Single Polypeptide Chain Research Articles

Development and Optimization of a Redox Enzyme-Based Fluorescence Biosensor for the Identification of MsrB1 Inhibitors.

MsrB1 is a thiol-dependent enzyme that reduces protein methionine-R-sulfoxide and regulates inflammatory response in macrophages. Therefore, MsrB1 could be a promising therapeutic target for the control of inflammation. To identify MsrB1 inhibitors, we construct a redox protein-based fluorescence biosensor composed of MsrB1, a circularly permutated fluorescent protein, and the thioredoxin1 in a single polypeptide chain. This protein-based biosensor, named RIYsense, efficiently measures protein methionine sulfoxide reduction by ratiometric fluorescence increase. We used it for high-throughput screening of potential MsrB1 inhibitors among 6868 compounds. A total of 192 compounds were selected based on their ability to reduce relative fluorescence intensity by more than 50% compared to the control. Then, we used molecular docking simulations of the compound on MsrB1, affinity assays, and MsrB1 activity measurement to identify compounds with reliable and strong inhibitory effects. Two compounds were selected as MsrB1 inhibitors: 4-[5-(4-ethylphenyl)-3-(4-hydroxyphenyl)-3,4-dihydropyrazol-2-yl]benzenesulfonamide and 6-chloro-10-(4-ethylphenyl)pyrimido[4,5-b]quinoline-2,4-dione. They are heterocyclic, polyaromatic compounds with a substituted phenyl moiety interacting with the MsrB1 active site, as revealed by docking simulation. These compounds were found to decrease the expression of anti-inflammatory cytokines such as IL-10 and IL-1rn, leading to auricular skin swelling and increased thickness in an ear edema model, effectively mimicking the effects observed in MsrB1 knockout mice. In summary, using a novel redox protein-based fluorescence biosensor, we identified potential MsrB1 inhibitors that can regulate the inflammatory response, particularly by influencing the expression of anti-inflammatory cytokines. These compounds are promising tools for understanding MsrB1's role during inflammation and eventually controlling inflammation in therapeutic approaches.

EuDockScore: Euclidean graph neural networks for scoring protein-protein interfaces.

Protein-protein interactions are essential for a variety of biological phenomena including mediating biochemical reactions, cell signaling, and the immune response. Proteins seek to form interfaces which reduce overall system energy. Although determination of single polypeptide chain protein structures has been revolutionized by deep learning techniques, complex prediction has still not been perfected. Additionally, experimentally determining structures is incredibly resource and time expensive. An alternative is the technique of computational docking, which takes the solved individual structures of proteins to produce candidate interfaces (decoys). Decoys are then scored using a mathematical function that assess the quality of the system, known as scoring functions. Beyond docking, scoring functions are a critical component of assessing structures produced by many protein generative models. Scoring models are also used as a final filtering in many generative deep learning models including those that generate antibody binders, and those which perform docking. In this work, we present improved scoring functions for protein-protein interactions which utilizes cutting-edge Euclidean graph neural network architectures, to assess protein-protein interfaces. These Euclidean docking score models are known as EuDockScore, and EuDockScore-Ab with the latter being antibody-antigen dock specific. Finally, we provided EuDockScore-AFM a model trained on antibody-antigen outputs from AlphaFold-Multimer (AFM) which proves useful in reranking large numbers of AFM outputs. The code for these models is available at https://gitlab.com/mcfeemat/eudockscore.

Enhancing protein aggregation prediction: a unified analysis leveraging graph convolutional networks and active learning.

Protein aggregation (PA) is a critical phenomenon associated with Alzheimer's and Parkinson's disease. Recent studies have suggested that factors like aggregation-prone regions (APRs) and β-strand interactions are crucial in understanding such behavior. While experimental methods have provided valuable insights, there has been a shift towards computational strategies, particularly machine learning, for their efficacy and speed. The challenge, however, lies in effectively incorporating structural information into these models. This study constructs a Graph Convolutional Network (GCN) to predict PA scores with the expanded and refined Protein Data Bank (PDB) and AlphaFold2.0 dataset. We employed AGGRESCAN3D 2.0 to calculate PA propensity and to enhance the dataset, we systematically separated multi polypeptide chains within PDB data into single polypeptide chains, removing redundancy. This effort resulted in a dataset comprising 302 032 unique PDB entries. Subsequently, we compared sequence similarity and obtained 22 774 Homo sapiens data from AlphaFold2.0. Using this expanded and refined dataset, the trained GCN model for PA prediction achieves a remarkable coefficient of determination (R 2) score of 0.9849 and a low mean absolute error (MAE) of 0.0381. Furthermore, the efficacy of the active learning process was demonstrated through its rapid identification of proteins with high PA propensity. Consequently, the active learning approach achieved an MAE of 0.0291 in expected improvement, surpassing other methods. It identified 99% of the target proteins by exploring merely 29% of the entire search space. This improved GCN model demonstrates promise in selecting proteins susceptible to PA, advancing protein science. This work contributes to the development of efficient computational tools for PA prediction, with potential applications in disease diagnosis and therapy.

