In this contribution I describe my personal memories of my work with Herman Berendsen and the Biophysical chemistry group in Groningen in the period of years from 1967 to 1975.

My memories of professor Herman Berendsen cover roughly two periods during which I had many contacts with him. Between 1966 and 1973 I was his MSc student and later his PhD student in the Department of Biophysical Chemistry at the University of Groningen. The second period started in 1991 when I returned to the University of Groningen as a professor of environmental sciences.

Exosomes are responsible for cell-to-cell communication and serves as a valuable drug delivery vehicle. However, exosome heterogeneity, non-standardized isolation methods and proteomics/bioinformatics approaches limit its clinical applications. To better understand exosome heterogeneity, biological function and molecular mechanism of its biogenesis, secretion and uptake, techniques in proteomics or bioinformatics were applied to investigate human embryonic kidney cell (293T cell line)-derived exosome proteome and enable an integrative comparison of exosomal proteins and protein-protein interaction (PPI) networks of eleven exosome proteomes extracted from diverse human samples, including 293T (two datasets), dermal fibroblast, mesenchymal stem cell, thymic epithelial primary cell, breast cancer cell line (MDA-MB-231), patient neuroblastoma cell, plasma, saliva, serum and urine. Mapping of exosome biogenesis/secretion/uptake-related proteins onto exosome proteomes highlights exosomal origin-specific routes of exosome biogenesis/secretion/uptake and exosome-dependent intercellular communication. The finding provides insight into comparative exosome proteomes and its biogenesis, secretion and uptake, and potentially contributes to clinical applications.

Xanthorhodopsin (XR) from Salinibacter ruber is a light-driven proton pump containing retinal and a light-harvesting carotenoid antenna salinixanthin. Previous structure-functional studies of XR were conducted using a protein isolated from the native host only due to the absence of heterologous expression in Escherichia coli. In this paper, we describe cell-free synthesis and incorporation in lipid-protein nanodiscs of the recombinant XR that demonstrated its principal compatibility with E. coli biosynthetic machinery. To produce XR in E. coli, three C-terminal deletion variants of this protein were constructed. In contrast to the full-length XR, their expression resulted in efficient synthesis in E. coli cells. However, cells producing recombinant XR variants bound retinal only upon growth in minimal medium, not in the rich one. The XR3 variant with deletion of ten C-terminal amino acid residues was obtained and characterized. Its absorption spectrum and photocycle kinetics were close to those reported for XR isolated from S. ruber membranes and bleached from salinixanthin. We have also constructed the first mutants of XR, H62M and D96N, and examined their properties.

This paper describes the start of the Biophysical Chemistry group of prof. Herman J.C. Berendsen (1934-2019) in Groningen, The Netherlands in the years from 1964 to 1974.

Analysing protein conformational ensembles whether from molecular dynamics (MD) simulation or other sources for functionally relevant conformational changes can be very challenging. In the nineteen nineties dimensional reduction methods were developed primarily for analysing MD trajectories to determine dominant motions with the aim of understanding their relationship to function. Coarse-graining methods were also developed so the conformational change between two structures could be described in terms of the relative motion of a small number of quasi-rigid regions rather than in terms of a large number of atoms. When these methods are combined, they can characterize the large-scale motions inherent in a conformational ensemble providing insight into possible functional mechanism. The dimensional reduction methods first applied to protein conformational ensembles were referred to as Quasi-Harmonic Analysis, Principal Component Analysis and Essential Dynamics Analysis. A retrospective on the origin of these methods is presented, the relationships between them explained, and more recent developments reviewed.

Bacterial biofilms are widespread in the environment, and bacteria in the biofilm are highly resistant to antibiotics and possess host immune defense mechanisms, which can lead to serious clinical and environmental health problems. The increasing problem of bacterial resistance caused by the irrational use of traditional antimicrobial drugs has prompted the search for better and novel antimicrobial substances. In this paper, we review the effects of phage endolysins, modified phage endolysins, and their combination with other substances on bacterial biofilms and provide an outlook on their practical applications. Phage endolysins can specifically and efficiently hydrolyze the cell walls of bacteria, causing bacterial lysis and death. Phage endolysins have shown superior bactericidal effects in vitro and in vivo, and no direct toxicity in humans has been reported to date. The properties of phage endolysins make them promising for the prevention and treatment of bacterial infections. Meanwhile, endolysins have been genetically engineered to exert a stronger scavenging effect on biological membranes when used in combination with antibiotics and drugs. Phage endolysins are powerful weapons for controlling bacterial biofilms.

This letter gives a description of my interactions with Herman over more than 30 years. His teaching, his tremendous insight in subjects newly presented to him, his generousness, his playful interactions with colleagues and students, the freedom in research direction that he allowed and also expected from his PhD students.

Due to the importance of protein-protein interactions in defence mechanism of living body, attempts were made to investigate its attributes, including, but not limited to, binding affinity, and binding region. Contemporary strategies for binding site prediction largely resort to deep learning techniques but turned out to be low precision models. As laboratory experiments for drug discovery tasks utilize this information, increased false positives devalue the computational methods. This emphasize the need to develop enhanced strategies. DeepBindPPI employs deep learning technique to predict the binding regions of proteins, particularly antigen-antibody interaction sites. The results obtained are applied in a docking environment to confirm their correctness. An integration of graph convolutional network with attention mechanism predicts interacting amino acids with improved precision. The model learns the determining factors in interaction from a general pool of proteins and is then fine-tuned using antigen-antibody data. Comparison of the proposed method with existing techniques shows that the developed model has comparable performance. The use of a separate spatial network clearly improved the precision of the proposed method from 0.4 to 0.5. An attempt to utilize the interface information for docking using the HDOCK server gives promising results, with high-quality structures appearing in the top10 ranks.

Alchemical free energy calculations have become a standard and widely used tool, in particular for calculating and comparing binding affinities of drugs. Although methods to compute such free energies have improved significantly over the last decades, the choice of path between the end states of interest is usually still the same as two decades ago. We will show that there is a fundamentally arbitrary, implicit choice of parametrization of this path. To address this, the notion of the length of a path or a metric is required. A metric recently introduced in the context of the accelerated weight histogram method also proves to be very useful here. We demonstrate that this metric can not only improve the efficiency of sampling along a given path, but that it can also be used to improve the actual choice of path. For a set of relevant use cases, the combination of these improvements can increase the efficiency of alchemical free energy calculations by up to a factor 16.

