Amino Acid Residues Research Articles

Phytochromobilin Binding and Specific Amino Acid Residues Near The Chromophore Contribute To Orange Light Perception By The Dualchrome Phytochrome Region.

A novel photoreceptor dualchrome 1 (DUC1), containing a fused structure of cryptochrome and phytochrome, was discovered in the marine green alga Pycnococcus provasolli. The DUC1 phytochrome region (PpDUC1-N) binds to the bilin (linear tetrapyrrole) chromophores, phytochromobilin (PΦB) or phycocyanobilin (PCB), and reversibly photoconverts between the orange-absorbing dark-adapted state and the far-red-absorbing photoproduct state. This contrasts with typical phytochromes, which photoconvert between the red-absorbing dark-adapted and far-red-absorbing photoproduct states. In this study, we examined the molecular mechanism of PpDUC1-N to sense orange light by identifying the chromophore species synthesized by P. provasolli and the amino acid residues within the PpDUC1-N responsible for sensing orange light in the dark-adapted state. We focused on the PcyA homolog of P. provasolli (PpPcyA). Coexpression with the photoreceptors followed by an enzymatic assay revealed that PpPcyA synthesized PCB. Next, we focused on the PpDUC1-N GAF domain responsible for chromophore binding and light sensing. Ten amino acid residues were selected as the mutagenesis target near the chromophore. Replacement of these residues with those conserved in typical phytochromes revealed that three mutations (F290Y/M304S/L353M) resulted in a 23-nm red-shift in the dark-adapted state. Finally, we combined these constructs to obtain the PΦB-binding F290Y/M304S/L353M mutant and a 38-nm red-shift was observed compared with the PCB-binding wild-type PpDUC1. The binding chromophore species and the key residues near the chromophore contribute to blue-shifted orange light sensing in the dark-adapted state of the PpDUC1-N.

Mono-ADP-ribosylation of peptides: an overview of synthetic and chemoenzymatic methodologies.

Adenosine diphosphate (ADP)-ribosylation is a ubiquitous post-translational modification that regulates vital biological processes like histone reorganization and DNA-damage repair through the modification of various amino acid residues. Due to advances in mass-spectrometry, the collection of long-known ADP-ribose (ADPr) acceptor sites, e.g. arginine, cysteine and glutamic acid, has been expanded with serine, tyrosine and histidine, among others. Well-defined ADPr-peptides are valuable tools for investigating the exact structures, mechanisms of action and interaction partners of the different flavors of this modification. This review provides a comprehensive overview of synthetic and chemoenzymatic methodologies that enabled the construction of peptides mono-ADP-ribosylated on various amino acids, and close mimetics thereof.

Hemoglobin Hydrolyzate Promotes Iron Absorption in the Small Intestine through Iron Binding Peptides.

Hemoglobin is an excellent source of iron supplements, and its hydrolyzate spontaneously binds iron during digestion and promotes iron absorption in vivo. However, the underlying mechanisms of what peptides bind and how they bind iron ions remain unclear. This study prepared the porcine hemoglobin hydrolyzate through enzymatic hydrolysis and acid treatment and investigated the mechanisms of hemoglobin hydrolyzate on iron absorption through the determination of iron levels in dietary intervention mice, iron binding site analyses, peptide digestion analyses, molecular simulation docking, and INT407 cell validation. The results showed that ingestion of the hemoglobin hydrolyzate diets increased iron levels in the blood of mice, accompanied by the upregulation of duodenal iron circulation-related genes such as ferritin, PCBP1, and HP. Carboxyl, imidazole groups, and aromatic amino acid residues were iron binding sites of hemoglobin hydrolyzate during digestion. VDEVGGEA and VDEVGGE were found to involve the spontaneous and efficient binding of hemoglobin hydrolyzate to iron ions in the intestinal cavity. In particular, the DEVGGE peptide was the typical sequence for hemoglobin hydrolytic peptides to exert iron binding activity.

Identification of Potent SFRP1 Inhibitors for Colorectal Cancer using a Comprehensive Computational Approach.

