Amino Acid Residues Research Articles

Evaluation of protein impurities in Ademetionine 1,4-Butanedisulfonate

Ademetionine 1,4-Butanedisulfonate (SAMe) is widely used as a prescription drug to treat cholestasis associated with liver disease, and is also used to treat depression, Alzheimer's disease and other diseases. Currently, the main way to produce SAMe is by fermentation of S-adenosylmethionine synthetase（SAMS）. However, during the fermentation process, host cells produce contaminants unrelated to the treatment. Among them, host cell protein (HCP), which is co-purified with the drug, is the main impurity in the production process. HCP-induced antigenic reactions can lead to allergies or other adverse reactions and affect drug quality, efficacy, and safety. Enzyme-linked immunosorbent assay (ELISA) and mass spectrometry (MS) are currently used as the main analytical methods for measuring HCP. The ELISA method may ignore some HCPs with low immunogenicity, while mass spectrometry is primarily employed for the characterization of proteomes. Orthogonal methods can more effectively evaluate impurities in drugs. In this study, ELISA was initially utilized to quantify the content of Saccharomyces cerevisiae host protein in 11 batches of SAMe active pharmaceutical ingredient (API) from two suppliers, then the above APIs were qualitatively analyzed by MS method. Following the identification of surrogate peptide of SAMS, an Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry（UPLC-MS/MS）method was established and validated, the SAMS protein residues in 11 batches of samples were subsequently determined. The results demonstrated that the levels of Saccharomyces cerevisiae host protein and SAMS in the samples were both low, with values below 20 ppm and 1.19 ppm, respectively. The qualitative results also indicate that the types of peptide residues in the samples are more diverse than those of protein residues. Furthermore, residues of proteins and peptides can be detected in all samples, which underscores the importance of evaluating the HCP in API. This study employs a range of complementary detection methods to conduct a comprehensive and sensitive evaluation of total HCP and specific proteins in drugs, thereby guiding more informed assessments of the risks posed by HCP impurities in raw materials during pharmaceutical processes.

L858R/L718Q and L858R/L792H Mutations of EGFR Inducing Resistance Against Osimertinib by Forming Additional Hydrogen Bonds.

Acquired resistance to first-line treatments in various cancers both promotes cancer recurrence as well as limits effective treatment. This is true for epidermal growth factor receptor (EGFR) mutations, for which secondary EGFR mutations are one of the principal mechanisms conferring resistance to the covalent inhibitor osimertinib. Thus, it is very important to develop a deeper understanding of the secondary mutational resistance mechanisms associated with EGFR mutations arising in tumors treated with osimertinib to expedite the development of innovative therapeutic drugs to overcome acquired resistance. This work uses all-atom molecular dynamics (MD) simulations to investigate the conformational variation of two reported EGFR mutants (L858R/L718Q and L858R/L792H) that resist osimertinib. The wild-type EGFR kinase domain and the L858R mutant are used as the reference. Our MD simulation results revealed that both the L718Q and L792H secondary mutations induce additional hydrogen bonds between the residues in the active pocket and the residues with the water molecules. These additional hydrogen bonds reduce the exposure area of C797, the covalent binding target of osimertinib. The additional hydrogen bonds also influence the binding affinity of the EGFR kinase domain by altering the secondary structure and flexibility of the amino acid residues in the domain. Our work highlights how the two reported mutations may alter both residue-residue and residue-solvent hydrogen bonds, affecting protein binding properties, which could be helpful for future drug discovery.

Structure and function of the proton channel OTOP1

OTOP1 belongs to the otopetrin family of membrane proteins that form proton channels in cells of diverse types. In mammals, OTOP1 is involved in sour transduction in taste cells and contributes to otoconia formation in the inner ear. From the structural point of view, otopetrins, including OTOP1, represent a quasi-tetramer consisting of four α-barrels. The exact transport pathways mediating proton flux through the OTOP1 channel and gating units modulating its activity are still a matter of debate. This review discusses current data on structural and functional features of OTOP1. Suggested proton transport pathways, regulatory mechanisms, and key amino acid residues determining functionality of the otopetrins are considered. The existing kinetic models of OTOP1 are discussed as well. Based on revealed functional properties, OTOP1 is suggested to operate as a logical XOR element that allows for proton flux only if transmembrane pH gradient exists.