Importin α is a nuclear transporter that binds to nuclear localization signals (NLSs), consisting of 7-20 positively charged amino acids found within cargo proteins. In addition to cargo binding, intramolecular interactions also occur within the importin α protein due to binding between the importin β-binding (IBB) domain and the NLS-binding sites, a phenomenon called auto-inhibition. The interactions causing auto-inhibition are driven by a stretch of basic residues, similar to an NLS, in the IBB domain. Consistent with this, importin α proteins that do not have some of these basic residues lack auto-inhibition; a naturally occurring example of such a protein is found in the apicomplexan parasite Plasmodium falciparum. In this report, we show that importin α from another apicomplexan parasite, Toxoplasma gondii, harbors basic residues (KKR) in the IBB domain and exhibits auto-inhibition. This protein has a long, unstructured hinge motif (between the IBB domain and the NLS-binding sites) that does not contribute to auto-inhibition. However, the IBB domain may have a higher propensity to form an α-helical structure, positioning the wild-type KKR motif in an orientation that results in weaker interactions with the NLS-binding site than a KRR mutant. We conclude that the importin α protein from T. gondii shows auto-inhibition, exhibiting a different phenotype from that of P. falciparum importin α. However, our data indicate that T. gondii importin α may have a low strength of auto-inhibition. We hypothesize that low levels of auto-inhibition may confer an advantage to these important human pathogens.

The inception of next generations sequencing technologies have exponentially increased the volume of biological sequence data. Protein sequences, being quoted as the 'language of life', has been analyzed for a multitude of applications and inferences. Owing to the rapid development of deep learning, in recent years there have been a number of breakthroughs in the domain of Natural Language Processing. Since these methods are capable of performing different tasks when trained with a sufficient amount of data, off-the-shelf models are used to perform various biological applications. In this study, we investigated the applicability of the popular Skip-gram model for protein sequence analysis and made an attempt to incorporate some biological insights into it. We propose a novel k-mer embedding scheme, Align-gram, which is capable of mapping the similar k-mers close to each other in a vector space. Furthermore, we experiment with other sequence-based protein representations and observe that the embeddings derived from Align-gram aids modeling and training deep learning models better. Our experiments with a simple baseline LSTM model and a much complex CNN model of DeepGoPlus shows the potential of Align-gram in performing different types of deep learning applications for protein sequence analysis.

Proteins can be oriented in the gas phase using strong electric fields, which brings advantages for structure determination using X-ray free electron lasers. Both the vacuum conditions and the electric-field exposure risk damaging the protein structures. Here, we employ molecular dynamics simulations to rehydrate and relax vacuum and electric-field exposed proteins in aqueous solution, which simulates a refinement of structure models derived from oriented gas-phase proteins. We find that the impact of the strong electric fields on the protein structures is of minor importance after rehydration, compared to that of vacuum exposure and ionization in electrospraying. The structures did not fully relax back to their native structure in solution on the simulated timescales of 200 ns, but they recover several features, including native-like intra-protein contacts, which suggests that the structures remain in a state from which the fully native structure is accessible. Our findings imply that the electric fields used in native mass spectrometry are well below a destructive level, and suggest that structures inferred from X-ray diffraction from gas-phase proteins are relevant for solution and in vivo conditions, at least after in silico rehydration.

To date, there have been no or just a few reports of successful cloning and expression to create biologically active ocins or bacteriocins. Cloning, expression, and production of class I ocins are problematic because of their structural arrangements, coordinated functions, size, and posttranslational modifications. Mass synthesis of these molecules is necessary for commercialization and to restrict the excessive use of conventional antibiotics, which encourages the development of antibiotic-resistant bacteria. In the case of class III ocins, there are no reports of obtaining biological active proteins to date. Being able to obtain biologically active proteins requires an understanding of mechanistic features due to their expanding importance and broad spectrum of activity. As a result, we intend to clone and express the class III type. The class I types that are devoid of posttranslational modifications were transformed into class III through fusion. Therefore, this construct resembles a class III type ocin. With the exception of Zoocin, expression of the proteins was found to be physiologically ineffective after cloning. But, few cell morphological changes such as elongation, aggregation, and the formation of terminal hyphae were observed. However, it was discovered that the target indicator had been altered to Vibrio spp. in a few. All the three ocins were subjected to in-silico structure prediction/analysis. Finally, we confirm the existence of unidentified additional intrinsic factors for successful expression to obtain biologically active protein.

There still is little treatment available for amyloid diseases, despite their significant impact on individuals and the social and economic implications for society. One reason for this is that the physical nature of amyloid formation is not understood sufficiently well. Therefore, fundamental research at the molecular level remains necessary to support the development of therapeutics. A few structures of short peptides from amyloid-forming proteins have been determined. These can in principle be used as scaffolds for designing aggregation inhibitors. Attempts to this end have often used the tools of computational chemistry, in particular molecular simulation. However, few simulation studies of these peptides in the crystal state have been presented so far. Hence, to validate the capability of common force fields (AMBER19SB, CHARMM36m, and OPLS-AA/M) to yield insight into the dynamics and structural stability of amyloid peptide aggregates, we have performed molecular dynamics simulations of twelve different peptide crystals at two different temperatures. From the simulations, we evaluate the hydrogen bonding patterns, the isotropic B-factors, the change in energy, the Ramachandran plots, and the unit cell parameters and compare the results with the crystal structures. Most crystals are stable in the simulations but for all force fields there is at least one that deviates from the experimental crystal, suggesting more work is needed on these models.

This paper describes the scientific work of Prof. Dr. Herman Berendsen on NMR spectroscopy and includes some personal notes. Since 1975, Berendsen and the author were colleagues in the Physical Chemistry group in Groningen for a period of 12 years.