The incidence of CRC has increased worldwide over the past decade. The statistics report from WHO highlights the increased severity and fatality rate of CRC among the populations. Wnt/β-catenin is recognized as the resource for cell regeneration and cancer signaling pathways driven by frizzled receptor cofactors. Aberrant regulation of Wnt/β- catenin suppression is an important challenge in treating CRC management. The SFRP1 comprises a cysteine-rich region that is homologous to the putative Wnt-binding sites of Frizzled proteins, with the potential to impede and alter the cascade of Wnt signaling. Indirect regulation, like targeting Wnt antagonist SFRP1, is an alternative strategy to suppress the cancer signals by enhancing the apoptotic activity. Hence, this study aimed to approach the SFRP1 protein as a therapeutic target to inhibit Wnt signaling in colorectal cancer. Further, it aimed to identify the lead compounds against the SFRP1 protein, which inhibit the oncogenic expression of CRC, which might be possible using computational approaches, recognizing the importance of the SFRP1 protein role in CRC. The homology-modeled SFRP1 structure was refined, and virtual screening was performed against the anti-cancer drugs and natural drug databases to find the best hit molecules. The molecular docking, MD, and MMGBSA analysis confirmed the firm binding of SFRP1 complexes to identify the potent CRC inhibitors. The amino acid residues Arg5, Arg11, Ala13, Lys 245, Lys274, Phe147, Pro99, and Ser277 are essential for ligand binding and show similar interactions for SFRP1 complexes. The ADME/T profile for top hits is acceptable in range and obtains the drug-likeness property. The 100ns run for MD simulation confirms the stability of protein complexes. Overall, the findings of this study reveal that the lead compounds screened are capable of inhibiting SFRP1 against CRC. Targeting SFRP1 paves the way for new platforms in the field of cancer and the therapeutic sector for new approachable finds.

Active Site Characterization of a Campylobacter jejuni Nitrate Reductase Variant Provides Insight into the Enzyme Mechanism.

Mo K-edge X-ray absorption spectroscopy (XAS) is used to probe the structure of wild-type Campylobacter jejuni nitrate reductase NapA and the C176A variant. The results of extended X-ray absorption fine structure (EXAFS) experiments on wt NapA support an oxidized Mo(VI) hexacoordinate active site coordinated by a single terminal oxo donor, four sulfur atoms from two separate pyranopterin dithiolene ligands, and an additional S atom from a conserved cysteine amino acid residue. We found no evidence of a terminal sulfido ligand in wt NapA. EXAFS analysis shows the C176A active site to be a 6-coordinate structure, and this is supported by EPR studies on C176A and small molecule analogs of Mo(V) enzyme forms. The SCys is replaced by a hydroxide or water ligand in C176A, and we find no evidence of a coordinated sulfhydryl (SH) ligand. Kinetic studies show that this variant has completely lost its catalytic activity toward nitrate. Taken together, the results support a critical role for the conserved C176 in catalysis and an oxygen atom transfer mechanism for the catalytic reduction of nitrate to nitrite that does not employ a terminal sulfido ligand in the catalytic cycle.

Toxin Homology Domain in Plant Type 2 Prolyl 4-Hydroxylases Acts as a Golgi Localization Domain

Prolyl 4-hydroxylase (P4H) generates hydroxyproline residues in proteins. Two classes of P4H have been found in plants. Type 1 P4H has a signal anchor at the N-terminus, while type 2 P4H has both an N-terminal signal peptide and a C-terminal toxin homology domain (Tox1 domain) with six conserved cysteine residues. We analyzed the localization of tobacco type 2 P4H (NtP4H2.2) in tobacco BY-2 cells. Cell fractionation studies, immunostaining of cells, and GFP fusion study indicated that NtP4H2.2 localizes predominantly to the Golgi apparatus and is a peripheral membrane protein associated with the luminal side of organelles. Expression of the GFP-Tox1 domains of NtP4H2.2 and another tobacco type 2 P4H NtP4H2.1 in BY-2 cells and Arabidopsis epidermal cells indicated that these proteins were targeted to the Golgi. The Tox1 domains from Arabidopsis and rice type 2 P4Hs also directed GFP to the Golgi in tobacco BY-2 cells. The Tox1 domain of NtP4H2.2 increased the membrane association of GFP, and mutation of the cysteine residues in this domain abolished Golgi localization. Furthermore, the catalytic domain of NtP4H2.2 also directed GFP to the Golgi. Thus, the Tox1 domains of plant P4Hs are the Golgi localization domains, and tobacco P4H2.2 localizes to the Golgi by the action of both this domain and the catalytic domain.