Recalibration of MARTINI-3 Parameters for Improved Interactions between Peripheral Proteins and Lipid Bilayers.

The MARTINI force field is one of the most used coarse-grained models for biomolecular simulations. Many limitations of the model including the protein-protein overaggregation have been improved in its latest version, MARTINI-3. In this study, we investigate the efficacy of the MARTINI-3 parameters for capturing the interactions of peripheral proteins with model plasma membranes. Particularly, we consider two classes of proteins, namely, annexin and epsin, which are known to generate negative and positive membrane curvatures, respectively. We find that current MARTINI-3 parameters are not able to correctly describe the protein-membrane interface and the protein-induced membrane curvatures for any of these proteins. The problem arises due to the lack of proper hydrophobic interactions between the protein residues and lipid tails. Making systematic adjustments, we show that a combination of reduction in the protein-water interactions and enhancement of protein-lipid hydrophobic interactions is essential for accurate prediction of the interfacial structure including the protein-induced membrane curvature. Next, we apply our model to a couple of other peripheral proteins, namely, Snf7, a core component of the ESCRT-III complex, and the PH domain of evectin-2. We find that our model captures the protein-membrane interfacial structure much more accurately than the MARTINI-3 model for all of the peripheral proteins considered in this study. However, the strategy described in this study may not be suitable for oligomeric transmembrane proteins where protein-protein hydrophobic interactions should be increased instead of protein-lipid hydrophobic interactions.

Powdered Medical Adhesive with Long Lasting Adhesion in Water Environment.

Medical adhesives have been used under surgical conditions. However, it is always a big challenge to maintain long-term adhesion in a water environment. Besides, it usually takes a long time to complete the adhesion, and the operation might be complicated. In this study, tannic acid and gelatin solution under acidic conditions were mixed, flocculated, lyophilized, and crushed; thus, a powdered medical adhesive (POWDER) was prepared with long-lasting adhesion in a water environment, convenience, and low price. Tannic acid bound gelatin and maintained adhesive force primarily through hydrogen bonding and reacted with amino sulfhydryl and other amino acid residues after oxidation into aldehyde, exhibiting excellent underwater adhesion. Oxidized dextran (ODex) powder rich in an aldehyde group was introduced to provide covalent binding in the adhesive. In vitro and in vivo studies showed that POWDER could quickly adhere to various tissues in the water environment. In vitro skin adhesion experiments demonstrated that it could achieve effective adhesion in a water environment for up to 60 days. Its blood compatibility, low cytotoxicity, and biodegradability were also verified. The POWDER developed in this study is of great significance for patients who need rapid wound treatments.

Molecular Modeling of Glycosylated Catalytic Domain of Human Carbonic Anhydrase IX.

Glycans exhibit significant structural diversity due to the flexibility of glycosidic bonds linking their constituent monosaccharides and the formation of numerous hydrogen bonds. The present work searches a simulated ensemble of glycan chain conformations attached to the catalytic domain of N-glycosylated human carbonic anhydrase IX (HCA IX-c) to identify conformations pointed away or back-folded toward the protein surface guided by different amino acid residues. A series of classical molecular dynamics (MD) simulation studies for a total of 30 μs followed by accelerated MD simulations for a total of 2 μs have been performed using two different force fields to capture varying degrees of fluctuations of both glycan chain and HCA IX. From the underlying free energy profile and kinetics derived using hidden Markov state model, several stable glycan orientations are identified that extend away from the protein surface and convert among each other with rate constants of the order 107-1010 S-1. Most importantly, we have identified a rare glycan conformation which reaches close to a catalytically important amino acid residue, Glu-106. We further enlist the protein residues that couple such less frequent event of the glycan chain back-folding toward the surface of the protein.