Heterologous expression, characterisation and 3D-structural insights of GH18 chitinases derived from sago palm (Metroxylon sagu)

Although plants don't have chitins, they produce chitinases to protect themselves from biotic and abiotic stressors. There are two forms of chitinases found in organisms: glycosyl hydrolase 18 (GH18) and 19 (GH19) families. Plant GH19 chitinases are well known for their role in protecting against pathogens, but the roles of GH18 chitinases have not been fully elucidated. This study aimed to produce and characterise two recombinant GH18 chitinases from Metroxylon sagu. Two GH18 chitinase genes, MsChi1 and MsChi2, were identified, with nucleotide sequences of 1009 and 1308 bp, respectively. The proteins encoded by MsChi1 and MsChi2 genes were single polypeptide chains of 310 and 300 amino acids with predicted molecular masses of 31.21 and 30.15 kDa, respectively. Both cDNAs were cloned and expressed in the GS115 strain of Pichia pastoris. Recombinant MsChi1 and MsChi2 exhibited optimal activity at 60 °C with acidic pH 4.0 and 5.0, respectively. Both recombinant enzymes could hydrolyze synthetic and natural substrates (colloidal chitin). rMsChi1 preferred 4-nitrophenol N,N′-diacetyl-β-D chitobioside, while rMsChi2 preferred 4-nitrophenol N,N′,N″-triacetyl-β-D chitotriose, suggesting they might function as exochitinase and endochitinase, respectively. They also demonstrated antifungal activities against tested fungi. Homology modeling indicated ASP and GLU as essential residues for proton donation and acceptance.

Expression, purification and application of a recombinant, membrane permeating version of the light chain of botulinum toxin B.

Botulinum neurotoxins (BoNTs) are valuable tools to unveil molecular mechanisms of exocytosis in neuronal and non-neuronal cells due to their peptidase activity on exocytic isoforms of SNARE proteins. They are produced by Clostridia as single-chain polypeptides that are proteolytically cleaved into light, catalytic domains covalently linked via disulfide bonds to heavy, targeting domains. This format of two subunits linked by disulfide bonds is required for the full neurotoxicity of BoNTs. We have generated a recombinant version of BoNT/B that consists of the light chain of the toxin fused to the protein transduction domain of the human immunodeficiency virus-1 (TAT peptide) and a hexahistidine tag. His6-TAT-BoNT/B-LC, expressed in Escherichia coli and purified by affinity chromatography, penetrated membranes and exhibited strong enzymatic activity, as evidenced by cleavage of the SNARE synaptobrevin from rat brain synaptosomes and human sperm cells. Proteolytic attack of synaptobrevin hindered exocytosis triggered by a calcium ionophore in the latter. The novel tool reported herein disrupts the function of a SNARE protein within minutes in cells that may or may not express the receptors for the BoNT/B heavy chain, and without the need for transient transfection or permeabilization.

FGF1

Fibroblast Growth Factor 1 (Fgf1), also known as acidic FGF (aFGF), is involved in the regulation of various biological processes, ranging from development to disease pathogenesis. It is a single chain polypeptide and is highly expressed in adult brain and kidney tissues. Its expression has been shown to be directed by multiple tissue-specific promoters, which generate transcripts of varying lengths. During development the Fgf1 gene is widely expressed, including in the neural tube, heart and lung. Mouse mutants for this gene are normal under standard laboratory conditions. However, when Fgf1 mutants are exposed to a high fat diet, an aggressive diabetic phenotype has been reported, along with aberrant adipose tissue expansion. Ongoing research on FGF1 and its signalling pathways holds promise for greater understanding of developmental processes as well as the development of novel therapeutic interventions for diseases including diabetes.