Unveiling Charge-Pair Salt-Bridge Interaction Between GAGs and Collagen Protein in Cartilage: Atomic Evidence from DNP-Enhanced ssNMR at Natural Isotopic Abundance.

The interactions between glycosaminoglycans (GAGs) and proteins are essential in numerous biochemical processes that involve ion-pair interactions. However, there is no evidence of direct and specific interactions between GAGs and collagen proteins in native cartilage. The resolution of solid-state NMR (ssNMR) can offer such information but the detection of GAG interactions in cartilage is limited by the sensitivity of the experiments when 13C and 15N isotopes are at natural abundance. In this communication, this limitation is overcome by taking advantage of dynamic nuclear polarization (DNP)-enhanced magic-angle spinning (MAS) experiments to obtain two-dimensional (2D) 15N-13C and 13C-13C correlations on native samples at natural abundance. These experiments unveiled inter-residue correlations in the aliphatic regions of the collagen protein previously unobserved. Additionally, our findings provide direct evidence of charge-pair salt-bridge interactions between negatively charged GAGs and positively charged arginine (Arg) residues of collagen protein. We also identified potential hydrogen bonding interactions between hydroxyproline (Hyp) and GAGs, offering atomic insights into the biochemical interactions within the extracellular matrix of native cartilage. Our approach may provide a new avenue for the structural characterization of other native systems.

In Silico Investigation against Inhibitors of Alpha-Amylase Using Structure-based Screening, Molecular Docking, and Molecular Simulations Studies.

Type-II diabetes mellitus is a chronic disorder that results from fluctuations in the glucose level leading to hyperglycemia with severe adverse effects increasing worldwide. Alpha-Amylase is the key enzyme involved in the mechanism of glucose formation therefore Alpha-Amylase inhibitors have become a therapeutic target in the development of new leads as they have the potential to suppress glucose levels. Existing drugs targeting Alpha-Amylase highlight major drawbacks in terms of poor absorption rate that causes several gastrointestinal issues. So, this research is aimed to develop novel inhibitors interacting with Alpha-Amylase's active site using structural-based screening, binding pattern analysis, and molecular dynamic simulation. Hence, to search for a potential lead, we analyzed a total of 133 valiolamine derivatives and 535 desoxynojirimycin derivatives that exhibited drug-like properties screened through Lipinski filters. Virtual screening followed by binding interaction analysis we identified ten compounds that exhibited better binding energy scores compared to the standard drugs voglibose and miglitol, used in our study. The docking analysis, ADMET and metabolic site prediction estimated the best top two compounds with good drug profiles. Further, top compounds VG9 and VG15 were promoted to simulation study using the Biovia Discovery study to access the stability at a time interval of 100 ns. MD simulation results revealed that our compound VG9 possesses better conformational stability in the complex to the active site residues of Alpha-Amylase target protein than standard drug voglibose. Thus, our investigation revealed that compound VG9 also exhibits the best pharmacokinetic as well as binding affinity results and could act as a potential lead compound targeting Alpha-Amylase for Type II diabetes.

Conjugation with the Carrier Helped to Reveal acidification-Induced Structural Shift in the Peptide from Phospholipase Domain of Parvovirus B19.

Spectroscopic studies on domains and peptides of large proteins are complicated because of the tendency of short peptides to form oligomers in aquatic buffers, but conjugation of a peptide with a carrier protein may be helpful. In this study we approved that a fragment of SK30 peptide from phospholipase A2 domain of VP1 Parvovirus B19 capsid protein (residues: 144-159; 164; 171-183; sequence: SAVDSAARIHDFRYSQLAKLGINPYTHWTVADEELLKNIK) turns from random coil to alpha helix in the acidic medium only in case if it had been conjugated with BSA (through additional N-terminal Cys residue, turning it into CSK31 peptide, and SMCC linker) according to CD-spectroscopy results. In contrast, unconjugated SK30 peptide does not undergo such shift because it forms stable oligomers connected by intermolecular antiparallel beta sheet, according to IR-spectroscopy, CD-spectroscopy, blue native gel electrophoresis and centrifugal ultrafiltration, as, probably, the whole isolated phospholipase domain of VP1 protein does. However, being a part of the long VP1 capsid protein, phospholipase domain may change its fold during the acidification of the medium in the endolysosome by the way of the formation of contacts between protonated His153 and Asp175, promoting the shift from random coil to alpha helix in its N-terminal part. This study opens up a perspective of vaccine development, since rabbit polyclonal antibodies against the conjugate of CSK31 peptide with BSA, in which the structure of the second alpha helix from the phospholipase A2 domain should be reproduced, can bind epitopes of the complete recombinant unique part of VP1 Parvovirus B19 capsid (residues: 1-227).