Revisiting Aspergillus terreus MTCC6324 recombinant alcohol oxidase (rAOx): enhanced in-silico insight into structure, function, and substrate sequestration mechanism

Alcohol oxidase (AOx) enzymes have gained significant attention for their potential in industrial applications due to their unique ability to catalyze irreversible oxidation of diverse alcohol substrates without external co-factors. This study revisits and enhances in-silico work on Aspergillus terreus MTCC6324 recombinant AOx (rAOx) enzyme, combining artificial intelligence (AlphaFold), molecular docking (AutoDock Vina) techniques and Molecular dynamics (MD) simulations (Desmond). Comprehensive sequence analysis revealed a high degree of conservation among 23 AOx amino acid sequences from various Aspergillus species highlights conserved regions, affirming its GXGXXG Rossmann fold motif. AlphaFold-predicted 3D structure of rAOx demonstrated improved stereo-chemical stability compared to I-TASSER predicted structure with 87.6% amino acid residues in most favourable region of Ramachandran plot compared to 79.5%, respectively. Molecular docking revealed the binding affinities of co-factors FAD and diverse alcohol substrates, with cinnamyl alcohol exhibiting robust interaction with rAOx holoenzyme. MD simulations further elucidate the stability and dynamics of rAOx-FAD-cinnamyl alcohol complex over 100 nanoseconds. The simulations showcase FAD’s stable binding within the protein core and highlights transient substrate interactions, dissociating within the active site after 75 ns suggesting a substrate sequestration mechanism. The study unveils substrate sequestration mechanism wherein cinnamyl alcohol exhibits temporary binding, leading to quick detachment from active site, mimicking reported exponential kinetics. This study not only validates previous findings but also offers a comprehensive understanding of intricate dynamics governing rAOx enzymatic activity. The improved sequence-to-structure prediction and detailed molecular insights into substrate sequestration provide a valuable foundation for future experimental investigations and rational design of bio-catalytic processes.

An in silico approach uncovering the competency of oncolytic human adenovirus 52 for targeted breast cancer virotherapy

Breast cancer remains a major health threat throughout the world specifically in women above 30 years of age however, it is rarely known to affect men as well. It is characterized by the abnormal division of cells in the breast tissue resulting in the development of breast malignancies. Various risk factors contributing to breast cancer include age, family history, genetic mutations (chiefly in BRCA1 and BRCA2 genes) along with hormonal imbalances (oestrogen, progesterone, HER2). Early detection which can be obtained through frequent rounds of self-examination, mammographic scanning, and clinical assessment plays a crucial role in the prevention of the disease. In addition, appropriate diagnosis assists in better therapeutic responses. This study highlights the considerable health risks associated with the conventional treatment procedures which arise and increased demand of advanced, secure, and risk-free treatment alternatives. Oncolytic viruses are potentially apparent for the aim of improving cancer therapeutics with reduced side effects. These viruses act as the fundamental therapeutic agent themselves that selectively target and kill malignant cells without harm to healthy tissues. The key objective of the research is to provide evidence that Human Adenovirus 52 is a potent oncolytic virus and to highlight its capacity to target and eliminate cancer cells with precision while causing the least amount of harm to healthy tissues. Validating the in-silico method entails evaluating the precision and dependability of the computational modelling by contrasting the in-silico predictions with the findings from the experiments rank as the secondary objective. The workflow of this research utilized in-silico computational drug designing approaches including retrieval of tertiary structures of both the target Breast Cancer Type 1 Susceptibility Protein (BRCA1) and the viral Human Adenovirus 52 protein, their validation generating Ramachandran Plots determining favoured amino acid residue angles and prediction of their active residues. Furthermore, the study focused on the molecular dynamics docking of proteins, interpretation of molecular interactions between the docked complex, as well as the assessment of the molecular dynamic simulations (MD) in addition to their MMGBSA binding energy calculations. A successful docking between BRCA1 and Adenovirus protein provided a significant score of 329.2 +/- 24.3, furthermore, MD simulations showed a high RMSD peak at 2.8 Å, RMSF were maximum at 3.5 Å with highest protein–protein interaction, the radius of gyration was stable throughout the simulation representing elastic stability along with a high energy interaction value of - 7882 kCal/mol. Moreover, the MMGBSA calculation results showed a notable release of binding free energy of - 68.96 kCal/mol demonstrating effective bond formation between the docked complex. These findings propose the effectiveness of Human Adenovirus 52 to treat cancer. The selected oncolytic Human Adenovirus 52 is a potential candidate for the target specific treatment of breast cancer through virotherapy. This computer-aided drug discovery presents significant potential in targeting cancer cells and would assist in the development of potent drug reagents for the cancer therapy.

