single-crystal X-ray crystallography, which works well with larger high-quality protein crystals. This article introduces a novel design for a 3D-printed in situ crystallization plate that serves a dual purpose: fostering crystal growth and allowing the concurrent examination of the effects of an EEF on crystals of varying sizes. The plate's compatibility with established X-ray crystallography techniques is evaluated.

Illuminating the mechanism and allosteric behavior of NanoLuc luciferase.

NanoLuc, a superior ß-barrel fold luciferase, was engineered 10 years ago but the nature of its catalysis remains puzzling. Here experimental and computational techniques are combined, revealing that imidazopyrazinone luciferins bind to an intra-barrel catalytic site but also to an allosteric site shaped on the enzyme surface. Structurally, binding to the allosteric site prevents simultaneous binding to the catalytic site, and vice versa, through concerted conformational changes. We demonstrate that restructuration of the allosteric site can boost the luminescent reaction in the remote active site. Mechanistically, an intra-barrel arginine coordinates the imidazopyrazinone component of luciferin, which reacts with O2 via a radical charge-transfer mechanism, and then it also protonates the resulting excited amide product to form a light-emitting neutral species. Concomitantly, an aspartate, supported by two tyrosines, fine-tunes the blue color emitter to secure a high emission intensity. This information is critical to engineering the next-generation of ultrasensitive bioluminescent reporters.

Domino-like effect of C112R mutation on ApoE4 aggregation and its reduction by Alzheimer's Disease drug candidate.

BACKGROUND: Apolipoprotein E (ApoE) ε4 genotype is the most prevalent risk factor for late-onset Alzheimer's Disease (AD). Although ApoE4 differs from its non-pathological ApoE3 isoform only by the C112R mutation, the molecular mechanism of its proteinopathy is unknown. METHODS: Here, we reveal the molecular mechanism of ApoE4 aggregation using a combination of experimental and computational techniques, including X-ray crystallography, site-directed mutagenesis, hydrogen-deuterium mass spectrometry (HDX-MS), static light scattering and molecular dynamics simulations. Treatment of ApoE ε3/ε3 and ε4/ε4 cerebral organoids with tramiprosate was used to compare the effect of tramiprosate on ApoE4 aggregation at the cellular level. RESULTS: We found that C112R substitution in ApoE4 induces long-distance (> 15 Å) conformational changes leading to the formation of a V-shaped dimeric unit that is geometrically different and more aggregation-prone than the ApoE3 structure. AD drug candidate tramiprosate and its metabolite 3-sulfopropanoic acid induce ApoE3-like conformational behavior in ApoE4 and reduce its aggregation propensity. Analysis of ApoE ε4/ε4 cerebral organoids treated with tramiprosate revealed its effect on cholesteryl esters, the storage products of excess cholesterol. CONCLUSIONS: Our results connect the ApoE4 structure with its aggregation propensity, providing a new druggable target for neurodegeneration and ageing.

Explanation of inconsistencies in the determination of human serum albumin thermal stability.

Thermal denaturation of human serum albumin has been the subject of many studies in recent decades, but the results of these studies are often conflicting and inconclusive. To clarify this, we combined different spectroscopic and calorimetric techniques and performed an in-depth analysis of the structural changes that occur during the thermal unfolding of different conformational forms of human serum albumin. Our results showed that the inconsistency of the results in the literature is related to the different quality of samples in different batches, methodological approaches and experimental conditions used in the studies. We confirmed that the presence of fatty acids (FAs) causes a more complex process of the thermal denaturation of human serum albumin. While the unfolding pathway of human serum albumin without FAs can be described by a two-step model, consisting of subsequent reversible and irreversible transitions, the thermal denaturation of human serum albumin with FAs appears to be a three-step process, consisting of a reversible step followed by two consecutive irreversible transitions.

Increased occurrence of Treponema spp. and double-species infections in patients with Alzheimer's disease.