Metamorphic, or fold-switching, proteins feature different folds that are physiologically relevant. The human chemokine XCL1 (or Lymphotactin) is a metamorphic protein that features two native states, an [Formula: see text] and an all[Formula: see text] fold, which have similar stability at physiological condition. Here, extended molecular dynamics (MD) simulations, principal component analysis of atomic fluctuations and thermodynamic modeling based on both the configurational volume and free energy landscape, are used to obtain a detailed characterization of the conformational thermodynamics of human Lymphotactin and of one of its ancestors (as was previously obtained by genetic reconstruction). Comparison of our computational results with the available experimental data show that the MD-based thermodynamics can explain the experimentally observed variation of the conformational equilibrium between the two proteins. In particular, our computational data provide an interpretation of the thermodynamic evolution in this protein, revealing the relevance of the configurational entropy and of the shape of the free energy landscape within the essential space (i.e., the space defined by the generalized internal coordinates providing the largest, typically non-Gaussian, structural fluctuations).

The number of disease states linked the aberrant regular protein conformations to oligomers and amyloid fibrils. Amyloid beta 1-42 (Aβ1-42) peptide is very hydrophobic and quickly forms the β-rich structure and fibrillar protein aggregates in some solutions and buffer conditions. Ultrasonication pulses can disrupt amyloid fibrils to smaller fragments and produce Aβ1-42 peptides of different sizes and oligomers. Herein, we investigated the effects of buffer and ultrasonication on Aβ1-42 structure at low and high concentrations. After ultrasonication, the Western blot results showed that Aβ1-42 fibrils were disaggregated into different sizes. The transmission electron microscopy results indicated Aβ1-42 at low concentration (25 µM) in Ham's/F12 phenol red-free culture medium formed short-size fragments and oligomers. In comparison, Aβ1-42 at higher concentration (100 µM) formed fibrils that break down into smaller fragments after ultrasonication. However, after regrowth, it formed mature fibrils again. Cell viability assay indicated that Aβ1-42 oligomers formed at a low concentration (25 µM) were more toxic to PC12 cells than other forms. In conclusion, by applying ultrasonication pulses and controlling peptide concentration and buffer condition, we can rich Aβ1-42 aggregates with a particular size and molecular structure.

Tryptic hydrolysates of protein fractions obtained by the Osborne method from chickpea (Cicer arietinum L.) seeds interacted with zinc ions and the results of chelation were monitored by the Energy Dispersive X-Ray (EDX) technique. The glutelin hydrolysate (GluHyd) reacted with zinc ions and depicted a relatively higher zinc content. For this reason, the zinc complex of the glutelin hydrolysate (GluHyd-Zn) was studied deeper, and 11 peptides were identified in its more zinc-containing second fraction obtained after gel filtration. The peptide HKERVQLHIIPTAVGK showed a relatively higher chelating capacity (57.86 ± 2.14%). According to the result of the ICP-OS analysis, 1 mg peptide could chelate 381.61 ± 133.39 µg zinc, and the molar ratio of peptide-zinc was about 1:4. Spectral methods proved that side chain and C-termini carboxyl groups of the peptide mostly were involved in chelation and N atoms of amino side chains, imidazole group of histidine, and N-termini at some extents were occupied by the metal ions. Modeling of zinc-peptide interaction was done using Molecular Operating Environment (MOE) software. The results of the docking correlate with the experimental data.ACE2 inhibitory effect of HKERVQLHIIPTAVGK-Zn complex (IC50 = 1.5 mg/mL) was better than that of HKERVQLHIIPTAVGK (IC50 = 2.2 mg/mL).

Due to its ability to reversibly bind O2, alongside a relatively low redox reactivity and a limited cytotoxicity, the oxygen-carrying protein hemerythrin has been considered as an alternative to hemoglobin in preparing blood substitutes. In order to increase the hydrodynamic volume and lower antigenicity, two site-directed variants, H82C and K92C, were engineered that contained a single cysteine residue on the surface of each hemerythrin octamer for the specific attachment of polyethylene glycol (PEG). A sulfhydryl-reactive PEGylation reagent with a 51.9 Å spacer arm was used for selective cysteine derivatization. The mutants were characterized by UV-vis spectroscopy, size-exclusion chromatography, oxygen affinity, and autooxidation rate measurements. The H82C variant showed altered oligomeric behavior compared to the wild-type and was unstable in the met form. The PEGylated K92C variant is reasonably stable, displays an oxygen affinity similar to that of the wild-type, and shows an increased rate of autoxidation; the latter disadvantage may be counteracted by further chemical modifications.

Amaranthaceae α-amylase inhibitors (AAIs) are knottin-type proteins with selective inhibitory potential against coleopteran α-amylases. Their small size and remarkable stability make them exciting molecules for protein engineering to achieve superior selectivity and efficacy. In this report, we have designed a set of AAI pro- and mature peptides chimeras. Based on in silico analysis, stable AAI chimeras having a stronger affinity with target amylases were selected for characterization. In vitro studies validated that chimera of the propeptide from Chenopodium quinoa α-AI and mature peptide from Beta vulgaris α-AI possess 3, 7.6, and 4.26 fold higher inhibition potential than parental counterparts. Importantly, recombinant AAI chimera retained specificity towards target coleopteran α-amylases. In addition, to improve the inhibitory potential of AAI, we performed in silico site-saturation mutagenesis. Computational analysis followed by experimental data showed that substituting Asparagine at the 6th position with Methionine had a remarkable increase in the specific inhibition potential of Amaranthus hypochondriacus α-AI. These results provide structural-functional insights into the vitality of AAI propeptide and a potential hotspot for mutagenesis to enhance the AAI activity. Our investigation will be a toolkit for AAI's optimization and functional differentiation for future biotechnological applications.

The mechanism by which glycoside hydrolases control the reaction specificity through hydrolysis or transglycosylation is a key element embedded in their chemical structures. The determinants of reaction specificity seem to be complex. We looked for structural differences in domain B between the 4-α-glucanotransferase from Thermotoga maritima (TmGTase) and the α-amylase from Thermotoga petrophila (TpAmylase) and found a longer loop in the former that extends towards the active site carrying a W residue at its tip. Based on these differences we constructed the variants W131G and the partial deletion of the loop at residues 120-124/128-131, which showed a 11.6 and 11.4-fold increased hydrolysis/transglycosylation (H/T) ratio relative to WT protein, respectively. These variants had a reduction in the maximum velocity of the transglycosylation reaction, while their affinity for maltose as the acceptor was not substantially affected. Molecular dynamics simulations allow us to rationalize the increase in H/T ratio in terms of the flexibility near the active site and the conformations of the catalytic acid residues and their associated pKas.