Design of Multivariate Biological Metal-Organic Frameworks: Toward Mimicking Active Sites of Enzymes.

Mimicking enzymatic processes carried out by natural enzymes, which are highly efficient biocatalysts with key roles in living organisms, attracts much interest but constitutes a synthetic challenge. Biological metal-organic frameworks (bioMOFs) are potential candidates to be enzyme catalysis mimics, as they offer the possibility to combine biometals and biomolecules into open-framework porous structures capable of simulating the catalytic pockets of enzymes. In this work, we first study the catalase activity of a previously reported bioMOF, derived from the amino acid L-serine, with formula {CaIICuII6[(S,S)-serimox]3(OH)2(H2O)} · 39H2O (1) (serimox = bis[(S)-serine]oxalyl diamide), which is indeed capable to mimic catalase enzymes, in charge of preventing cell oxidative damage by decomposing, efficiently, hydrogen peroxide to water and oxygen (2H2O2 → 2 H2O + O2). With these results in hand, we then prepared a new multivariate bioMOF (MTV-bioMOF) that combines two different types of bioligands derived from L-serine and L-histidine amino acids with formula CaIICuII6[(S,S)-serimox]2[(S,S)-hismox]1(OH)2(H2O)}·27H2O (2) (hismox = bis[(S)-histidine]oxalyl diamide ligand). MTV-bioMOF 2 outperforms 1 degrading hydrogen peroxide, confirming the importance of the amino acid residue from the histidine amino acid acting as a nucleophile in the catalase degradation mechanism. Despite displaying a more modest catalytic behavior than other reported MOF composites, in which the catalase enzyme is immobilized inside the MOF, this work represents the first example of a MOF in which an attempt is made to replicate the active center of the catalase enzyme with its constituent elements and is capable of moderate catalytic activity.

Delivery of rapamycin by biomimetic peptide nanoparticles targeting oxidized low-density lipoprotein in atherosclerotic plaques.

Drug delivery systems based on biomimetic peptide nanoparticles are steadily gaining prominence in the treatment of diverse medical conditions. This study focused on the development of peptides that depend on ligand-receptor interactions to load rapamycin (RAPA). Furthermore, a multifunctional peptide was engineered to target oxidized low-density lipoprotein (oxLDL) within atherosclerotic plaques, facilitating the localized delivery of RAPA. The interactions between peptides and RAPA/oxLDL were analyzed by simulations and experimental approaches. Results show that the main amino acid residues on the mammalian target of rapamycin that bind to RAPA are constructed as peptides (P1 and P2), which have specific interactions with RAPA and can effectively improve the loading efficiency of RAPA. The encapsulation and drug loading efficiencies of P1/P2 were 68.0/47.9% and 48.3/36.5%, respectively. In addition, the interaction force of the multifunctional peptide (P3) and oxLDL surpassed that of their interaction with human umbilical vein endothelial cells by a factor of 3.6, conclusively establishing the specific targeting of oxLDL by these nanoparticles. The encapsulation and drug loading efficiencies of P3 for RAPA were determined to be 60.2% and 41.5%. P3 can effectively load RAPA and target oxLDL within the plaque, suggesting that P3 has potential as a therapeutic agent for atherosclerotic disease.

EThcD as a Unique Tool for the Top-Down De Novo Sequencing of Intact Natural Ranid Amphibian Peptides.

De novo sequencing of any novel peptide/protein is a difficult task. Full sequence coverage, isomeric amino acid residues, inter- and intramolecular S-S bonds, and numerous other post-translational modifications make the investigators employ various chemical modifications, providing a variety of specific fragmentation MSn patterns. The chemical processes are time-consuming, and their yields never reach 100%, while the subsequent purification often leads to the loss of minor components of the initial peptide mixture. Here, we present the advantages of the EThcD method that enables establishing the full sequence of natural intact peptides of ranid frogs in de novo top-down mode without any chemical modifications. The method provides complete sequence coverage, including the cyclic disulfide section, and reliable identification of isomeric leucine/isoleucine residues. The proposed approach demonstrated its efficiency in the analysis of peptidomes of ranid frogs from several populations of Rana arvalis, Rana temporaria, and Pelophylax esculentus complexes.