Although the link between microbial infections and Alzheimer's disease (AD) has been demonstrated in multiple studies, the involvement of pathogens in the development of AD remains unclear. Here, we investigated the frequency of the 10 most commonly cited viral (HSV-1, EBV, HHV-6, HHV-7, and CMV) and bacterial (Chlamydia pneumoniae, Helicobacter pylori, Borrelia burgdorferi, Porphyromonas gingivalis, and Treponema spp.) pathogens in serum, cerebrospinal fluid (CSF) and brain tissues of AD patients. We have used an in-house multiplex PCR kit for simultaneous detection of five bacterial and five viral pathogens in serum and CSF samples from 50 AD patients and 53 healthy controls (CTRL). We observed a significantly higher frequency rate of AD patients who tested positive for Treponema spp. compared to controls (AD: 62.2 %; CTRL: 30.3 %; p-value = 0.007). Furthermore, we confirmed a significantly higher occurrence of cases with two or more simultaneous infections in AD patients compared to controls (AD: 24 %; CTRL 7.5 %; p-value = 0.029). The studied pathogens were detected with comparable frequency in serum and CSF. In contrast, Borrelia burgdorferi, human herpesvirus 7, and human cytomegalovirus were not detected in any of the studied samples. This study provides further evidence of the association between microbial infections and AD and shows that paralleled analysis of multiple sample specimens provides complementary information and is advisable for future studies.

Purification of MBP fusion proteins using engineered DARPin affinity matrix.

Maltose binding protein (MBP) has a long history as an expression tag with the ability to increase the solubility of fused proteins. A critical step for obtaining a sufficient amount of the MBP fusion protein is purification. Commercially available amylose matrix for the affinity purification of MBP fusion proteins has two main issues: (i) low (micromolar) affinity and (ii) the limited number of uses due to the cleavage of polysaccharide matrix by the amylases, present in the crude cell extract. Here, we present a new affinity purification approach based on the protein-protein interaction. We developed the affinity matrix which contains immobilized Designed Ankyrin Repeat Protein off7 (DARPin off7) - previously identified MBP binder with nanomolar affinity. The functionality of the DARPin affinity matrix was tested on the purification of MBP-tagged green fluorescent protein and flavodoxin. The affinity purification of the MBP fusion proteins, based on the MBP-DARPin off7 interaction, enables the purification of the fusion proteins in a simple two-steps procedure. The DARPin affinity matrix - easy to construct, resistant to amylase, insensitive to maltose contamination, and reusable for multiple purification cycles - provides an alternative approach to commercially available affinity matrices for purification of proteins containing the MBP tag.

Multi-pathogen infections and Alzheimer's disease.

Alzheimer's disease (AD) is a chronic neurodegenerative disease associated with the overproduction and accumulation of amyloid-ß peptide and hyperphosphorylation of tau proteins in the brain. Despite extensive research on the amyloid-based mechanism of AD pathogenesis, the underlying cause of AD is not fully understood. No disease-modifying therapies currently exist, and numerous clinical trials have failed to demonstrate any benefits. The recent discovery that the amyloid-ß peptide has antimicrobial activities supports the possibility of an infectious aetiology of AD and suggests that amyloid-ß plaque formation might be induced by infection. AD patients have a weakened blood-brain barrier and immune system and are thus at elevated risk of microbial infections. Such infections can cause chronic neuroinflammation, production of the antimicrobial amyloid-ß peptide, and neurodegeneration. Various pathogens, including viruses, bacteria, fungi, and parasites have been associated with AD. Most research in this area has focused on individual pathogens, with herpesviruses and periodontal bacteria being most frequently implicated. The purpose of this review is to highlight the potential role of multi-pathogen infections in AD. Recognition of the potential coexistence of multiple pathogens and biofilms in AD's aetiology may stimulate the development of novel approaches to its diagnosis and treatment. Multiple diagnostic tests could be applied simultaneously to detect major pathogens, followed by anti-microbial treatment using antiviral, antibacterial, antifungal, and anti-biofilm agents.

Conformational properties of LOV2 domain and its C450A variant within broad pH region.