Hydrazoic acid (HN3) and its deprotonated form azide ion (N3-) (AHA) are toxic because they inhibit the cytochrome c oxidase complex IV (CoX IV) embedded in the inner mitochondrial membrane that forms part of the enzyme complexes involved in cellular respiration. Critical to its toxicity is the inhibition of CoX IV in the central nervous system and cardiovascular system. Hydrazoic acid is an ionizable species and its affinity for membranes, and the associated permeabilities, depend on the pH values of aqueous media on both sides of the membranes. In this article, we address the permeability of AHA through the biological membrane. In order to understand the affinity of the membrane for the neutral and ionized form of azide, we measured the octanol/water partition coefficients at pH values of 2.0 and 8.0, which are 2.01 and 0.00034, respectively. Using a Parallel Artificial Membrane Permeability Assay (PAMPA) experiment, we measured the effective permeability through the membrane, which is logPe - 4.97 and - 5.26 for pH values of 7.4 and pH 8.0, respectively. Experimental permeability was used to validate theoretical permeability, which was estimated by numerically solving a Smoluchowski equation for AHA diffusion through the membrane. We demonstrated that the rate of permeation through the cell membrane of 8.46·104 s-1 is much higher than the rate of the chemical step of CoX IV inhibition by azide of 200 s-1. The results of this study show that transport through the membrane does not represent the rate-limiting step and therefore does not control the rate of CoX IV inhibition in the mitochondria. However, the observed dynamics of azide poisoning is controlled by circulatory transport that takes place on a time scale of minutes.

The rise of New Delhi metallo beta-lactamase (NDM) producing bacteria imposes a significant threat to the treatment of bacterial infections due to their broad spectrum against beta-lactams. The activity of metallo beta-lactamases is affected by active site residues as well as residues near the active site. Therefore, we aimed to identify the amino acid residues around the active site of NDM-4 which influence its function. To achieve that, seven substitution mutations (S191A, D192A, S213A, K216A, S217A, D223A and D225A) of NDM-4 were generated through site-directed mutagenesis. Out of these, expression of NDM-4_D192A and NDM-4_S217A in Escherichia coli cells increased the beta-lactam susceptibility as compared to NDM-4. Further, proteins were purified to assess the effect of substitution mutations on zinc content, in vitro catalytic efficiency, and stability of NDM-4. The catalytic efficiency was reduced for these mutants (D192A and S217A) towards beta-lactam substrates, while the thermal stability remained insubstantial as compared to NDM-4. However, the purified NDM-4_D192A exhibited altered zinc content. In silico studies reveal that these changes might be the outcomes of alterations in hydrogen bonding networks and substrate interactions. Taken together, we infer that the D192 and the S217 residues play a substantial role in the activity of NDM-4.

Biological control to prevent fungal plant diseases offers an alternative approach to facilitate sustainable agriculture. Since the chitin in fungal cell walls is a target for biocontrol agents, chitinases are one of the important antifungal molecules. In this study, the aim was to investigate a new chitinase isolated from a fluvial soil bacterium and to show the antifungal activity of the characterized chitinase by comparing the three common methods. The bacterium with the highest chitinase activity was identified as Aeromonas sp. by 16 S rRNA sequence analysis. Following the determination of the optimum enzyme production time, the enzyme was partially purified, and the physicochemical parameters of the enzyme were investigated. In the antifungal studies, direct Aeromonas sp. BHC02 cells or partially purified chitinase were used. As a result, in the first method in which the Aeromonas sp. BHC02 cells were spread on the surface of petri dishes, no zone formation was observed around the test fungi spotted on the surface. However, zone formation was observed in the methods in which the antifungal activity was investigated using the partially purified chitinase enzyme. For example, in the second method, the enzyme was spread on the surface of PDA, and zone formation was observed only around Penicillum species among the test fungi spotted on the surface. In the third method, in which the necessary time was given for the formation of mycelium of the test fungi, it was observed that the growth of Fusarium solani, Alternaria alternata and Botrytis cinerea was inhibited by the partially purified chitinase. This study concludes that the results of the antifungal activities depend on the method used and all fungal chitins cannot be degraded with one strain's chitinase. Depending on the variety of chitin, some fungi can be more resistant.

Tuberculosis caused by Mycobacterium tuberculosis (M.tb) has killed millions worldwide. Antibiotic resistance leads to the ineffectiveness of the current therapies. Aminoacyl tRNA synthetase (aaRS) class of proteins involved in protein synthesis are promising bacterial targets for developing new therapies. Here, we carried out a systematic comparative study on the aaRS sequences from M.tb and human. We listed important M.tb aaRS that could be explored as potential M.tb targets alongside the detailed conformational space analysis of methionyl-tRNA synthetase (MetRS) in apo- and substrate-bound form, which is among the proposed targets. Understanding the conformational dynamics is central to the mechanistic understanding of MetRS, as the substrate binding leads to the conformational changes causing the reaction to proceed. We performed the most complete simulation study of M.tb MetRS for 6 microseconds (2 systems × 3 runs × 1 microsecond) in the apo and substrate-bound states. Interestingly, we observed differential features, showing comparatively large dynamics for the holo simulations, whereas the apo structures became slightly compact with reduced solvent exposed area. In contrast, the ligand size decreased significantly in holo structures possibly to relax ligand conformation. Our findings correlate with experimental studies, thus validating our protocol. Adenosine monophosphate moiety of the substrate exhibited quite higher fluctuations than the methionine. His21 and Lys54 were found to be the important residues forming prominent hydrogen bond and salt-bridge interactions with the ligand. The ligand-protein affinity decreased during simulations as computed by MMGBSA analysis over the last 500 ns trajectories, which indicates the conformational changes upon ligand binding. These differential features could be further explored for designing new M.tb inhibitors.