Using Attention-UNet Models to Predict Protein Contact Maps.

Proteins are essential to life, and understanding their intrinsic roles requires determining their structure. The field of proteomics has opened up new opportunities by applying deep learning algorithms to large databases of solved protein structures. With the availability of large data sets and advanced machine learning methods, the prediction of protein residue interactions has greatly improved. Protein contact maps provide empirical evidence of the interacting residue pairs within a protein sequence. Template-free protein structure prediction systems rely heavily on this information. This article proposes UNet-CON, an attention-integrated UNet architecture, trained to predict residue-residue contacts in protein sequences. With the predicted contacts being more accurate than state-of-the-art methods on the PDB25 test set, the model paves the way for the development of more powerful deep learning algorithms for predicting protein residue interactions. The source codes are available in the GitHub link: (https://github.com/jisnava/UNet CON).

Exploring the central region of amylin and its analogs aggregation: the influence of metal ions and residue substitutions

Human amylin (hIAPP) is found in the form of amyloid deposits within the pancreatic cells of nearly all patients diagnosed with type 2 diabetes mellitus (T2DM). However, rat amylin (rIAPP) and pramlintide - hIAPP analogs - are both non-toxic and non-amyloidogenic. Their primary sequences exhibit only slight variations in a few amino acid residues, primarily concentrated in the central region, spanning residues 20 to 29. This inspired us to study this fragment and investigate the impact on the aggregation properties of substituting residues within the central region of amylin and its analogs. Six fragments derived from amylin have undergone comprehensive testing against various metal ions by implementing a range of analytical techniques, including Nuclear Magnetic Resonance (NMR) spectroscopy, Thioflavin T (ThT) assays, Atomic Force Microscopy (AFM), and cytotoxicity assays. These methodologies serve to provide a thorough understanding of how the substitutions and interactions with metal ions impact the aggregation behavior of amylin and its analogs.

Elucidating the complex membrane binding of a protein with multiple anchoring domains using extHMMM.

Membrane binding is a crucial mechanism for many proteins, but understanding the specific interactions between proteins and membranes remains a challenging endeavor. Coagulation factor Va (FVa) is a large protein whose membrane interactions are complicated due to the presence of multiple anchoring domains that individually can bind to lipid membranes. Using molecular dynamics simulations, we investigate the membrane binding of FVa and identify the key mechanisms that govern its interaction with membranes. Our results reveal that FVa can either adopt an upright or a tilted molecular orientation upon membrane binding. We further find that the domain organization of FVa deviates (sometimes significantly) from its crystallographic reference structure, and that the molecular orientation of the protein matches with domain reorganization to align the C2 domain toward its favored membrane-normal orientation. We identify specific amino acid residues that exhibit contact preference with phosphatidylserine lipids over phosphatidylcholine lipids, and we observe that mostly electrostatic effects contribute to this preference. The observed lipid-binding process and characteristics, specific to FVa or common among other membrane proteins, in concert with domain reorganization and molecular tilt, elucidate the complex membrane binding dynamics of FVa and provide important insights into the molecular mechanisms of protein-membrane interactions. An updated version of the HMMM model, termed extHMMM, is successfully employed for efficiently observing membrane bindings of systems containing the whole FVa molecule.

Light-responsive transcription factors VvHYH and VvGATA24 mediate wax terpenoid biosynthesis in Vitis vinifera.

