Synthesis of taste active γ-glutamyl peptides with pea protein hydrolysate and their taste mechanism via in silico study.

Pea (Pisum sativum L.) protein hydrolysate (PPH) has a bitter taste, which has limited its use in food industry. γ-Glutamylation is used to debitter PPH. Results showed that the bitterness of PPH was decreased significantly due to the formation of γ-glutamyl peptides, including 16 γ-[Glu](n=1/2)-amino acids (AAs) and 8 newly discovered γ-glutamyl tripeptides (γ-Glu-Asn-Phe, γ-Glu-Leu-Val, γ-Glu-Leu-Tyr, γ-Glu-Gly-Leu, γ-Glu-Gly-Phe, γ-Glu-Gly-Tyr, γ-Glu-Val-Val, and γ-Glu-Gln-Tyr). Their total production concentrations were 27.25 µmol/L and 77.76 µmol/L, respectively. The γ-Glu-AA-AAs presented an umami-enhancing, salty-enhancing, and kokumi taste when their concentration reached 1.67 ± 0.20 âˆ¼ 2.07 ± 0.20, 1.65 ± 0.25 âˆ¼ 2.29 ± 0.45 and 0.68 ± 0.19 âˆ¼ 1.03 ± 0.22 mmol/L, respectively. The γ-Glu-AA-AAs exhibited a kokumi taste by entering the Venus flytrap (VFT) of the calcium-sensing receptor and interacting with Ser147, Ala168, and Ser170. γ-Glu-AA-AAs can enhance the umaminess of Monosodium Glutamate (MSG) as they can enter the binding pocket of the taste receptor type 1 subunit 3 (T1R3)-MSG complex.

Chromosome-level Dinobdella ferox genome provided a molecular model for its specific parasitism.

BACKGROUND: Dinobdella ferox is the most frequently reported leech species parasitizing the mammalian nasal cavity. However, the molecular mechanism of this special parasitic behavior has remained largely unknown. METHODS: PacBio long-read sequencing, next-generation sequencing (NGS), and Hi-C sequencing were employed in this study to generate a novel genome of D. ferox, which was annotated with strong certainty using bioinformatics methods. The phylogenetic and genomic alterations of D. ferox were then studied extensively alongside the genomes of other closely related species. The obligatory parasitism mechanism of D. ferox was investigated using RNA-seq and proteomics data. RESULTS: PacBio long-read sequencing and NGS yielded an assembly of 228 Mb and contig N50 of 2.16 Mb. Along Hi-C sequencing, 96% of the sequences were anchored to nine linkage groups and a high-quality chromosome-level genome was generated. The completed genome included 19,242 protein-coding genes. For elucidating the molecular mechanism of nasal parasitism, transcriptome data were acquired from the digestive tract and front/rear ends of D. ferox. Examining secretory proteins in D. ferox saliva helped to identify intimate connections between these proteins and membrane proteins in nasal epithelial cells. These interacting proteins played important roles in extracellular matrix (ECM)-receptor interaction, tight junction, focal adhesion, and adherens junction. The interaction between D. ferox and mammalian nasal epithelial cells included three major steps of pattern recognition, mucin connection and breakdown, and repair of ECM. The remodeling of ECM between epithelial cells of the nasal mucosa and epithelial cells of D. ferox may produce a stable adhesion environment for parasitism. CONCLUSIONS: Our study represents the first-ever attempt to propose a molecular model for specific parasitism. This molecular model may serve as a practical reference for parasitism models of other species and a theoretical foundation for a molecular process of parasitism.

Investigation of Potential cGMP-Specific PDE V and Aminopeptidase N Inhibitors of Allium ampeloprasum L. and Its Bioactive Components: Kinetic and Molecular Docking Studies.

The primary objectives of this study were to assess the inhibitory effects of Allium ampeloprasum L. extract (AAE) and its derived organosulfur and polyphenolic compounds on the enzymatic activities of cGMP-specific PDE V (PDE5) and aminopeptidase N (APN). Additionally, the study aimed to investigate their potential as inhibitors against these two target enzymes through kinetic analyses and molecular docking studies. The in vitro enzyme assays demonstrated that both AAE and its derived compounds significantly decreased the activity of PDE5 and APN. Further analyses involving kinetics and molecular docking provided insights into the specific inhibitor types of AAE and its derived compounds along with the proposed molecular docking models illustrating the interactions between the ligands (the compounds) and the enzymes (PDE5 and APN). In particular, AAE-derived polyphenolic compounds showed relatively stable binding affinity (-7.2 to -8.3 kcal/mol) on PDE5 and APN. Our findings proved the potential as an inhibitor against PDE5 and APN of AAE and AAE-derived organosulfur and polyphenolic compounds as well as a functional material for erectile dysfunction improvement.