COVID-19 is a disease that have affected the entire world, and it continues to spread with new variants. A patient's innate immune system plays a critical role in the mild and severe transition of COVID-19. Antimicrobial peptides (AMPs), which are important components of the innate immune system, are potential molecules to fight pathogenic bacteria, fungi, and viruses. Human β-defensin 2 (hBD-2), a 41-amino-acid antimicrobial peptide, is one of the defensins inducibly expressed in the skin, lungs, and trachea in humans. In this study, it was aimed to investigate the interaction of hBD-2 produced recombinantly in Pichia pastoris with the human angiotensin-converting enzyme 2 (ACE-2) under in vitro conditions. First, hBD-2 was cloned in P. pastoris X-33 via the pPICZαA vector, a yeast expression platform, and its expression was confirmed by SDS-PAGE, western blotting, and qRT-PCR. Then, the interaction between recombinant hBD-2 and ACE-2 proteins was revealed by a pull-down assay. In light of these preliminary experiments, we suggest that the recombinantly produced hBD-2 may be protective against SARS-CoV-2 and be used as a supplement in treatment. However, current findings need to be supported by cell culture studies, toxicity analyses, and in vivo experiments.

The complete enzymatic degradation of lignocellulosic biomass requires the cooperative action of cellulosic, hemicellulosic, and lignolytic enzymes such as cellulase, xylanase, laccase, galactosidase, and arabinofuranosidase. Arabinofuranosidases (E.C 3.2.1.55), which belong to the glycoside hydrolase family of enzymes, hydrolyze the 1,3- and 1,5-α-arabinosyl bonds in L-arabinose- containing molecules. L-arabinoses are present in hemicellulosic part of lignocellulosic biomass. Arabinofuranosidases also play an important role in the complete hydrolysis of arabinoxylans. Analysis of the genome project and CAZY database revealed two putative arabinofuranosidase genes in the A. acidocaldarius genome. The aim of the study was cloning, heterologous expression, purification and biochemical characterization of the arabinofuranosidase enzyme encoded in A. acidocaldarius genome. For this purpose, the AbfA gene of the arabinofuranosidase protein was cloned into the pQE-40 vector, heterologously expressed in E. coli BL21 GOLD (DE3) and successfully purified using His-Tag. Biochemical characterization of the purified enzyme revealed that A. acidocaldarius arabinofuranosidase exhibited activity over a wide pH and temperature range with optimum activity at 45 ºC and pH 6.5 in phosphate buffer towards 4-nitrophenyl-α-L-arabinofuranoside as the substrate. In addition, the enzyme is highly stable over wide range of temperature and maintaining 60% of its activity after 90 min of incubation at 80 ºC. Through the bioinformatics studies, the homology model of A. acidocaldarius arabinofuranosidase was generated and the substrate binding site and residues located in this site were identified. Further molecular docking analysis revealed that the substrate located in the catalytically active pose and, residues N174, E175, and E294 have direct interaction with 4-nitrophenyl-α-L-arabinofuranoside. Moreover, based on phylogenetic analysis, A. acidocaldarius arabinofuranosidase exists in the sub-group of intracellular arabinofuranosidases, and G. stearothermophilus and B.subtilis arabinofuranosidases are close relatives of A. acidocaldarius arabinofuranosidase. This is the first study to report the gene cloning, recombinant expression and biochemical and bioinformatic characterization of an auxiliary GH51 arabinofuranosidase from an acidothermophilic bacterium A. acidocaldarius.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder that affects approximately 1/68 children, with a more recent study suggesting numbers as high as 1/36. According to Diagnostic and Statistical Manual of Mental Disorders, the etiology of ASD is unknown and diagnosis of this disorder is behavioral. There is currently no biomarker signature for ASD, however, identifying a biomarker signature is crucial as it would aid in diagnosis, identifying treatment targets, monitoring treatments, and identifying the etiology of the disorder. Here we used nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) to investigate the saliva from individuals with ASD and matched controls in a 14 vs 14 study. We found numerous proteins to have statistically significant dysregulations, including lactotransferrin, transferrin, polymeric immunoglobulin receptor, Ig A L, Ig J chain, mucin 5 AC, and lipocalin 1 isoform X1. These findings are consistent with previous studies by our lab, and others, and point to dysregulations in the immune system, lipid metabolism and/or transport, and gastrointestinal disturbances, which are common and reoccurring topics in ASD research.

Small structural E protein of coronaviruses uses its C-terminal PDZ motif to compromise the cellular PDZ interactome. In this work we compared core PDZ interactivity of small (seven amino acids) peptide PDZ motifs, originating from the envelope proteins of recently transmitted coronaviruses SARS-CoV, SARS-CoV2, and MERS-CoV. As the interaction targets we used 23 domains of the largest PDZ proteins MUPP1/MPDZ and PATJ/INAD. Results revealed exceptional affinity and interaction promiscuity of MERS-CoV PDZ motif in vitro, suggesting an increased probability of potential PDZ targets in vivo. We hypothesize that together with its known ability to enter the cells from both apical and basolateral sites, this might further contribute to its elevated disruption of cellular PDZ pathways and higher virulence.

Antimicrobial peptides (AMPs) are a kind of small molecular peptide that an organism produces to resist the invasion of foreign microorganisms. AMP BSN-37 is a bovine AMP that exhibits high antibacterial activity. In this paper, the optimized gene AMP BSN-37 was cloned into pCold-SUMO for fusion expression by recombinant DNA technology. The gene sequence of AMP BSN-37 was obtained by codons reverse translation, and the codons were optimized according to the codons preference of Escherichia coli (E. coli). The recombinant plasmid was constructed and identified by PCR, enzyme digestion and sequencing. Then the recombinant plasmid was transformed into BL21 E. coli to induce expression, and the IPTG concentration and time were optimized. The expressed soluble fusion protein SUMO-BSN-37 was purified by chromatography and then cleaved by SUMO proteases to release BSN-37. SDS-PAGE electrophoresis and Western blotting were used for identification. The recombinant plasmid pCold-SUMO-BSN-37 was obtained, and the fusion AMP BSN-37 was preliminarily expressed in BL21. After optimization, the optimal expression condition was 37 ℃ with 0.4 µM IPTG and 6 h incubation. Under optimal conditions, a large amount of fusion AMP BSN-37 was obtained by purification. Western blotting showed that the fusion peptide was successfully expressed and had good activity. The expressed BSN-37 showed antimicrobial activity similar to that of synthesized BSN-37. In this study, soluble expression products of AMP BSN-37 were obtained, and the problem regarding the limited source of AMP BSN-37 could be effectively solved, laying a foundation for further research on AMP BSN-37.