Comparing the Soluble Form of Recombinant Human Insulin-like Growth Factor-1 (rhIGF-1) in Escherichia coli Using Thioredoxin as Fused and Co-expressed Protein.

Insulin-like growth factor-1 (IGF-1) is a single-chain polypeptide with various physiological functions. Escherichia coli is one of the most desirable hosts for recombinant protein production, especially for human proteins whose post-translation modifications are not essential for their bioactivity, such as hIGF-1. In this study, bacterial thioredoxin (Trx) was studied as a fused and non-fused protein to convert the insoluble form of recombinant human IGF-1 (rhIGF-1) to its soluble form in E. coli. The rhIGF-1 was expressed in the E. coli Origami strain in the form of fused-Trx. It was co-expressed with Trx and then purified and quantified. In the next step, the biological activity of rhIGF-1 was evaluated by alkaline phosphatase (ALP) activity assay in human adipose-derived stem cells (hASCs) regarding the differentiation enhancement effect of IGF-1 through the osteogenic process. Results showed that Trx in both the fused and non-fused forms had a positive effect on the production of the soluble form of rhIGF-1. A significant increase in ALP activity in hASCs after rhIGF-1 treatment was observed, confirming protein bioactivity. It was strongly suggested that the overproduction of Trx could increase the solubility of co-expressed recombinant proteins by changing the redox state in E. coli cells.

In Silico Analyses of Vertebrate G-Protein-Coupled Receptor Fusions United With or Without an Additional Transmembrane Sequence Indicate Classification into Three Groups of Linkers.

Natural G-protein-coupled receptors (GPCRs) rarely have an additional transmembrane (TM) helix, such as an artificial TM-linker that can unite two class A GPCRs in tandem as a single-polypeptide chain (sc). Here, we report that three groups of TM-linkers exist in the intervening regions of natural GPCR fusions from vertebrates: (1) the original consensus (i.e., consensus 1) and consensus 2~4 (related to GPCR itself or its receptor-interacting proteins); (2) the consensus but GPCR-unrelated ones, 1~7; and (3) the inability to apply 1/2 that show no similarity to any other proteins. In silico analyses indicated that all natural GPCR fusions from Amphibia lack a TM-linker, and reptiles have no GPCR fusions; moreover, in either the GPCR-GPCR fusion or fusion protein of (GPCR monomer) and non-GPCR proteins from vertebrates, excluding tetrapods, i.e., so-called fishes, TM-linkers differ from previously reported mammalian and are avian sequences and are classified as Groups 2 and 3. Thus, previously reported TM-linkers were arranged: Consensus 1 is [T(I/A/P)(A/S)-(L/N)(I/W/L)(I/A/V)GL(L/G)(A/T)(S/L/G)(I/L)] first identified in invertebrate sea anemone Exaiptasia diaphana (LOC110241027) and (330-SPSFLCI-L-SLL-340) identified in a tropical bird Opisthocomus hoazin protein LOC104327099 (XP_009930279.1); GPCR-related consensus 2~4 are, respectively, (371-prlilyavfc fgtatg-386) in the desert woodrat Neotoma lepida A6R68_19462 (OBS78147.1), (363-lsipfcll yiaallgnfi llfvi-385) in Gavia stellate (red-throated loon) LOC104264164 (XP_009819412.1), and (479-ti vvvymivcvi glvgnflvmy viir-504) in a snailfish GPCR (TNN80062.1); In Mammals Neotoma lepida, Aves Erythrura gouldiae, and fishes protein (respectively, OBS83645.1, RLW13346.1 and KPP79779.1), the TM-linkers are Group 2. Here, we categorized, for the first time, natural TM-linkers as rare evolutionary events among all vertebrates.

Crystal structure of the tegument protein UL82 (pp71) from human cytomegalovirus.