Modulation of translational decoding by m6A modification of mRNA.

N6-methyladenosine (m6A) is an abundant, dynamic mRNA modification that regulates key steps of cellular mRNA metabolism. m6A in the mRNA coding regions inhibits translation elongation. Here, we show how m6A modulates decoding in the bacterial translation system using a combination of rapid kinetics, smFRET and single-particle cryo-EM. We show that, while the modification does not impair the initial binding of aminoacyl-tRNA to the ribosome, in the presence of m6A fewer ribosomes complete the decoding process due to the lower stability of the complexes and enhanced tRNA drop-off. The mRNA codon adopts a π-stacked codon conformation that is remodeled upon aminoacyl-tRNA binding. m6A does not exclude canonical codon-anticodon geometry, but favors alternative more dynamic conformations that are rejected by the ribosome. These results highlight how modifications outside the Watson-Crick edge can still interfere with codon-anticodon base pairing and complex recognition by the ribosome, thereby modulating the translational efficiency of modified mRNAs.

Pulse EPR spectroscopy and molecular modeling reveal the origins of the local heterogeneity of dietary fibers.

Optimizing human diet by including dietary fibers would be more efficient when the fibers' chain interactions with other molecules are understood in depth. Thereby, it is important to develop methods for characterizing the fiber chain to be able to monitor its structural alterations upon intermolecular interactions. Here, we demonstrate the utility of the electron paramagnetic resonance (EPR) spectroscopy, complemented by simulations in probing the atomistic details of the chain conformations for spin-labeled fibers. Barley ß-glucan, a native polysaccharide with linear chain, was utilized as a test fiber system to demonstrate the technique's capabilities. Pulse dipolar EPR data show good agreement with results of the fiber chain modeling, revealing sinuous chain conformations and providing polymer shape descriptors: the gyration tensor, spin-spin distance distribution function, and information about proton density near the spin probe. Results from EPR measurements point to the fiber aggregation in aqueous solution, which agrees with the results of the dynamic light scattering. We propose that the combination of pulse EPR measurements with modeling can be a perfect experimental tool for in-depth structural investigation of dietary fibers and their interaction under such conditions, and that the presented methodology can be extended to other weakly ordered or disordered macromolecules.

Discovery and biological evaluation of cholic acid derivatives as potent TGR5 positive allosteric modulators.

In this study, twenty-two novel cholic acid (CA) derivatives were designed and synthesized as potential Takeda G protein-coupled receptor 5 (TGR5) positive allosteric modulators (PAMs) using structure-based drug design (SBDD). GloSensor cAMP accumulation assay was employed to assess the functional activity and allosteric mechanism of final compounds. Biological results showed that all target compounds were able to activate the TGR5 in the cAMP formation assay. Remarkably, compound B1, selective methylation of 7-OH in CA, exhibited 5-fold higher activity for TGR5 compared to that of CA. Moreover, B1 positively modulate the functional activity of chenodeoxycholic acid (CDCA) in TGR5, indicating that B1 is a TGR5 PAM. On the other hand, 12-carbonyl derivative A1 displayed 7-fold higher potency for TGR5 relative to CA. Unexpectedly, compound A1 exhibited the same positive allosteric effect as B1, suggesting that A1 is a TGR5 PAM as well. Molecular modeling study revealed that 12-carbonyl in A1 and 12-OH in B1 formed H-bolds with the key amino acid Thr131, which are significant for TGR5 allosteric property. Taken together, we found two potent TGR5 PAMs A1 and B1 through SBDD, which could be used as lead compounds to further study TGR5 allosteric functionality.

Theoretical study on efficient HF gas sensing by functionalized, decorated, and doped nanocone strategy.