LOV2 (Light-Oxygen-Voltage) domain from Avena sativa phototropin 1 (AsLOV2) belongs to the superfamily of PAS (Per-Arnt-Sim) domains, members of which function as signaling sensors. AsLOV2 undergoes a conformational change upon blue-light absorption by its FMN cofactor. AsLOV2 wild type (wt) is intensively studied as a photo-switchable element in conjugation with various proteins. On the other hand, its variant AsLOV2 with replaced cysteinyl residue C450, which is critical for the forming a covalent adduct with FMN upon irradiation, forms a precursor for some recently developed genetically encoded photosensitizers. In the presented work, we investigated conformational properties of AsLOV2 wt and its variant C450A by circular dichroism, tryptophan and FMN fluorescence, and differential scanning calorimetry in dependence on pH and temperature. We show that both variants are similarly sensitive towards pH of solvent. On the other hand, the mutation C450A leads to a more stable AsLOV2 variant in comparison with the wild type. Thermal transitions of the AsLOV2 proteins monitored by circular dichroism indicate the presence of significant residual structure in thermally-denatured states of both proteins in the pH range from 4 to 9. Both pH- and thermal- transitions of AsLOV2 are accompanied by FMN leaching to solvent. Higher stability, reversibility of thermal transitions, and efficiency of FMN rebinding in the case of C450A variant suggest that the cofactor release may be modulated by suitable mutations in combination with a suitable physicochemical perturbation. These findings can have implications for a design of genetically encoded photosensitizers.

Dual effect of non-ionic detergent Triton X-100 on insulin amyloid formation.

Atomic force microscopy, Thioflavin T (ThT) fluorescence assay, circular dichroism spectroscopy, differential scanning calorimetry, and molecular modeling techniques have been employed to investigate the amyloid aggregation of insulin in the presence of non-ionic detergent, Triton X-100 (TX-100). In contrast to recently described inhibition of lysozyme amyloid formation by non-ionic detergents (Siposova, 2017), the amyloid aggregation of insulin in the presence of sub-micellar TX-100 concentration exhibits two dissimilar phases. The first, inhibition phase, is observed at the protein to detergent molar ratio of 1:0.1 to 1:1. During this phase, the insulin amyloid fibril formation is inhibited by TX-100 up to ∼60%. The second, "morphological" phase, is observed at increasing detergent concentration, corresponding to protein:detergent molar ratio of ∼1:1 - 1:10. Under these conditions a significant increase of the steady-state ThT fluorescence intensities and a dramatically changed morphology of the insulin fibrils were observed. Increasing of the detergent concentration above the CMC led to complete inhibition of amyloidogenesis. Analysis of the experimental and molecular modeling results suggests an existence of up to six TX-100 binding sites within dimer of insulin with different binding energy. The physiological relevance of the results is discussed.

Modification of the kinetic stability of immunoglobulin G by solvent additives.

Biophysical properties of antibody-based biopharmaceuticals are a critical part of their release criteria. In this context, finding the appropriate formulation is equally important as optimizing their intrinsic biophysical properties through protein engineering, and both are mutually dependent. Most previous studies have empirically tested the impact of additives on measures of colloidal stability, while mechanistic aspects have usually been limited to only the thermodynamic stability of the protein. Here we emphasize the kinetic impact of additives on the irreversible denaturation steps of immunoglobulins G (IgG) and their antigen-binding fragments (Fabs), as these are the key committed steps preceding aggregation, and thus especially informative in elucidating the molecular parameters of activity loss. We examined the effects of ten additives on the conformational kinetic stability by differential scanning calorimetry (DSC), using a recently developed three-step model containing both reversible and irreversible steps. The data highlight and help to rationalize different effects of the additives on the properties of full-length IgG, analyzed by onset and aggregation temperatures as well as by kinetic parameters derived from our model. Our results further help to explain the observation that stabilizing mutations in the antigen-binding fragment (Fab) significantly affect the kinetic parameters of its thermal denaturation, but not the aggregation properties of the full-length IgGs. We show that the proper analysis of DSC scans for full-length IgGs and their corresponding Fabs not only helps in ranking their stability in different formats and formulations, but provides important mechanistic insights for improving the conformational kinetic stability of IgGs.

The molten-globule residual structure is critical for reflavination of glucose oxidase.