Human cytomegalovirus (HCMV) is an opportunistic pathogen that infects a majority of the world population. It may cause severe disease in immunocompromised people and lead to pregnancy loss or grave disabilities of the fetus upon congenital infection. For effective replication and lifelong persistence in its host, HCMV relies on diverse functions of its tegument protein UL82, also known as pp71. Up to now, little is known about the molecular mechanisms underlying the multiple functions of this crucial viral protein. Here, we describe the X-ray structure of full-length UL82 to a resolution of 2.7 Å. A single polypeptide chain of 559 amino acids mainly folds into three ß-barrels. We show that UL82 forms a dimer in the crystal as well as in solution. We identify point mutations that disturb the dimerization interface and show that the mutant protein is monomeric in solution and upon expression in human cells. On the basis of the three-dimensional structure, we identify structural homologs of UL82 from other herpesviruses and analyze whether their functions are preserved in UL82. We demonstrate that UL82, despite its structural homology to viral deoxyuridinetriphosphatases (dUTPases), does not possess dUTPase activity. Prompted by the structural homology of UL82 to the ORF10 protein of murine herpesvirus 68 (MHV68), which is known to interact with the RNA export factor ribonucleic acid export 1 (Rae1), we performed coimmunoprecipitations and demonstrated that UL82 indeed interacts with Rae1. This suggests that HCMV UL82 may play a role in mRNA export from the nucleus similar to ORF10 encoded by the gammaherpesviruses MHV68.

An Innovative Topical Medical Device with Hyaluronic Acid and Polypeptides in Patients with Reduced Knee Function.

A topical medical device, AI500®, constituted of a single-chain polypeptide embedded in hyaluronic acid, was tested and evaluated in patients with reduced knee function due to osteoarthritis and other knee conditions. A total of 35 participants with reduced knee function assessed by the WOMAC Physical Function score were recruited. Four study visits were planned, from the first application at V0 to 1 week follow up at V3. Patient symptomatology was evaluated after 24 h (V1) and after 48 h (V2) through phone contact, and after 1 week from V0, on site (V3). The overall duration of the follow up was one week. An amelioration of 40% in WOMAC Physical Functional scores after 1 week of treatment was recorded, thus achieving the primary endpoint of 20%. Furthermore, a reduction of 29% in Physical Functional scores and of 28% in total WOMAC scores between V0-V2 was registered, together with a decrease of 39% between V0 and V3. The NRS scale showed a 29% and 37% reduction in pain between V0-V1 and V0-V2, respectively. Product safety was confirmed by the very low rate of adverse effects, non-device related, observed in only 2 patients out of 35, resolved spontaneously within 24-48 h. No safety concerns or risks associated with the use of the device were highlighted. There are few the studies on the topical use of HA-based gels for the treatment of knee problems. Compared to invasive intra-articular injections and oral pharmacological therapies used in cases of knee pain, the topical application of AI500® is non-invasive, safe, and appreciated by patients. Good results in terms of functional improvement and symptoms resolution were obtained in less than 1 week.

Investigating vulnerability of the conserved SARS-CoV-2 spike's heptad repeat 2 as target for fusion inhibitors using chimeric miniproteins

Inhibition of SARS-CoV-2 membrane fusion is a highly desired target to combat COVID-19. The interaction between the spike's heptad repeat (HR) regions 1 (HR1) and 2 (HR2) is a crucial step during the fusion process and these highly conserved HR regions constitute attractive targets for fusion inhibitors. However, the relative importance of each subregion of the long HR1-HR2 interface for viral inhibition remains unclear. Here, we designed, produced, and characterized a series of chimeric miniproteins that mimic two different half subdomains of HR1. The proteins were designed as single polypeptide chains that spontaneously fold into antiparallel trimeric helical bundles aimed at structurally imitate the molecular surface of each HR1 half subregion. All the miniproteins folded stably as helical structures and could bind complementary HR2 peptides with moderate affinity. However, only the miniproteins mimicking the N-terminal HR1 half subdomain, but not those imitating C-terminal one, could inhibit cell infection by SARS-COV-2 real viruses in cell cultures. Most interestingly, the inhibitory activity of the miniproteins correlated with their structural stability, but not with their relative binding affinity for HR2 peptides. These results are highly relevant for designing more focused and active fusion inhibitors targeting the highly conserved HR2 region of the Spike.

The distal-proximal relationships among the human moonlighting proteins: Evolutionary hotspots and Darwinian checkpoints

Moonlighting proteins, known for their ability to perform multiple, often unrelated functions within a single polypeptide chain, challenge the traditional “one gene, one protein, one function” paradigm. As organisms evolved, their genomes remained relatively stable in size, but the introduction of post-translational modifications and sub-strategies like protein promiscuity and intrinsic disorder enabled multifunctionality. Enzymes, in particular, exemplify this phenomenon, engaging in unrelated processes alongside their primary catalytic roles. This study employs a systematic, quantitative informatics approach to shed light on human moonlighting protein sequences. Phylogenetic analyses of human moonlighting proteins are presented, elucidating the distal-proximal relationships among these proteins based on sequence-derived quantitative features. The findings unveil the captivating world of human moonlighting proteins, urging further investigations in the emerging field of moonlighting proteomics, with the potential for significant contributions to our understanding of multifunctional proteins and their roles in diverse cellular processes and diseases.