The landscape of intrinsically disordered proteins in Leishmania parasite: Implications for drug discovery

Proteins that lack three-dimensional structures are known as Intrinsically disordered proteins (IDPs). In this study, we aimed to identify intrinsically disordered proteins in the Leishmania donovani proteome using various predictors that can identify IDPs based on amino acid residues and charge hydropathy. Top identified IDPs are analyzed using STRING, PSP-Hunter, Deep Loc-2.0, and Alpha fold to understand the protein-protein interaction, phase separation, localization, and structural assessment of those proteins. From this study, we found that >50 % of Leishmania donovani proteome has proteins or regions of proteins that are intrinsically disordered with VSL2 score >0.5; most proteins interact with many other proteins with PPI enrichment p-value <1.0e-16. Few proteins, such as Protein phosphatase inhibitor, UMSBP, and Zinc knuckle, have redox-sensitive regions. Functional disorder profiles of identified IDPs showed MoRFs and predicted protein domains. HASPB, UTP11, Nucleolar protein 12, and UMSBP have a high probability of undergoing phase separation. Localization studies showed that most of these proteins are in the cytoplasm and nucleus. Our present study of identifying IDPs in Leishmania proteome yields significant information on druggable targets and can serve as a basis for further studies to identify unexplored pathways.

Preparation and characteristics of tea water-insoluble protein/chitosan composite particles

The emulsifying properties of tea water-insoluble protein particles (TWIPs) are insufficient to expand their applicability in food emulsifying systems. This study aimed to explore the emulsification characteristics and application of tea water-insoluble protein/chitosan composite particles (TCPs). Scanning electron microscopy showed the TWIPs were tightly coupled with chitosan (CS). The transform of secondary structure and amino acid residues were beneficial to the formation of TCPs and improvement of emulsification performance. Increasing the CS concentration improved the viscoelasticity, as verified by rheological property, and decreased the droplet size of Pickering emulsions (PEs) at a TWIP-CS mass ratio of 50:50. In addition, when the fixed TWIP-CS ratio was 50:50, the TCPs concentration of 0.5% favored the formation of bovine bone white soup with extraordinary emulsification stability (p < 0.05). In conclusion, the emulsification property of TWIPs was improved at a TWIP-CS ratio of 50:50, which was also verified in PEs and bovine bone white soup. This work provided a theoretical basis for the development and further deep processing of tea by-products, and broadened the application of tea protein in liquid emulsification system.

NnM469, a novel recombinant jellyfish venom metalloproteinase from Nemopilema nomurai, disrupted the cell matrix

Molecular cloning and functional characterization of Nemopilema nomurai venom metalloproteinases have provided deeper insights into the pathogenesis of jellyfish dermatitis. This study reports a new cDNA clone from N. nomurai tentacle venom (Transcript sequence: ID469) encoding 362 amino acid residues, belonged to astacin family and capable of disrupting the cell matrix. The N. nomurai metalloproteinase 469 (NnM469) comprises a signal peptide and propeptide, followed by metalloproteinase domain containing a zinc-binding motif, and two ShKT domains. Notably, NnM469 features a zinc-binding motif (HEXXH) at the active site, within an extended sequence of HEXXHXXGFXHE, which is unique to astacin. Immunocytochemistry revealed that NnM469 is located in the stab tube and envelope of jellyfish nematocysts. Western blot and LC-MS/MS analysis confirmed that the NnM469 protein was successfully expressed using the Pichia pastoris expression system. The recombinant NnM469 could degrade the cell matrix, resulting in the death of HaCaT cells with an IC50 of 26.34 μg/mL. Finally, I-TASSER-generated structure and function predictions indicated that conserved Asp53, His168, His172, His178, and Tyr227 serve as key amino acid residues for the Zn2+ ion binding in the catalytic center. In summary, the study of the molecular characteristics and function of NnM469 presents an opportunity to develop therapeutic interventions for jellyfish venom-induced dermatitis.