Hydrogen fluoride (HF) is a highly dangerous and corrosive gas that can cause severe burns and respiratory damage. The density functional theory method (DFT) used to study the interaction between the HF gas and the surface of a carbon nanocone (CNC) doped with gallium atom as a chemical sensor. The results showed that CNC wasn't a good candidate to sense the HF gas and consequently its electrical properties are changed insignificant. To improve the properties of the CNC, several strategies were tried: functionalizing by pyridinol (Pyr) and pyridinol oxide (PyrO), decorated with metals (M = B, Al, and Ga), and doped with element of third group (M = B, Al, and Ga). The obtained data demonstrated that the promising results were obtained by doping the CNC with Ga atom. After full optimization, we achieved one stable configuration between the HF gas and CNC-Ga structure (S15 configuration) with Eads = -19.86 kcal/mol. The electronic properties of the CNC-Ga structure is sensible changed after the HF molecule is adsorbed. According to calculated the energy gap between HOMO and LUMO orbitals of S15 configuration are increased which could be applied a chemical signal. Eventually, one could propose that the CNC-Ga has the ability to act as a Φ-type sensor based on its physical adsorption energy and quick recovery time and doped with gallium atom is a promising strategy.

Combining Experimental Restraints and RNA 3D Structure Prediction in RNA Nanotechnology.

Precise RNA tertiary structure prediction can aid in the design of RNA nanoparticles. However, most existing RNA tertiary structure prediction methods are limited to small RNAs with relatively simple secondary structures. Large RNA molecules usually have complex secondary structures, including multibranched loops and pseudoknots, allowing for highly flexible RNA geometries and multiple stable states. Various experiments and bioinformatics analyses can often provide information about the distance between atoms (or residues) in RNA, which can be used to guide the prediction of RNA tertiary structure. In this chapter, we will introduce a platform, iFoldNMR, that can incorporate non-exchangeable imino protons resonance data from NMR as restraints for RNA 3D structure prediction. We also introduce an algorithm, DVASS, which optimizes distance restraints for better RNA 3D structure prediction.

Effector-dependent structural transformation of a crystalline framework with allosteric effects on molecular recognition ability.

Structurally flexible porous crystals that combine high regularity and stimuli responsiveness have received attracted attention in connection with natural allostery found in regulatory systems of activity and function in biological systems. Porous crystals with molecular recognition sites in the inner pores are particularly promising for achieving elaborate functional control, where the local binding of effectors triggers their distortion to propagate throughout the structure. Here we report that the structure of a porous molecular crystal can be allosterically controlled by local adsorption of effectors within low-symmetry nanochannels with multiple molecular recognition sites. The exchange of effectors at the allosteric site triggers diverse conversion of the framework structure in an effector-dependent manner. In conjunction with the structural conversion, it is also possible to switch the molecular affinity at different recognition sites. These results may provide a guideline for the development of supramolecular materials with flexible and highly-ordered three-dimensional structures for biological applications.

Histamine-bound magnesium porphyrins: diverse coordination modes, inhibitory role in photodegradation of chlorophyll a and antioxidant activity.

The neurotransmitter histamine exists in two isomeric forms and could be an interesting ligand due to three nitrogen atoms with the possibility of binding to metals in different ways besides its crucial role in biological systems. However, no metal-histamine interaction is known in the literature. Therefore, two histamine-bound magnesium porphyrins [MgT(4-Cl)PP(hist)2] 1 and [MgT(4-Br)PP(hist)] 2 have been synthesized and structurally characterized. Interestingly, 1 is a hexa-coordinated magnesium porphyrin due to the axial coordination of two histamine molecules via the nitrogen of the aliphatic amino group with the Mg-Nhistamine distance of 2.300 Å, while 2 is penta-coordinated due to the axial coordination of one histamine molecule through the imidazole nitrogen atom with the Mg-Nhistamine distance of 2.145 Å. The diverse coordination modes of this unique ligand are explored for the first time. Theoretical studies at the level of DFT supported the binding of histamine via imidazole nitrogen atoms for complex 2. Histamine-bound magnesium porphyrins are found to be stable against the photodegradation of magnesium porphyrin in the presence of light and oxygen. Freshly isolated chlorophyll a from spinach showed similar resistivity against photodegradation. Moreover, the histamine-bound complexes showed higher antioxidant activity for 1 (92.45%) compared to the free base porphyrin (73.11%) and MgT(4-Cl)PP (75.89%).