Methods for the preparation and analysis of the diterpene cyclase fusicoccadiene synthase.

Prenyltransferases are terpene synthases that combine 5-carbon precursor molecules into linear isoprenoids of varying length that serve as substrates for terpene cyclases, enzymes that catalyze fascinating cyclization reactions to form diverse terpene natural products. Terpenes and their derivatives comprise the largest class of natural products and have myriad functions in nature and diverse commercial uses. An emerging class of bifunctional terpene synthases contains both prenyltransferase and cyclase domains connected by a disordered linker in a single polypeptide chain. Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is one of the most well-characterized members of this subclass and serves as a model system for the exploration of structure-function relationships. PaFS has been structurally characterized using a variety of biophysical techniques. The enzyme oligomerizes to form a stable core of six or eight prenyltransferase domains that produce a 20-carbon linear isoprenoid, geranylgeranyl diphosphate (GGPP), which then transits to the cyclase domains for the generation of fusicoccadiene. Cyclase domains are in dynamic equilibrium between randomly splayed-out and prenyltransferase-associated positions; cluster channeling is implicated for GGPP transit from the prenyltransferase core to the cyclase domains. In this chapter, we outline the methods we are developing to interrogate the nature of cluster channeling in PaFS, including enzyme activity and product analysis assays, approaches for engineering the linker segment connecting the prenyltransferase and cyclase domains, and structural analysis by cryo-EM.

Insulin-like growth factor-1 (IGF-1) levels in multiple sclerosis patients: A systematic review and meta-analysis.

Multiple sclerosis (MS) is a chronic progressive autoimmune disorder of the central nervous system (CNS) that can cause inflammation, demyelination, and axon degeneration. Insulin-like growth factor-1 (IGF-1) is a single-chain polypeptide mainly synthesized in the liver and brain. IGF-1 causes neuronal and non-neuronal cell proliferation, survival, and differentiation. Therefore, it can be used in treating neuro-demyelinating diseases such as MS. The current systematic review and meta-analysis aims to compare the levels of IGF-1 in MS patients and healthy controls and also investigates IGF binding proteins (IGF-BP) and growth hormone (GH) levels between MS patients and healthy controls. In this study, we systematically searched electronic databases of PubMed, Scopus, Web of Science (WOS), and Google Scholar, up to December 2022. Studies that measured IGF-1, GH, IGFBP-1, IGFBP-2, or IGFBP-3 in MS patients and healthy controls in either blood or cerebral spinal fluid (CSF) were identified. We calculated Standardized mean differences (SMD) to compare levels of IGF-1, GH, IGFBP-1, IGFBP-2, or IGFBP-3 in MS patients and controls. Finally, we included 11 eligible studies from 1998 to 2018. The sample size of included studies varied from 20 to 200 resulting in a total sample size of 1067 individuals, 531 MS patients, and 536 healthy controls. The mean age of the patient and control groups were 38.96 and 39.38, respectively. The average EDSS among patients was 4.56. We found that blood levels of IGF-1 (SMD = 0.20, 95% CI = -0.20 to 0.59, I2 = 82.4%, K = 8, n = 692), CSF level of IGF-1 (SMD = 0.25, 95% CI = -0.06 to 0.56, I2 = 0.0%, K = 3 n = 164) and blood levels of GH were not significantly higher in MS patients than controls (SMD = 0.08, 95% CI = -0.33 to 0.49, I2 = 77.0% K = 3, n = 421). Moreover, the blood levels of IGFBP-1 (SMD = 0.70, 95% CI = 0.01 to 1.40, I2 = 77%, K = 4, n = 255) were significantly higher in MS cases than in controls. However, the blood levels of IGFBP-2 (SMD = 0.43, 95% CI = -0.34 to 1.21, I2 = 64.2%, K = 3, n = 78) and blood levels of IGFBP-3 (SMD = 1.04, 95% CI = -0.09 to 2.17, I2 = 95.6%, K = 6, n = 443) were not significantly higher in patients than controls. Our meta-analysis revealed no significant difference in serum levels of IGF-1, GH, IGFBP-2, and IGFBP-3 between the MS group and healthy controls, except for IGFBP1. However, our systematic review showed that the studies were controversial for IGFBP-3 serum levels. Some studies found an increase in serum level of IGFBP-3 in MS patients compared to the healthy group, while others showed a decrease.