The cuticular wax that covers the surfaces of plants is the first barrier against environmental stresses and increasingly accumulates with light exposure. However, the molecular basis of light-responsive wax biosynthesis remains elusive. In grape (Vitis vinifera), light exposure resulted in higher wax terpenoid content and lower decay and abscission rates than controls kept in darkness. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA-seq data were integrated to draw the chromatin accessibility and cis-elements regulatory map to identify the potential action sites. Terpenoid synthase 12 (VvTPS12) and 3-Hydroxy-3-methylglutaryl-CoA reductase 2 (VvHMGR2) were identified as grape wax biosynthesis targets, while VvHYH and VvGATA24 were identified as terpenoid biosynthesis activators, as more abundant wax crystals and higher wax terpenoid content were observed in transiently overexpressed grape berries and Nicotiana benthamiana leaves. The interaction between VvHYH and the open chromatin of VvTPS12 was confirmed qualitatively using a dual luciferase assay and quantitatively using surface plasma resonance, with an equilibrium dissociation constant of 2.81 nM identified via the latter approach. Molecular docking simulation implied the structural nature of this interaction, indicating that 24 amino acid residues of VvHYH, including Arg106A, could bind to the VvTPS12 G-box cis-element. VvGATA24 directly bound to the open chromatin of VvHMGR2, with an equilibrium dissociation constant of 8.59 nM. 12 amino acid residues of VvGATA24, including Pro218B, interacted with the VvHMGR2 GATA-box cis-element. Our work characterizes the mechanism underlying light-mediated wax terpenoid biosynthesis and provides gene targets for future molecular breeding.

Insights into the role of FoxL2 in tebuconazole-induced male- biased sex differentiation of zebrafish

Tebuconazole (TEB) is a commonly used fungicide that inhibits the aromatase Cyp19A and downregulates the transcription factor forkhead box L2 (FoxL2), leading to male-biased sex differentiation in zebrafish larvae. However, the specific mechanism by which FoxL2 functions following TEB exposure remains unclear. In this study, the phosphorylation sites and kinase-specific residues in zebrafish FoxL2 protein (zFoxL2) were predicted. Subsequently, recombinant zFoxL2 was prepared via prokaryotic expression, and a polyclonal rabbit-anti-zFoxL2 antibody was generated. Zebrafish fibroblast (ZF4) cells were exposed to 100-μM TEB alone for 8 h, after which changes in the expression of genes involved in the foxl2 regulatory pathway (akt1, pi3k, cyp19a1b, c/ebpb and sox9a) were detected. When co-exposed to 1-μM estradiol and 100-μM TEB, the expression of these key genes tended to be restored. Interestingly, TEB did not affect the expression of the foxl2 gene or protein but it significantly suppressed the phosphorylation of FoxL2 (pFoxL2) at serine 238 (decreased by 43.64 %, p = 0.009). Co-immunoprecipitation assays showed that, following exposure to 100-μM TEB, the total precipitated proteins in ZF4 cells decreased by 17.02 % (p = 0.029) and 31.39 % (p = 0.027) in the anti-zFoxL2 antibody group and anti-pFoxL2 (ser238) antibody group, respectively, indicating that TEB suppressed the capacity of the FoxL2 protein to bind to other proteins via repression of its own phosphorylation. The pull-down assay confirmed this conclusion. This study preliminarily elucidated that the foxl2 gene functions via post-translational regulation through hypophosphorylation of its encoded protein during TEB-induced male-biased sex differentiation.

Interaction mechanism of oseltamivir phosphate with bovine serum albumin: multispectroscopic and molecular docking study

Oseltamivir phosphate (OP) is an antiviral drug with potential risks to human health due to overuse, leading to serious consequences such as gastrointestinal disturbances, abnormal neuropsychiatric symptoms, and sudden death. Therefore, gaining an in-depth understanding of its interaction with proteins is crucial. We investigated the interaction between OP and bovine serum albumin (BSA) utilizing multispectral methods (i.e., fluorescence, ultraviolet absorption, circular dichroism) combined with molecular docking techniques. Fluorescence spectroscopy indicated that OP quenched BSA fluorescence by forming the OP-BSA complex. The Stern-Volmer constants (KSV) between OP and BSA were determined to be 3.06 × 103 L/mol, 2.36 × 103 L/mol, and 1.86 × 103 L/mol at 293 K, 298 K, and 303 K, respectively. OP occupies exclusively one binding site on BSA, and the fluorescent probe displacement measurements revealed that this is BSA site I. Thermodynamic data (∆H, ∆S, and ∆G) obtained by fitting the van’t Hoff equation were − 77.49 kJ/mol, -176.54 J/(mol∙K), and − 24.88 kJ/mol, respectively, suggesting that hydrogen bonding and van der Waals forces mainly participate in OP-BSA complex stabilization. Moreover, the reaction occurs spontaneously at room temperature. Synchronous fluorescence spectra indicated that OP interacts with tryptophan residue of BSA. The results of ultraviolet (UV) and 3D fluorescence spectroscopy indicated that the OP-BSA complex formation altered the microenvironment around amino acid residues. Circular dichroism spectra revealed that the addition of OP decreased the α-helix content of BSA by 7.13%. Docking analysis confirmed that OP binds to BSA site I through hydrogen bonding with amino acids VAL342, SER453, and ASP450. Finally, ADMET studies were conducted to explore the pharmacokinetics of OP as an antiviral drug.