Using Data Science for Mechanistic Insights and Selectivity Predictions in a Non-Natural Biocatalytic Reaction.

The study of non-natural biocatalytic transformations relies heavily on empirical methods, such as directed evolution, for identifying improved variants. Although exceptionally effective, this approach provides limited insight into the molecular mechanisms behind the transformations and necessitates multiple protein engineering campaigns for new reactants. To address this limitation, we disclose a strategy to explore the biocatalytic reaction space and garner insight into the molecular mechanisms driving enzymatic transformations. Specifically, we explored the selectivity of an "ene"-reductase, GluER-T36A, to create a data-driven toolset that explores reaction space and rationalizes the observed and predicted selectivities of substrate/mutant combinations. The resultant statistical models related structural features of the enzyme and substrate to selectivity and were used to effectively predict selectivity in reactions with out-of-sample substrates and mutants. Our approach provided a deeper understanding of enantioinduction by GluER-T36A and holds the potential to enhance the virtual screening of enzyme mutants.

Leveraging AI Advances and Online Tools for Structure-Based Variant Analysis.

Understanding how a gene variant affects protein function is important in life science, as it helps explain traits or dysfunctions in organisms. In a clinical setting, this understanding makes it possible to improve and personalize patient care. Bioinformatic tools often only assign a pathogenicity score, rather than providing information about the molecular basis for phenotypes. Experimental testing can furnish this information, but this is slow and costly and requires expertise and equipment not available in a clinical setting. Conversely, mapping a gene variant onto the three-dimensional (3D) protein structure provides a fast molecular assessment free of charge. Before 2021, this type of analysis was severely limited by the availability of experimentally determined 3D protein structures. Advances in artificial intelligence algorithms now allow confident prediction of protein structural features from sequence alone. The aim of the protocols presented here is to enable non-experts to use databases and online tools to investigate the molecular effect of a genetic variant. The Basic Protocol relies only on the online resources AlphaFold, Protein Structure Database, and UniProt. Alternate Protocols document the usage of the Protein Data Bank, SWISS-MODEL, ColabFold, and PyMOL for structure-based variant analysis. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: 3D Mapping based on UniProt and AlphaFold Alternate Protocol 1: Using experimental models from the PDB Alternate Protocol 2: Using information from homology modeling with SWISS-MODEL Alternate Protocol 3: Predicting 3D structures with ColabFold Alternate Protocol 4: Structure visualization and analysis with PyMOL.

Direct observation of the conformational states of PIEZO1.

PIEZOs are mechanosensitive ion channels that convert force into chemoelectric signals1,2 and have essential roles in diverse physiological settings3. In vitro studies have proposed that PIEZO channels transduce mechanical force through the deformation of extensive blades of transmembrane domains emanating from a central ion-conducting pore4-8. However, little is known about how these channels interact with their native environment and which molecular movements underlie activation. Here we directly observe the conformational dynamics of the blades of individual PIEZO1 molecules in a cell using nanoscopic fluorescence imaging. Compared with previous structural models of PIEZO1, we show that the blades are significantly expanded at rest by the bending stress exerted by the plasma membrane. The degree of expansion varies dramatically along the length of the blade, where decreased binding strength between subdomains can explain increased flexibility of the distal blade. Using chemical and mechanical modulators of PIEZO1, we show that blade expansion and channel activation are correlated. Our findings begin to uncover how PIEZO1 is activated in a native environment. More generally, as we reliably detect conformational shifts of single nanometres from populations of channels, we expect that this approach will serve as a framework for the structural analysis of membrane proteins through nanoscopic imaging.

Optical Control of Protein Functions via Genetically Encoded Photocaged Aspartic Acids.

Site-specific protein decaging by light has become an effective approach for in situ manipulation of protein activities in a gain-of-function fashion. Although successful decaging of amino acid side chains of Lys, Tyr, Cys, and Glu has been demonstrated, this strategy has not been extended to aspartic acid (Asp), an essential amino acid residue with a range of protein functions and protein-protein interactions. We herein reported a genetically encoded photocaged Asp and applied it to the photocontrolled manipulation of a panel of proteins including firefly luciferase, kinases (e.g., BRAF), and GTPase (e.g., KRAS) as well as mimicking the in situ phosphorylation event on kinases. As a new member of the increasingly expanded amino acid-decaging toolbox, photocaged Asp may find broad applications for gain-of-function study of diverse proteins as well as biological processes in living cells.