Linear and ring polypeptides complexed with oppositely charged surfactants: the cohesion of the complexes as revealed in atomistic simulations.

The use of linear supercharged unfolded polypeptides (SUPs) and oppositely charged surfactants in aqueous solution has demonstrated impressive adhesive properties. These substances possess biocompatibility, biodegradability and other necessary properties for practical application as a biomedical glue in wound repair. The success of these substances, coupled with limited knowledge about such systems, provides hope for enhancing the performance of the final product. One potential approach involves altering the topology of the polypeptide chain. In this article, we conduct a comparative analysis to examine the behavior of the ring and linear chains of a polypeptide in aqueous solution. This analysis utilizes full-atomic computer modeling to monitor the properties of the chains. We investigate the temperature dependence of the shape and size of individual polypeptides in the solution, as well as the formation of complexes via mixing the polypeptide chains with oppositely charged sodium dodecylbenzene sulfonate (SDBS) surfactant molecules in a stoichiometric ratio. Additionally, we explore the cohesive properties of the resulting complex through power experiments involving the extraction of single polypeptide chains out of the SUP-SDBS complexes.

Expression, Purification, and Determination of Sensitivity to Calcium Ions of Ctenophore Photoproteins.

Light-sensitive Ca2+-regulated photoproteins of ctenophores are single-chain polypeptide proteins of 206-208 amino acids in length comprising three canonical EF-hand Ca2+-binding sites, each of 12 contiguous residues. These photoproteins are a stable complex of apoprotein and 2-hydroperoxy adduct of coelenterazine. Addition of calcium ions to photoprotein is only required to trigger bright bioluminescence. However, in contrast to the related Ca2+-regulated photoproteins of jellyfish their capacity to bioluminescence disappears on exposure to light over the entire absorption spectral range of ctenophore photoproteins. Here, we describe protocols for expression of gene encoding ctenophore photoprotein in Escherichia coli cells, obtaining of the recombinant apoprotein of high purity and its conversion into active photoprotein with synthetic coelenterazine as well as determination of its sensitivity to calcium ions using light-sensitive Ca2+-regulated photoprotein berovin from ctenophore Beroe abyssicola as an illustrative case.

Strategies for the production of isotopically labelled Fab fragments of therapeutic antibodies in Komagataella phaffii (Pichia pastoris) and Escherichia coli for NMR studies.

The importance and fast growth of therapeutic monoclonal antibodies, both innovator and biosimilar products, have triggered the need for the development of characterization methods at high resolution such as nuclear magnetic resonance (NMR) spectroscopy. However, the full power of NMR spectroscopy cannot be unleashed without labelling the mAb of interest with NMR-active isotopes. Here, we present strategies using either Komagataella phaffii (Pichia pastoris) or Escherichia coli that can be widely applied for the production of the antigen-binding fragment (Fab) of therapeutic antibodies of immunoglobulin G1 kappa isotype. The E. coli approach consists of expressing Fab fragments as a single polypeptide chain with a cleavable linker between the heavy and light chain in inclusion bodies, while K. phaffii secretes a properly folded fragment in the culture media. After optimization, the protocol yielded 10-45 mg of single chain adalimumab-Fab, trastuzumab-Fab, rituximab-Fab, and NISTmAb-Fab per liter of culture. Comparison of the 2D-1H-15N-HSQC spectra of each Fab fragment, without their polyhistidine tag and linker, with the corresponding Fab from the innovator product showed that all four fragments have folded into the correct conformation. Production of 2H-13C-15N-adalimumab-scFab and 2H-13C-15N-trastuzumab-scFab (>98% enrichment for all three isotopes) yielded NMR samples where all amide deuterons have completely exchanged back to proton during the refolding procedure.