Investigation on the interaction mechanism of trilobatin with pepsin and trypsin by multi-spectroscopic and molecular docking methods

Pepsin and trypsin are noteworthy for the digestion of proteins in the human body. Trilobatin is a dihydrochalone that has anti-obesity, antioxidant, and anti-diabetes functions. The interaction of trilobatin and pepsin or trypsin is not currently being explored. Therefore, in this research multiple methods involving fluorescence spectroscopy, synchronous fluorescence spectroscopy, ultraviolet–visible (UV–Vis) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) and molecular docking were employed for studying the interaction between trilobatin and pepsin or trypsin. During the interaction between trilobatin and pepsin or trypsin, the quenching type was static quenching and a single binding site existed. The binding constants (Ka) values were 2.177 and 3.028 × 104 L/mol in trilobatin-pepsin and trilobatin-trypsin system at 298 K, respectively. However, the presense of metal ions (Zn2+, Ca2+ and K+) decreased the binding of trilobatin to pepsin and trypsin. The complex between trilobatin and pepsin was generated typically through hydrogen bonding and van der Waals force (ΔH° < 0 and ΔS° < 0), in comparison the complex between trilobatin and trypsin was established chiefly through hydrogen bond and hydrophobic force (ΔH° > 0 and ΔS° > 0). The data from UV–Vis, synchronous fluorescence, FT-IR and CD spectra indicated that trilobatin reduced the hydrophobicity of Tyrosine (Tyr) residues of pepsin and trypsin. Furthermore, trilobatin altered the secondary structures of pepsin and trypsin. Meanwhile, trilobatin changed the conformation of pepsin and trypsin so that the viscosity of the interaction systems decreased with increased of trilobatin concentration. The molecular docking modeling identified that the trilobatin interacted with amino acid residues around pepsin and trypsin. This work consistently showed the interaction between trilobatin and pepsin or trypsin, in addition to offering valuable information for the application of trilobatin.

A single phosphorylatable amino acid residue is essential forthe recognition of multiple potyviral HCPro effectors by potato Nytbr.

Potato virus Y (PVY, Potyviridae) is among the most important viral pathogens of potato. The potato resistance gene Nytbr confers hypersensitive resistance to the ordinary strain of PVY (PVYO), but not the necrotic strain (PVYN). Here, we unveil that residue 247 of PVY helper component proteinase (HCPro) acts as a central player controlling Nytbr strain-specific activation. We found that substituting the serine at 247 in the HCPro of PVYO (HCProO) with an alanine as in PVYN HCPro (HCProN) disrupts Nytbr recognition. Conversely, an HCProN mutant carrying a serine at position 247 triggers defence. Moreover, we demonstrate that plant defences are induced against HCProO mutants with a phosphomimetic or another phosphorylatable residue at 247, but not with a phosphoablative residue, suggesting that phosphorylation could modulate Nytbr resistance. Extending beyond PVY, we establish that the same response elicited by the PVYO HCPro is also induced by HCPro proteins from other members of the Potyviridae family that have a serine at position 247, but not by those with an alanine. Together, our results provide further insights in the strain-specific PVY resistance in potato and infer a broad-spectrum detection mechanism of plant potyvirus effectors contingent on a single amino acid residue.

The negative electrostatic potential of the coordination between calcium and carboxyl/oxygen group in calcium-peptide complexes contributes to calcium utilization

The coordination electrostatic potential has significantly influenced the binding energy of calcium-peptide coordination complexes. However, its impact on calcium bioavailability is unclear. A calcium-binding peptide (CBP) DEDEQIPSHPPR, purified from soybean yogurt was selected. The study selected tetrapeptide residues of acidic amino acids that play an important role in CBP and designed sixteen tetrapeptides rich in carboxyl oxygen atoms to investigate the influence of electrostatic potential on calcium-peptide binding. The results indicated that DEDE exhibited the strongest binding energy (−375.27 kcal/mol) and binding capacity (464.11 ± 2.14 mg/g) with calcium ions compared to other peptides. Moreover, DEDE also increased intracellular calcium concentration the most, being 2.04 times higher than CaCl2. Calcium ions coordinated with carboxyl oxygen atoms possessing negative electrostatic potential, which correlated positively with the bioavailability of calcium ions. This electrostatic potential analysis provides a theoretical basis for developing and screening bioactive peptides with high calcium bioavailability.