Visualizing single-molecule conformational transition and binding dynamics of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) play crucial roles in cellular processes and hold promise as drug targets. However, the dynamic nature of IDPs remains poorly understood. Here, we construct a single-molecule electrical nanocircuit based on silicon nanowire field-effect transistors (SiNW-FETs) and functionalize it with an individual disordered c-Myc bHLH-LZ domain to enable label-free, in situ, and long-term measurements at the single-molecule level. We use the device to study c-Myc interaction with Max and/or small molecule inhibitors. We observe the self-folding/unfolding process of c-Myc and reveal its interaction mechanism with Max and inhibitors through ultrasensitive real-time monitoring. We capture a relatively stable encounter intermediate ensemble of c-Myc during its transition from the unbound state to the fully folded state. The c-Myc/Max and c-Myc/inhibitor dissociation constants derived are consistent with other ensemble experiments. These proof-of-concept results provide an understanding of the IDP-binding/folding mechanism and represent a promising nanotechnology for IDP conformation/interaction studies and drug discovery.

Correspondence on "Structural Insight into the Catalytic Mechanism of the Endoperoxide Synthase FtmOx1".

In their recent Angewandte Chemie publication (doi: 10.1002/anie.202112063), Cen, Wang, Zhou et al. reported the crystal structure of a ternary complex of the non-heme iron endoperoxidase FtmOx1 (PDB entry 7ETK). The biochemical data assessed in this study were from a retracted study (doi: 10.1038/nature15519) by Zhang, Liu, Zhang et al.; no additional biochemical data were included, yet there was no discussion on the source of the biochemical data in the report by Cen, Wang, Zhou et al. Based on this new crystal structure and subsequent QM/MM-MD calculations, Cen, Wang, Zhou et al. concluded that their work provided evidence supporting the CarC-like mechanistic model for FtmOx1 catalysis. However, the authors did not accurately describe either the CarC-like model or the COX-like model, and they did not address the differences between them. Further, and contrary to their interpretations in the manuscript, the authors' data are consistent with the COX-like model once the details of the CarC-like and COX-like models have been carefully analyzed.

Pathogenic mutation hotspots in protein kinase domain structure.

Control of eukaryotic cellular function is heavily reliant on the phosphorylation of proteins at specific amino acid residues, such as serine, threonine, tyrosine, and histidine. Protein kinases that are responsible for this process comprise one of the largest families of evolutionarily related proteins. Dysregulation of protein kinase signaling pathways is a frequent cause of a large variety of human diseases including cancer, autoimmune, neurodegenerative, and cardiovascular disorders. In this study, we mapped all pathogenic mutations in 497 human protein kinase domains from the ClinVar database to the reference structure of Aurora kinase A (AURKA) and grouped them by the relevance to the disease type. Our study revealed that the majority of mutation hotspots associated with cancer are situated within the catalytic and activation loops of the kinase domain, whereas non-cancer-related hotspots tend to be located outside of these regions. Additionally, we identified a hotspot at residue R371 of the AURKA structure that has the highest number of exclusively non-cancer-related pathogenic mutations (21) and has not been previously discussed.

Enhancing cryo-EM maps with 3D deep generative networks for assisting protein structure modeling.

MOTIVATION: The tertiary structures of an increasing number of biological macromolecules have been determined using cryo-electron microscopy (cryo-EM). However, there are still many cases where the resolution is not high enough to model the molecular structures with standard computational tools. If the resolution obtained is near the empirical borderline (3-4.5 Å), improvement in the map quality facilitates structure modeling. RESULTS: We report EM-GAN, a novel approach that modifies an input cryo-EM map to assist protein structure modeling. The method uses a 3D generative adversarial network (GAN) that has been trained on high- and low-resolution density maps to learn the density patterns, and modifies the input map to enhance its suitability for modeling. The method was tested extensively on a dataset of 65 EM maps in the resolution range of 3-6 Å and showed substantial improvements in structure modeling using popular protein structure modeling tools. AVAILABILITY AND IMPLEMENTATION: https://github.com/kiharalab/EM-GAN, Google Colab: https://tinyurl.com/3ccxpttx.