Unconventional structure and mechanisms for membrane interaction and translocation of the NF-κB-targeting toxin AIP56

Bacterial AB toxins are secreted key virulence factors that are internalized by target cells through receptor-mediated endocytosis, translocating their enzymatic domain to the cytosol from endosomes (short-trip) or the endoplasmic reticulum (long-trip). To accomplish this, bacterial AB toxins evolved a multidomain structure organized into either a single polypeptide chain or non-covalently associated polypeptide chains. The prototypical short-trip single-chain toxin is characterized by a receptor-binding domain that confers cellular specificity and a translocation domain responsible for pore formation whereby the catalytic domain translocates to the cytosol in an endosomal acidification-dependent way. In this work, the determination of the three-dimensional structure of AIP56 shows that, instead of a two-domain organization suggested by previous studies, AIP56 has three-domains: a non-LEE encoded effector C (NleC)-like catalytic domain associated with a small middle domain that contains the linker-peptide, followed by the receptor-binding domain. In contrast to prototypical single-chain AB toxins, AIP56 does not comprise a typical structurally complex translocation domain; instead, the elements involved in translocation are scattered across its domains. Thus, the catalytic domain contains a helical hairpin that serves as a molecular switch for triggering the conformational changes necessary for membrane insertion only upon endosomal acidification, whereas the middle and receptor-binding domains are required for pore formation.

TetR and OmpR family regulators in natural product biosynthesis and resistance.

This article provides a comprehensive review and sequence-structure analysis of transcription regulator (TR) families, TetR and OmpR/PhoB, involved in specialized secondary metabolite (SSM) biosynthesis and resistance. Transcription regulation is a fundamental process, playing a crucial role in orchestrating gene expression to confer a survival advantage in response to frequent environmental stress conditions. This process, coupled with signal sensing, enables bacteria to respond to a diverse range of intra and extracellular signals. Thus, major bacterial signaling systems use a receptor domain to sense chemical stimuli along with an output domain responsible for transcription regulation through DNA-binding. Sensory and output domains on a single polypeptide chain (one component system, OCS) allow response to stimuli by allostery, that is, DNA-binding affinity modulation upon signal presence/absence. On the other hand, two component systems (TCSs) allow cross-talk between the sensory and output domains as they are disjoint and transmit information by phosphorelay to mount a response. In both cases, however, TRs play a central role. Biosynthesis of SSMs, which includes antibiotics, is heavily regulated by TRs as it diverts the cell's resources towards the production of these expendable compounds, which also have clinical applications. These TRs have evolved to relay information across specific signals and target genes, thus providing a rich source of unique mechanisms to explore towards addressing the rapid escalation in antimicrobial resistance (AMR). Here, we focus on the TetR and OmpR family TRs, which belong to OCS and TCS, respectively. These TR families are well-known examples of regulators in secondary metabolism and are ubiquitous across different bacteria, as they also participate in a myriad of cellular processes apart from SSM biosynthesis and resistance. As a result, these families exhibit higher sequence divergence, which is also evident from our bioinformatic analysis of 158 389 and 77 437 sequences from TetR and OmpR family TRs, respectively. The analysis of both sequence and structure allowed us to identify novel motifs in addition to the known motifs responsible for TR function and its structural integrity. Understanding the diverse mechanisms employed by these TRs is essential for unraveling the biosynthesis of SSMs. This can also help exploit their regulatory role in biosynthesis for significant pharmaceutical, agricultural, and industrial applications.

Purification and Characterization of Glucosamine Oxidase from Aspergillus Terreus NUK-1204 for Production of Glucosaminic Acid

D-Glucosaminic acid (2-amino-2-deoxy-D-gluconic acid) is a valuable glucosamine derivative from the oxidation of the glucosamine (2-amino-2-deoxy-D-glucose). It can be produced via the fermentation of certain microorganisms, which involves complicated purification task, though. In an attempt to find an enzyme suited to glucosaminic acid production, the study presented a purified homogeneous glucosamine oxidase deriving from the wheat bran culture of a soil-isolated fungal strain, Aspergillus terreus NUK-1204. Maximum activity was observed in the wheat bran solid culture after five days of NUK-1204 growth, following its release from cells at 0.011 unitmL -1culture filtrate. The new kind of oxidase comprises a single polypeptide chain with a molecular mass of 54 kDa, containing FAD and exhibiting absorption maxima at 274, 372 and 439 nm. The enzyme was stable in 4.5-11 pH range, with an optimal reaction pH at 10.5 and optimal reaction temperature at 30°C. It remained stable at 20-50°C for 1 hr. The relative activity of glucosamine oxidase towards glucosamine and N-acetyl-glucosamine was 100:7, demonstrating high substrate specificity. It doesn’t inhibit glucosamine. To the best of our knowledge, this is the first report ever on the discovery of a glucosamine oxidase based on enzyme substrate specificity.