HIV-1 protease is essential for the production of mature, infectious virions and is a major target in antiretroviral therapy. We successfully purified a HIV-1 subtype C variant, L38↑N↑L- 4, containing an insertion of asparagine and leucine at position 38 without the four background mutations - K20R, E35D, R57K, V82I using a modified purification protocol. Isothermal titration calorimetry indicated that 50% of the variant protease sample was in the active conformation compared to 62% of the wild type protease. The secondary structure composition of the variant protease was unaffected by the double insertion. The specific activity and kcat values of the variant protease were approximately 50% lower than the wild type protease values. The variant protease also exhibited a 1.6-fold increase in kcat/KM when compared to the wild type protease. Differential scanning calorimetry showed a 5 °C increase in Tm of the variant protease, indicating the variant was more stable than the wild type. Molecular dynamics simulations indicated the variant was more stable and compact than the wild type protease. A 3-4% increase in the flexibility of the hinge regions of the variant protease was observed. In addition, increased flexibility of the flaps, cantilever and fulcrum regions of the variant protease B chain was observed. The variant protease sampled only the closed flap conformation indicating a potential mechanism for drug resistance. The present study highlights the direct impact of a double amino acid insertion in hinge region on enzyme kinetics, conformational stability and dynamics of an HIV-1 subtype C variant protease.

Glucose-6-phosphate isomerase (GPI) is a highly conserved glycolytic enzyme in nature, and less information was available for GPI from hens. In this study a newly discovered selenocysteine (Sec)-containing GPI in common chicken breast meat was first isolated, purified and identified. Data about LC-MS/MS, FTIR and Se species analyses show that the molecular weight of the enzyme is 62,091 Da and only one Sec is inserted at the 403rd position in the highly conserved primary domain SIS_PGI with sugar conversion function. The enzyme shows excellent activity against hydroxyl radicals as vitamin C (Vc) in vitro. It is deduced that the Sec-containing GPI in the chicken meat may depend on Sec in its molecular structure to resist reactive oxygen species (ROS) stress produced by the accompanying biochemical reactions in cells, to protect its stability and maintain its efficient function that catalyzes the conversion of glucose-6-phosphate to fructose-6-phosphate in the critical glycolytic pathway.

A personal perspective on the scientific and social dimensions of interactions with Herman Berendsen from 1969 through 2019 shows how his intellect and character had an influence beyond his laboratory.

Glutathione S-Transferase (GST) enzyme is abundant in mammals, insects, fish and microorganisms, as well as in various tissues of these species, particularly in tissues exposed to xenobiotics from the environment. As a result, the enzyme execute detoxifying function by scavenging a diverse range of xenobiotics, such as chemotherapeutic medicines, environmental carcinogens and endogenous compounds. In this study, GST enzyme was partially purified from mallow (Malva slyvestris L.) seed for the first time and the kinetic parameters were determined. The optimum ionic intensity was found in 400 mM Tris-Buffer, optimum pH: 7.0, and optimum substrate concentration was determined as 0.2 mM. One of the biggest reasons for deterioration of ecological balance in nature is heavy metal accumulation in soil, air and water which becomes a major threat to the vital activities of living things. In this study, inhibitory effects of Cd+ 2, Ag+, Zn+ 2 and Fe+ 3 heavy metals, which are common in nature, on mallow seed glutathione S-transferase enzyme were investigated. Each heavy metal showed micromolar inhibitory effects on enzyme activity. IC50 values of the metals were calculated as 60.93, 74.602, 178.22 and 369 µM, respectively.

Quorum sensing (QS) is the process by which microorganisms employ chemicals called autoinducers (AIs) to communicate with their population. The QS mechanism generally controls the expression of the virulence related genes in bacteria. N-acyl homoserine lactones (AHLs) are the most widespread QS molecules. Due to their diverse AHL-lactonase activities, Bacillus species make particularly suitable candidates for procedures such as demolition of pathogenic bacterial QS signals and bioremediation of β-lactam antibiotics from contaminated environments. In this study, seven Bacillus strains with Quorum quenching (QQ) activity were isolated using an enrichment medium supplemented with Penicillin G (PenG). The AHL-lactonase encoding gene (aiiA) was amplified by PCR and sequenced. Amino acid sequences underwent multiple sequence alignment. Docking studies were carried out with both C6HSL and PenG ligand using AutoDock tools. The aiiA amino acid sequences of the isolates were found to be well conserved. Furthermore, amino acid sequence alignment revealed that 74.9% of amino acid sequences were conserved in the genus Bacillus. Docking of the C6HSL to wild type (3DHA) and H97D variant reduced the docking score by only 0.1 kcal/mol for the mutated protein. When PenG docked with a higher (1.5 kcal/mol) score as a ligand to wild-type and mutant receptors, the docking score for the mutated protein likewise decreased by 0.1 kcal/mol. This research contributed to the diversification of organisms with QQ activity and beta-lactam antibiotic resistance. It also clarified the binding score of the PenG ligand to the Bacillus AHL lactonase molecule for the first time.