Ligand-Copper(I) Primary O2-Adducts: Design, Characterization, and Biological Significance of Cupric-Superoxides.

In this Account, we overview and highlight synthetic bioinorganic chemistry focused on initial adducts formed from the reaction of reduced ligand-copper(I) coordination complexes with molecular oxygen, reactions that produce ligand-CuII(O2•-) complexes (O2•- ≡ superoxide anion). We provide mostly a historical perspective, starting in the Karlin research group in the 1980s, emphasizing the ligand design and ligand effects, structure, and spectroscopy of these O2 adducts and subsequent further reactivity with substrates, including the interaction with a second ligand-CuI complex to form binuclear species. The Account emphasizes the approach, evolution, and results obtained in the Karlin group, a synthetic bioinorganic research program inspired by the state of knowledge and insights obtained on enzymes possessing copper ion active sites which process molecular oxygen. These constitute an important biochemistry for all levels/types of organisms, bacteria, fungi, insects, and mammals, including humans.Copper is earth abundant, and its redox properties in complexes allow for facile CuII/CuI interconversions. Simple salts or coordination complexes have been well known to serve as oxidants for the stoichiometric or catalytic oxidation or oxygenation (i.e., O-atom insertion) of organic substrates. Thus, copper dioxygen- or peroxide-centered synthetic bioinorganic studies provide strong relevance and potential application to synthesis or even the development of cathodic catalysts for dioxygen reduction to hydrogen peroxide or water, as in fuel cells. The Karlin group's focus however was primarily oriented toward bioinorganic chemistry with the goal to provide fundamental insights into the nature of copper-dioxygen adducts and further reduced and/or protonated derivatives, species likely occurring in enzyme turnover or related in one or more aspects of formation, structure, spectroscopic properties, and scope of reactivity toward organic/biochemical substrates.Prior to this time, the 1980s, O2 adducts of redox-active first-row transition-metal ions focused on iron, such as the porphyrinate-Fe centers occurring in the oxygen carrier proteins myoglobin and hemoglobin and that determined to occur in cytochrome P-450 monooxygenase turnover. Deoxy (i.e., reduced Fe(II)) heme proteins react with O2, giving FeIII-superoxo complexes (preferably referred to by traditional biochemists as ferrous-oxy species). And, it was in the 1970s that great strides were made by synthetic chemists in generating hemes capable of forming O2 adducts, their physiochemical characterization providing critical insights to enzyme (bio)chemistry and providing ideas and important goals leading to countless person years of future research.

CDMS Analysis of Intact 19S, 20S, 26S, and 30S Proteasomes: Evidence for Higher-Order 20S Assemblies at a Low pH†.

Charge detection mass spectrometry (CDMS) was examined as a means of studying proteasomes. To this end, the following masses of the 20S, 19S, 26S, and 30S proteasomes from Saccharomyces cerevisiae (budding yeast) were measured: m(20S) = 738.8 ± 2.9 kDa, m(19S) = 926.2 ± 4.8 kDa, m(26S) = 1,637.0 ± 7.6 kDa, and m(30S) = 2,534.2 ± 10.8 kDa. Under some conditions, larger (20S)x (where x = 1 to ∼13) assemblies are observed; the 19S regulatory particle also oligomerizes, but to a lesser extent, forming (19S)x complexes (where x = 1 to 4, favoring the x = 3 trimer). The (20S)x oligomers are favored in vitro, as the pH of the solution is lowered (from 7.0 to 5.4, in a 20 mM ammonium acetate solution) and may be related to in vivo proteasome storage granules that are observed under carbon starvation. From measurements of m(20S)x (x = 1 to ∼13) species, it appears that each multimer retains all 28 proteins of the 20S complex subunit. Several types of structures that might explain the formation of (20S)x assemblies are considered. We stress that each structural type [hypothetical planar, raft-like geometries (where individual proteasomes associate through side-by-side interactions); elongated, rodlike geometries (where subunits are bound end-to-end); and geometries that are roughly spherical (arising from aggregation through nonspecific subunit interactions)] is highly speculative but still interesting to consider, and a short discussion is provided. The utility of CDMS for characterizing proteasomes and related oligomers is discussed.