Isoleucine Residues Research Articles

Structural modeling of phosphatidylinositol 3-kinase-γ with novel derivatives of stilbenoids

Phosphatidylinositol 3-kinases (PI3K) form a family of lipid kinases that catalyze the phosphorylation of 3-hydroxyl group of the inositol ring of phosphatidylinositol and its derivatives. It is implicated in inflammatory disorders and cancer thus making it an attractive drug target. Crystal structure of human PI3Kγ was taken and structure was completed using MODELLER and validated using PROCHECK. Stilbenoid molecules, piceatannol and resveratrol, were docked to kinase domain of PI3Kγ using AutoDock Vina and docked complexes were subjected to molecular dynamic simulations using Desmond suite of programmes. Based on the structural analysis of these complexes, modified derivatives of the native molecules were designed, docked and molecular dynamic simulations were performed. Kinase domain has a bi-lobar structure with ATP binding site lying in the cleft connecting the two lobes that are primarily composed of 12 α-helices and 8 β-strands. Piceatannol and resveratrol bind at the ATP binding site, with one its rings in a position primarily occupied by adenine of ATP making a hydrogen bond with backbone of Val882. Molecules also make interactions with Lys833 and several isoleucine residues. Interactions with Ser806 appear to be crucial for the loop conformation and compactness. Derivative molecules of stilbenoids also occupy the ATP binding cleft and the chemical modifications result in hydrogen bonded interactions to Glu880, and ionic interactions to Lys833 and Lys808 thereby enhancing their potencies in comparison to native molecules. Biophysical parameters and quality of interactions of stilbenoid derivatives augurs well for development of potent and specific inhibitory molecules against PI3Kγ enzyme.

Isoleucine gate blocks K+ conduction in C-type inactivation.

Many voltage-gated potassium (Kv) channels display a time-dependent phenomenon called C-type inactivation, whereby prolonged activation by voltage leads to the inhibition of ionic conduction, a process that involves a conformational change at the selectivity filter toward a non-conductive state. Recently, a high-resolution structure of a strongly inactivated triple-mutant channel kv1.2-kv2.1-3m revealed a novel conformation of the selectivity filter that is dilated at its outer end, distinct from the well-characterized conductive state. While the experimental structure was interpreted as the elusive non-conductive state, our molecular dynamics simulations and electrophysiological measurements show that the dilated filter of kv1.2-kv2.1-3m is conductive and, as such, cannot completely account for the inactivation of the channel observed in the structural experiments. The simulation shows that an additional conformational change, implicating isoleucine residues at position 398 along the pore lining segment S6, is required to effectively block ion conduction. The I398 residues from the four subunits act as a state-dependent hydrophobic gate located immediately beneath the selectivity filter. By mutating I398 to Asparagine, ion permeation can be resumed in the kv1.2-kv2.1-3m channel, which was not a reversion C-type inactivation, since AgTxII fails to block the ionic permeation of kv1.2-kv2.1-3m_I398N. As a critical piece of the C-type inactivation machinery, this structural feature is the potential target of a broad class of QA blockers and negatively charged activators thus opening new research directions towards the development of drugs that specifically modulate gating-states of Kv channels.

Mutation of a highly conserved isoleucine residue in loop 2 of several β-coronavirus macrodomains indicates that enhanced ADP-ribose binding is detrimental for replication.

The conserved coronavirus (CoV) macrodomain (Mac1) counters the activity of host ADP-ribosyltransferases and is critical for CoV replication and pathogenesis. As such, Mac1 is a potential therapeutic target for CoV-induced disease. However, we lack a basic knowledge of how several residues in its ADP-ribose binding pocket contribute to its biochemical and virological functions. We engineered mutations into two highly conserved residues in the ADP-ribose binding pocket of Mac1, both as recombinant proteins and viruses for Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Interestingly, a Mac1 isoleucine-to-alanine mutant protein had enhanced ADP-ribose binding which proved to be detrimental for virus replication, indicating that this isoleucine controls ADP-ribose binding and is beneficial for virus replication and pathogenesis. These results provide unique insight into how macrodomains control ADP-ribose binding and will be critical for the development of novel inhibitors targeting Mac1 that could be used to treat CoV-induced disease.

Biallelic TYR and TKFC variants in Egyptian patients with OCA1 and new expanded TKFC features

BackgroundOculocutaneous albinism type1 (OCA1) is caused by the TYR gene's homozygous and compound heterozygous variants. TKFC gene variants cause triokinase & FMN cyclase deficiency syndrome with variable multisystemic disorders.ObjectivesTo determine the potential disease-causing variants in two deceased patients presenting atypical OCA1 features by demonstrating three generations for a single family. The two deceased neonates had severe skeletal abnormalities and fatal hypertrophic cardiomyopathy. We also explored the potential mechanisms for the causative relationship between TKFC and multisystem disorders.Patients and methodsDue to the new emerging symptoms that weren’t reported before with the TYR gene, the following methods were performed: Sanger sequencing for the TYR gene, followed by whole exome sequencing, co-segregation, and computational analyses.ResultsExtensive parental consanguinity was found, and consequently an autosomal recessive mode of inheritance was prioritized. Upon performing sequencing and segregation data, the following has been confirmed: positive co-segregation of nonsense homozygous NM_000372.5:c.346C > T p.(Arg116*) variant in TYR gene and multisystem disease-missense homozygous NM_015533.4:c.598G > A p.(Val200Ile) variant in TKFC gene in the two affected index patients who deceased due to hypertrophic cardiomyopathy. Using computational analysis, we found that c.598G > A p.(Val200Ile) pathogenicity has led to the failure of L2-K1 active site closure due to the potential differential fluctuation between valine and isoleucine residues. Subsequently, disruption of endogenous DHA phosphorylation was found. Two potential mechanisms exploring the causative relationship between TKFC gene and multisystem disorders have been suggested.ConclusionsThis study presented a first family with the co-existence of biallelic variants in TYR and TKFC genes associating severe skeletal abnormalities and lethal hypertrophic cardiomyopathy. Neither of these genes would have been pursued in the standard genetic counseling. Such discovery is paving the way for more efficient genetic counseling. Comparing TKFC results with literature data showed that our relevant expanded TKFC variant is the 3rd worldwide.