Two amino acid pairs in the Gc Glycoprotein of Severe Fever with Thrombocytopenia Syndrome Virus responsible for the enhanced virulence

Severe fever with thrombocytopenia syndrome (SFTS) is a significant public health concern, with a high fatality rate in humans and cats. In this study, we explored the genetic determinants that contribute to the different virulence of SFTS virus (SFTSV) based on Tk-F123 and Ng-F264 strains isolated from cats. Tk-F123 was 100% lethal in type I interferon receptor-knockout mice, whereas Ng-F264 exhibited no fatality. We identified a pair of amino acid residues in the Gc protein, glycine and serine, at residues 581 and 934, respectively, derived from Tk-F123, leading to a fatal infection. Those in Ng-F264 were arginine and asparagine. These results suggest that this pair of residues affects the Gc protein function and regulates SFTSV virulence. Our findings provide useful clues for the elucidation of viral pathogenicity and the development of effective live-attenuated vaccines and antiviral strategies.

Interplay of CDKs and Cyclins with glycolytic regulatory enzymes PFK and PK.

In plants, as in all eukaryotes, the cell cycle is regulated by the heterodimer formed by cyclins (Cycs) and cyclin-dependent kinases (CDKs), that phosphorylate serine/threonine residues in target proteins. The extensive involvement of these heterodimers in nuclear cell cycle-related processes has been demonstrated. However, recent findings have linked Cyc-CDK complexes to the regulation of cytosolic processes, including various metabolic pathways, suggesting close coordination between the cell cycle and catabolic/anabolic processes to maintain cellular energy homeostasis.This study extends the analysis of Cyc-CDK complex regulation in maize to two key regulators of glycolysis: phosphofructose kinase (PFK) and pyruvate kinase (PK). Both are cytosolic enzymes, highly regulated positively and negatively by different metabolites, showing a similar activation pattern in their homotetrameric form and low activity when as dimers/monomers. Each enzyme exhibits two putative minimal phosphorylation motives for Cyc-CDKs, conserved in some plant species and in four (PFK) and three (PK) isoforms in maize. This work demonstrates that both enzymes are active with fluctuating levels of activity along maize germination; also, that they associate with different maize Cycs and CDKs as demonstrated by pull-down assays, as well as their in vitro phosphorylation by recombinant CycD;2-CDKA or CycD2;2-CDKB complexes. Additionally, the inhibition of PFK and PK activity following phosphorylation by active Cycs-CDKB complexes obtained by immunoprecipitation from imbibed embryonic axis protein extracts suggests a narrow and negative regulation of glycolysis as the cell cycle progresses. A decreased carbon flow through this pathway is proposed to divert carbon from sugars towards the oxidative pentose phosphate pathway, thereby promoting de novo nucleic acid synthesis precursors to stimulate cell cycle progression.

Maggot kinase: A novel and cost-effective fibrinolytic enzyme from maggots

Maggot kinase, a novel fibrinolytic enzyme source, has been isolated from fly maggots and comprehensively characterized. Using CM-52 ion exchange chromatography and affinity chromatography, we obtained a highly purified and active form of the enzyme. In particular, the fibrinolytic activity of maggot kinase, evaluated using the fibrin plate method, was found to be 8.98 ± 0.08 × 105 U/mg, demonstrating significant efficacy. Further structural analysis using mass spectrometry revealed that maggot kinase consists of a primary sequence of 226 amino acid residues with a molecular weight of 22.91 kDa. In vitro thrombolytic assays demonstrated the enzyme's remarkable ability to degrade the essential fibrinogen subunits (α, β, and γ), thereby facilitating clot lysis. Notably, our studies in a mouse model underscored the significant in vivo thrombolytic activity of maggot kinase, demonstrating its potential to inhibit thrombosis. The finding is particularly significant considering the widespread use of fly maggots in agriculture and animal husbandry due to their rapid growth cycle and minimal nutritional requirements. Our research highlights the untapped potential of fly maggots as a source for maggot kinase development for antithrombotic drug and functional food applications.