Glucose oxidase (GOX) is a homodimeric glycoprotein with tightly bound one molecule of FAD cofactor per monomer of the protein. GOX has numerous applications, but the preparation of biotechnologically interesting GOX sensors requires a removal of the native FAD cofactor. This process often leads to unwanted irreversible deflavination and, as a consequence, to the low enzyme recovery. Molecular mechanisms of reversible reflavination are poorly understood; our current knowledge is based only on empiric rules, which is clearly insufficient for further development. To develop conceptual understanding of flavin-binding competent states, we studied the effect of deflavination protocols on conformational properties of GOX. After deflavination, the apoform assembles into soluble oligomers with nearly native-like holoform secondary structure but largely destabilized tertiary structure presumambly due to the packing density defects around the vacant flavin binding site. The reflavination is cooperative but not fully efficient; after the binding the flavin cofactor, the protein directly disassembles into native homodimers while the fraction of oligomers remains irreversibly inactivated. Importantly, the effect of Hofmeister salts on the conformational properties of GOX and reflavination efficiency indicates that the native-like residual tertiary structure in the molten-globule states favorably supports the reflavination and minimizes the inactivated oligomers. We interpret our results by combining the ligand-induced changes in quaternary structure with salt-sensitive, non-equilibrated conformational selection model. In summary, our work provides the very first steps toward molecular understanding the complexity of the GOX reflavination mechanism.

Analysis of IgG kinetic stability by differential scanning calorimetry, probe fluorescence and light scattering.

Monoclonal antibodies of the immunoglobulin G (IgG) type have become mainstream therapeutics for the treatment of many life-threatening diseases. For their successful application in the clinic and a favorable cost-benefit ratio, the design and formulation of these therapeutic molecules must guarantee long-term stability for an extended period of time. Accelerated stability studies, e.g., by employing thermal denaturation, have the great potential for enabling high-throughput screening campaigns to find optimal molecular variants and formulations in a short time. Surprisingly, no validated quantitative analysis of these accelerated studies has been performed yet, which clearly limits their application for predicting IgG stability. Therefore, we have established a quantitative approach for the assessment of the kinetic stability over a broad range of temperatures. To this end, differential scanning calorimetry (DSC) experiments were performed with a model IgG, testing chaotropic formulations and an extended temperature range, and they were subsequently analyzed by our recently developed three-step sequential model of IgG denaturation, consisting of one reversible and two irreversible steps. A critical comparison of the predictions from this model with data obtained by an orthogonal fluorescence probe method, based on 8-anilinonaphthalene-1-sulfonate binding to partially unfolded states, resulted in very good agreement. In summary, our study highlights the validity of this easy-to-perform analysis for reliably assessing the kinetic stability of IgGs, which can support accelerated formulation development of monoclonal antibodies by ranking different formulations as well as by improving colloidal stability models.

Negligible interaction of [Ru(Phen)3]2+ with human serum albumin makes it promising for a reliable invivo assessment of the tissue oxygenation.

The interaction between a ruthenium - based water soluble oxygen probe ([Ru(Phen)3]2+, phen - phenanthroline) and human serum albumin (HSA) was investigated with the aim of describing the influence of HSA on the [Ru(Phen)3]2+ luminescence properties. Nowadays, several oxygen sensitive luminescent probes are used to determine the oxygen level in different compartments of living organisms. However, they can interact, depending on their hydrophilic/hydrophobic characters, with various serum proteins, and/or lipids, during their utilization for invivo oxygen measurement. Since HSA is the most abundant serum protein in most biological organisms, its presence may affect the spectral properties of the employed probes and, consequently, the determination of the oxygen concentration. Having this in mind, we have applied several spectroscopic and calorimetric techniques to study [Ru(Phen)3]2+ - HSA mixtures. Only a negligible effect of HSA on the absorption and luminescence spectra of [Ru(Phen)3]2+ was observed. In addition, differential scanning calorimetric studies showed that [Ru(Phen)3]2+ does not significantly influence HSA thermal stability. Importantly, [Ru(Phen)3]2+ retained a reliable luminescence lifetime sensitivity to the oxygen concentration in solutions supplemented with HSA and in U87 MG cancer cells. Finally, the biodistribution of [Ru(Phen)3]2+ in the presence of serum proteins in the blood stream of chick embryo's chorioallantoic membrane (CAM) was investigated. Fast [Ru(Phen)3]2+ and similar extravasations were observed in the presence or absence of CAM-serum. We can conclude that HSA-[Ru(Phen)3]2+ complex interaction does not significantly influence the potential of [Ru(Phen)3]2+ to be a suitable candidate for a reliable oxygen probe in living organisms.