Computational and in-vitro Investigation of Phytochemicals from Allamanda cathartica as a Potential Candidate for the Treatment of Type 2 Diabetes mellitus

Diabetes, a chronic metabolic disease, has become a severe health problem worldwide. According to the latest data from the International Diabetes Federation (IDF), there were 537 million diabetic mellitus (DM) patients worldwide in 2021, expected to increase to 783 million by 2045. Over 90% of people with diabetes have type 2 diabetes, which is driven by socio-economic, demographic, environmental, and genetic factors. Diabetes is still a major global health issue, with prevalence rates rising all across the world. This not only imposes a significant burden on healthcare systems but also impacts the quality of life of affected individuals and their families. Therefore, there is a continuous high demand worldwide to develop novel, more effective, and easily affordable medications against diabetes. This paper illustrates the in-vitro antidiabetic activity and probable binding mechanism prediction of phytoconstituents of Allamanda cathartica against type 2 diabetes molecular targets. Through the application of bioactivity score prediction, molecular docking, and ADMET prediction approach, the potential and selective phytoconstituents with the highest binding affinity and lower toxicity were gained from the curated datasets. Further molecular dynamics simulations and DFT calculations were carried out to identify the favorable binding conformations when the top-scored phytoconstituents bind with the PPAR[Formula: see text] receptors compared to the rosiglitazone. Compound AC2 interacts with the PPAR[Formula: see text] proteins by forming seven hydrogen bonds with the amino acid residues Phe282, His449, Tyr327, His323, and Ser289. The ligand was bound to the protein during the simulation since none of the complex conformations was unstable, and no unfolding or folding occurred. Our results provided an approach to further design and optimize the natural product-inspired small druglike molecules as potential antidiabetic agents. This study highlighted that the phytoconstituents of Allamanda cathartica have antidiabetic potential through the binding of PPAR[Formula: see text], [Formula: see text]-amylase and [Formula: see text]-glucosidase proteins that can be further explored for novel antidiabetic drug development.

A single-sample workflow for joint metabolomic and proteomic analysis of clinical specimens

Understanding the interplay of the proteome and the metabolome helps to understand cellular regulation and response. To enable robust inferences from such multi-omics analyses, we introduced and evaluated a workflow for combined proteome and metabolome analysis starting from a single sample. Specifically, we integrated established and individually optimized protocols for metabolomic and proteomic profiling (EtOH/MTBE and autoSP3, respectively) into a unified workflow (termed MTBE-SP3), and took advantage of the fact that the protein residue of the metabolomic sample can be used as a direct input for proteome analysis. We particularly evaluated the performance of proteome analysis in MTBE-SP3, and demonstrated equivalence of proteome profiles irrespective of prior metabolite extraction. In addition, MTBE-SP3 combines the advantages of EtOH/MTBE and autoSP3 for semi-automated metabolite extraction and fully automated proteome sample preparation, respectively, thus advancing standardization and scalability for large-scale studies. We showed that MTBE-SP3 can be applied to various biological matrices (FFPE tissue, fresh-frozen tissue, plasma, serum and cells) to enable implementation in a variety of clinical settings. To demonstrate applicability, we applied MTBE-SP3 and autoSP3 to a lung adenocarcinoma cohort showing consistent proteomic alterations between tumour and non-tumour adjacent tissue independent of the method used. Integration with metabolomic data obtained from the same samples revealed mitochondrial dysfunction in tumour tissue through deregulation of OGDH, SDH family enzymes and PKM. In summary, MTBE-SP3 enables the facile and reliable parallel measurement of proteins and metabolites obtained from the same sample, benefiting from reduced sample variation and input amount. This workflow is particularly applicable for studies with limited sample availability and offers the potential to enhance the integration of metabolomic and proteomic datasets.Graphical