Exploration of 1,3,5-trisubstituted pyrazoline derivatives as human carbonic anhydrase inhibitors: Synthesis, biological evaluation and in silico studies

The human carbonic anhydrase (hCA) IX and XII isoforms are overexpressed in hypoxic conditions, contributing to cancer. Lack of isoform selectivity has been one of the main challenges associated with the existing drugs targeting hCAs. Hence, the development of alternative approaches, such as tail approach to develop more selective hCA IX and XII inhibitors is need of the hour. In the present work, we designed and synthesized 24 new 1,3.5-trisubstituted-pyrazoline derivatives with diverse substitutions. The synthesized analogs were evaluated for their hCA inhibitory activities against hCA I, II, IX, and XII isoforms. Among the tested compounds, derivative 8 displayed good inhibitory activity against hCA IX (Ki = 331 nM) and XII (Ki = 96.7 nM). In addition, 9a-g also exhibited some inhibitory activities against hCA IX and XII, with Kis ranging from 574 to 799 nM and 137–369 nM, respectively. Molecular modelling studies of compound 8 displayed metal coordination with zinc ion and hydrophobic, hydrophilic interactions with adjacent amino acid residues, and maintained stable interactions throughout 100 ns. In addition, ADMET studies demonstrated that compound 8 obeyed the Lipinski's rule of five and was found to be druggable and non-toxic. Hence, compound 8 was identified as potential lead for further development.

Interfacial structure modification and enhanced emulsification stability of microalgae protein through interaction with anionic polysaccharides

Microalgae protein (MP) have emerged as a focal point of research within food processing due to its nutritional value and foaming properties. However, its isoelectric point around pH 4 leads to it susceptible to collision, binding, and precipitation. Additionally, MP has poor emulsification properties and only shows stability under strongly alkaline conditions. This study investigated the effects of food-grade anionic polysaccharides (guar gum (GG), gum arabic (GA), low acyl gellan gum (LG), and pectin (PT)) on the molecular structure and emulsification properties of MP. Results indicated that these anionic polysaccharides enhanced the UV absorption of MP near 620 nm, especially at 14 % content, while fluorescence intensity decreased due to amino acid residues masking without structural changes. The addition of polysaccharides resulted in bimodal or multimodal particle size distributions, with LG and PT showing larger particle sizes. At pH 4, negatively charged polysaccharides formed stable complexes with near-neutral MP, improving solution stability via electrostatic repulsion and diminishing turbidity. The droplet distribution analysis indicated that higher anionic polysaccharide ratios (1:4, 1:2, and 1:1) correlated with smaller droplet sizes and increased emulsion stability. Zeta-potential measurements revealed negative charges for emulsions, with LG-MP and PT-MP complexes displaying higher absolute values (15.0 to 20.7 mV) compared to GG-MP and GA-MP complexes, indicating superior stability. Storage stability analysis showed that LG-MP and PT-MP complexes stabilized emulsions had minimal delamination over two weeks. Rheological assessments showed that increasing GG and GA contents from 14 % to 50 % had negligible effects on apparent viscosity, while LG-MP (1:1) complexes stabilized emulsion displayed higher viscosity compared to PT-MP emulsions. Frequency sweep results showed that GG-MP, GA-MP, and LG-MP emulsions had greater elastic moduli (G') than viscous moduli (G"), indicating elastic behavior, whereas PT-MP emulsions transitioned from liquid-like to solid-like behavior as frequency increased. This study illustrates the advantages of high LG and PT content in preventing particle aggregation and enhancing emulsion stability, providing a theoretical and practical foundation for MP applications in food processing.

Three Stages of Nascent Protein Translocation Through the Ribosome Exit Tunnel.

All proteins in living organisms are produced in ribosomes that facilitate the translation of genetic information into a sequence of amino acid residues. During translation, the ribosome undergoes initiation, elongation, termination, and recycling. In fact, peptide bonds are formed only during the elongation phase, which comprises periodic association of transfer RNAs and multiple auxiliary proteins with the ribosome and the addition of an amino acid to the nascent polypeptide one at a time. The protein spends a considerable amount of time attached to the ribosome. Here, we conceptually divide this portion of the protein lifetime into three stages. We define each stage on the basis of the position of the N-terminus of the nascent polypeptide within the ribosome exit tunnel and the context of the catalytic center. We argue that nascent polypeptides experience a variety of forces that determine how they translocate through the tunnel and interact with the tunnel walls. We review current knowledge about nascent polypeptide translocation and identify several white spots in our understanding of the birth of proteins.