Bacteriophage endolysins have been shown to hold great promise as new antibacterial agents for animal and human health in food preservation. In the present study, endolysin from Staphylococcus aureus subsp. aureus ATCC 27692-B1 bacteriophage 52 (LysSA52) was cloned, expressed, and characterized for its antimicrobial properties. Following DNA extraction from bacteriophage 52, a 1446-bp DNA fragment containing the endolysin gene (lysSA52) was obtained by PCR amplification and cloned into pET SUMO expression vector. The positive clone was validated by sequencing and open-reading frame analysis. The LysSA52 sequence shared high homology with staphylococcal phage endolysins of the SA12, SA13, and DSW2 phages and others. The cloned lysSA52 gene encoding 481 amino acids endolysin was expressed in Escherichia coli BL21 with a calculated molecular mass of 66 kDa (LysSA52). This recombinant endolysin LysSA52 exhibited lytic activity against 8 of 10 Gram-positive bacteria via agar spot-on lawn antimicrobial assay, including methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Streptococcus pneumonia, Streptococcus pyogenes, Enterococcus faecium, Enterococcus faecalis, and Bacillus atrophaeus. In addition, the 0.50 mg/mL, LysSA52 endolysins reduced about 60% of the biofilms of S. aureus and S. epidermidis established on a microtiter plate in 12 h treatment. The data from this study indicate that LysSA52 endolysin could be used as an antibacterial protein to prevent and treat infections caused by staphylococci and several other Gram-positive pathogenic bacteria irrespective of their antibiotic resistance.

The efficacy of human recombinant insulin can be affected by its aggregation. Effects of acetylation were observed on insulin structure, stability, and aggregation at 37 and 50 °C and pH of 5.0 and 7.4 with the use of spectroscopy, circular dichroism (CD), dynamic light scattering (DLS), and atomic force microscopy (AFM). Raman and FTIR results were indicative of structural changes in AC-INS, and CD analyses showed a slight increase in β-sheet content in AC-INS. Melting temperature (Tm) measurements indicated an overall more stable structure and spectroscopic assessment showed a more compact one. Formation of amorphous aggregates was followed over time and kinetics parameters showed a longer nucleation phase (higher t* amount) and lower aggregates amount (lower Alim) for acetylated insulin (AC-INS) compared to native (N-INS) in all tested conditions. The results of amyloid-specific probes approved the formation of amorphous aggregates. Size particle and microscopic analysis suggested that AC-INS was less prone to form aggregates, which were smaller if formed. In conclusion, this study has demonstrated that controlled acetylation of insulin may lead to its higher stability and lower propensity toward amorphous aggregation and has provided insight into the result of this type of post-translational protein modification.

In many bacteria, the High Temperature requirement A (HtrA) protein functions as a chaperone and protease. HtrA is an important factor in stress tolerance and plays a significant role in the virulence of several pathogenic bacteria. Camostat, gabexate and nafamostat mesylates are serine protease inhibitors and have recently shown a great impact in the inhibition studies of SARS-CoV2. In this study, the inhibition of Listeria monocytogenes HtrA (LmHtrA) protease activity was analysed using these three inhibitors. The cleavage assay, using human fibrinogen and casein as substrates, revealed that the three inhibitors effectively inhibit the protease activity of LmHtrA. The agar plate assay and spectrophotometric analysis concluded that the inhibition of nafamostat (IC50 value of 6.6 ± 0.4 µM) is more effective compared to the other two inhibitors. Previous studies revealed that at the active site of the protease, these inhibitors are hydrolysed and one of the hydrolysates is covalently bound to the active site serine. To understand the mode of binding of these inhibitors at the active site of LmHtrA, docking of the inhibitors followed by molecular dynamics simulations were carried out. Analysis of the LmHtrA-inhibitor complex structures revealed that the covalently bound inhibitor is unable to occupy the S1 pocket of the LmHtrA which is in contrast to the previously determined camostat and nafamostat complex structures. This observation provides the first glimpse of the substrate specificity of LmHtrA which is not known. The obtained results also suggest that the development of novel inhibitors of LmHtrA and its homologs with active site architecture similar to LmHtrA can be pursued with suitable modification of these inhibitors. To date, only a very few studies have been carried out on identifying the inhibitors of HtrA proteolytic activity.

The majority of the clotting factors involved in blood coagulation pathways are serine proteases and thrombin is one of the key serine proteases involved in blood clotting. Many synthetic and chemical drugs targeting these proteases as therapeutics are known. However, they are associated with serious side effects such as bleeding, haemorrhage, edema etc. Serine protease inhibitors from plants have been suggested as one of the potential anticoagulant molecules against thrombosis. In the present work, a direct thrombin inhibitor from Moringa oleifera was isolated, purified and characterized. The homogeneity of the inhibitor is confirmed on native- PAGE. The purified inhibitor (5 µg) showed 63% thrombin inhibition at pH 7.2 at 37 °C. The IC50 value of the isolated inhibitor was determined as 4.23 µg. The inhibitor on SDS-PAGE appeared as a single protein-stained band corresponding to 50 kDa thereby indicating its molecular weight as 50 kDa. Purified thrombin inhibitor (5 µg) showed 12% inhibition of trypsin, and 17% inhibition of chymotrypsin. This suggests more specificity of purified inhibitor towards thrombin. The isolated inhibitor showed a non-competitive mode of inhibition against thrombin as determined by the Dixon plot. The inhibition constant (Ki) was calculated as 4.35 × 10-7 M. The present work reports for the first time a direct thrombin inhibitor from M. oleifera which may be further explored as an antithrombotic drug.

Spider venom contains various peptides and proteins, which can be used for pharmacological applications. Finding novel therapeutic strategies against neurodegenerative diseases with the use of purified peptides and proteins, extracted from spiders can be greatly precious. Neurodegenerative diseases are rapidly developing and expanding all over the world. Excitotoxicity is a frequent condition amongst neuro-degenerative disorders. This harmful process is usually induced through hyper-activation of N-Methyl-D-Aspartate (NMDA) receptor, and P/Q-type voltage-gated calcium channels (VGCCs). The omega-agatoxin-Aa4b is a selective and strong VGCCblocker. This study aimed to investigate the effects of this blocker on the NMDA-induced memory and learning defect in rats. For this purpose, nineteen spiders of the funnel-weaver Agelena orientalis species were collected. The extracted venom was lyophilized andpurified through gel-filtration chromatography, and capillary electrophoresis techniques. Subsequently, mass spectrometry (HPLC-ESI-MS) was used for identification of this bio-active small protein. Afterward, the effect of the omega-agatoxin-Aa4b (2 μg, intra-cornu ammonis-3 of the hippocampus) on the NMDA-induced learning and memory deficits in rats was evaluated. Learning and memory performances were evaluated by the use of passive avoidance test. For synaptic quantification and memory function the amount of calcium/calmodulin-dependent protein kinase ІІ (CaCdPKІІ) gene expression was measured using the Real-time PCR technique. To compare the experimental groups, hematoxylin and eosin (H&E) staining of hippocampus tissues was performed. Our results rendered that the omega-Agatoxin-Aa4b treatment can ameliorate and reverse the learning and memory impairment caused by NMDA-induced excitotoxicity in rat hippocampus.

Muscle weakness as a secondary feature of attenuated neuronal input often leads to disability and sometimes death in patients with neurogenic neuromuscular diseases. These impaired muscle function has been observed in several diseases including amyotrophic lateral sclerosis, Charcot-Marie-Tooth, spinal muscular atrophy and Myasthenia gravis. This has spurred the search for small molecules which could activate fast skeletal muscle troponin complex as a means to increase muscle strength. Discovered small molecules have however been punctuated by off-target and side effects leading to the development of the second-generation small molecule, Reldesemtiv. In this study, we investigated the impact of Reldesemtiv binding to the fast skeletal troponin complex and the molecular determinants that condition the therapeutic prowess of Redesemtiv through computational techniques. It was revealed that Reldesemtiv binding possibly potentiates troponin C compacting characterized by reduced exposure to solvent molecules which could favor the slow release of calcium ions and the resultant sensitization of the subunit to calcium. These conformational changes were underscored by conventional and carbon hydrogen bonds, pi-alkyl, pi-sulfur and halogen interactions between Reldesemtiv the binding site residues. Arg113 (-3.96 kcal/mol), Met116 (-2.23 kcal/mol), Val114 (-1.28 kcal/mol) and Met121 (-0.63 kcal/mol) of the switch region of the inhibitory subunit were among the residues that contributed the most to the total free binding energy of Reldesemtiv highlighting their importance. These findings present useful insights which could lay the foundation for the development of fast skeletal muscle small molecule activators with high specificity and potency.

Pfu DNA polymerase is one of the most preferred molecular enzymes that is isolated from the hyperthermophilic Pyrococcus furiosus and used for high-throughput DNA synthesis by the polymerase chain reaction. Therefore, an efficient Pfu DNA polymerase production method is necessary for molecular techniques. In the present study, Pfu DNA polymerase was expressed in recombinant Escherichia coli BL21(DE3) and significant parameters for the biomass production were optimized using the central composite design which is the most popular method of response surface methodology. Induction conditions including cell density prior induction (OD600nm), post-induction temperature, IPTG concentration, and post-induction time and their interactions on biomass production were investigated. The maximum biomass production (14.1 g/L) in shake flasks was achieved using the following predicted optimal conditions: OD600nm before induction of 0.4 and the induction at 32 °C for 7.7 h, with 0.6 mM IPTG. Optimized culture conditions were implemented to scale up experiments. 22% and 70% increase in biomass production was achieved in 3 L and 10 L bioreactors, respectively as compared to initial biomass production observed in unoptimized conditions. Similary, a 30% increase of Pfu DNA polymerase production was obtained after the optimization. The polymerase activity of the purifed Pfu DNA polymerase was assessed by PCR amplification and determined as 2.9 U/μl by comparison with commercial Pfu DNA polymerase. The findings of this study indicated that the proposed fermentation conditions will contribute to further scale‑up studies to enhance the biomass for the production of other recombinant proteins.

BACKGROUND: The health benefits of natural products have a long history. Chaga (Inonotus obliques) is used in traditional medicine and is an essential antioxidant for protecting the body from oxidants. Reactive oxygen species (ROS) are produced routinely due to metabolic processes. However, environmental pollution factors such as methyl tert-butyl ether (MTBE) can increase oxidative stress in the human body. MTBE is widely used as a fuel oxygenator that can harm health. The widespread use of MTBE has posed significant threats to the environment by polluting environmental resources, including groundwater. This compound can accumulate in the bloodstream by inhaling polluted air, with a strong affinity for blood proteins. The primary mechanism of MTBE's harmful effects is ROS production. The use of antioxidants may help reduce MTBE oxidation conditions. The present study proposes that biochaga, as an antioxidant, can reduce MTBE damage in the bovine serum albumin (BSA) structure. METHODS AND RESULTS: This study investigated the role of different concentrations of biochaga in the structural change of BSA in the presence of MTBE by biophysical methods such as UV-Vis, fluorescence, FTIR spectroscopy, DPPH radical inhibition method, aggregation test, and molecular docking. Research at the molecular level is critical to investigate the structural change of proteins by MTBE and the protective effect of the ideal dose (2.5 µg/ml) of biochaga. CONCLUSION: the results of spectroscopic examinations showed that the concentration of 2.5 µg/ml of biochaga has the least destructive effect on the structure of BSA in the presence and absence of MTBE, and it can play as an antioxidant.

Influenza A virus hemagglutinin (HA) is a major virus antigen. No cryo-electron microscopy or X-ray data can be obtained for the HA intraviral (cytoplasmic) domain (CT) post-translationally modified with long fatty acid residues bound to three highly conserved cysteines. We recently proposed a model of HA CT of Influenza A/H1N1 virus possessing an antiparallel beta structure based on the experimental secondary structure analysis of four 14-15 amino acid long synthetic peptides, corresponding to the HA CT sequence, with free or acetaminomethylated cysteines. To dispel doubts about possible non-specific "amyloid-like" aggregation of those synthetic peptides in phosphate buffer solution, we have determined the order of oligomers based on blue native gel electrophoresis, membrane filtration, fluorescence spectroscopy and molecular modeling approaches. We have found that unmodified peptides form only low molecular weight oligomers, while modified peptides form both oligomers of low order similar to those found for unmodified peptides and high order conglomerates, which however are not of beta-amyloid-like fold. This study confirms that the beta structure previously detected by circular dichroism spectroscopy analysis is more likely the result of intrinsic propensity of the HA CT amino acid sequence than the consequence of aggregation. The structures of low order oligomers of the synthetic peptides were used for in silico experiments on modeling of HA CT interactions with matrix protein M1 at physiological and acidic pH levels and revealed two different areas of binding. Finally, tripeptides capable of blocking interactions between HA CT and M1 were proposed.