In Silico Analysis of Rpp1 Locus-Derived NB-ARC-LRR Domains Reveals Insights into Rust Resistance in Soybean

Soybean rust poses significant economic challenges to soybean producers, resulting in decreased seed size, weight, oil content, and overall crop yield. This study aimed to comprehensively characterize the rust resistance locus Rpp1 identified in soybean genotype EC 241780 and its associated genes within the soybean genome. Our investigation revealed distinctive features of three genes (Glyma18G281700, Glyma18G281600, and Glyma18G281500) located within the Rpp1 locus on chromosome 18. Phylogenetic and domain analyses confirmed the presence of the ULP1 domain in these genes, while motif analysis identified unique patterns exclusive to them. Notably, these genes possessed exclusive isoleucine and glutamic acid residues at positions 836 and 904, along with an unusual expansion of 11 glutamic acid residues at position 861. Expression-data showed significantly higher expression levels of Glyma18G281700 and Glyma18G281600 during leaf trifoliate stages I and II, suggesting their active involvement under adverse conditions. Protein structure analysis revealed that Glyma18G281500 exhibited distinct characteristics compared to the other two genes and displayed a more stable protein structure. This observation may indicate a distinct functional role for Glyma18G281500. In conclusion, our findings suggest that the genes within the Rpp1 locus may contribute to rust resistance, particularly against specific rust races, due to their unique characteristics associated with the ULP1 domain.

Anomalous Temperature Dependence of the Triplet-Triplet Energy Transfer in Cereibacter sphaeroides I(L177)H Mutant Reaction Centers.

In photosynthetic reaction centers, quenching of the primary donor triplet state by energy transfer to the carotenoid molecule provides efficient suppression of generation of singlet-excited oxygen, potent chemical oxidant. This process in the Cereibacter sphaeroides reaction centers is thermoactivated, and discontinues at temperatures below 40K. In these reaction centers, substitution of amino acid residue isoleucine at the 177position of the L-subunit with histidine results in the sharp decrease of activation energy, so that the carotenoid triplets are populated even at 10K. Activation energy of the T-T energy transfer was estimated as 7.5cm-1, which is more than 10-fold lower than activation energy in the wild type reaction centers. At certain temperatures, the energy transfer in the mutant is decelerated, which is related to the increase of effective distance of the triplet-triplet transfer. To the best of our knowledge, the described mutation presents the first reaction center modification leading to the significant decrease in activation energy of the T-T energy transfer to carotenoid molecule. TheI(L177)H mutant reaction centers present a considerable interest for further studies of the triplet state quenching mechanisms, and of other photophysical and photochemical processes in the reaction centers of bacterial photosynthesis.

Cyclic-di-GMP induces inflammation and acute lung injury through direct binding to MD2.

Severe bacterial infections can trigger acute lung injury (ALI) and acute respiratory distress syndrome, with bacterial pathogen-associated molecular patterns (PAMPs) exacerbating the inflammatory response, particularly in COVID-19 patients. Cyclic-di-GMP (CDG), one of the PAMPs, is synthesized by various Gram-positve and Gram-negative bacteria. Previous studies mainly focused on the inflammatory responses triggered by intracellular bacteria-released CDG. However, how extracellular CDG, which is released by bacterial autolysis or rupture, activates the inflammatory response remains unclear. The interaction between extracellular CDG and myeloid differentiation protein 2 (MD2) was investigated using in vivo and in vitro models. MD2 blockade was achieved using specific inhibitor and genetic knockout mice. Site-directed mutagenesis, co-immunoprecipitation, SPR and Bis-ANS displacement assays wereused to identify the potential binding sites of MD2 on CDG. Our data show that extracellular CDG directly interacts with MD2,leading to activation of the TLR4signalling pathway and lung injury. Specific inhibitors or genetic knockout of MD2 in mice significantly alleviated CDG-induced lung injury. Moreover, isoleucine residues at positions 80 and 94, along with phenylalanine at position 121, are essential for the binding of MD2 to CDG. These results reveal that extracellular CDG induces lung injury through direct interaction with MD2 and activation of the TLR4 signalling pathway, providing valuable insights into bacteria-induced ALI mechanisms and new therapeutic approaches for the treatment of bacterial co-infection in COVID-19 patients.

Importance of isoleucine residue in ion channel formation ability of 11-residue peptaibols

Peptaibols are a class of short peptides, typically 7 to 20 amino acids long, characterized by noncanonical amino acid residues such as aminoisobutyric acid (Aib). Although the helix length is shorter than the membrane thickness, the 11-residue peptaibol trichorovin-XII (TV-XII) can form ion channels in membranes. Assuming that a higher proportion of isoleucine (Ile) relative to leucine (Leu) residues is crucial for maintaining the ion channel activity of TV-XII, peptide analogs of TV-XII with varying Ile content were designed, synthesized, and evaluated. The secondary structure of all derivatives under hydrophobic conditions was confirmed by CD measurement as an α-helix-like β-bend ribbon spiral structure. The most stable ion channel activity was found in compound 4a with maximum Ile. Furthermore, the C-terminal Ile analog showed greater ion channel activity compared to the Leu analog. This suggests that the choice between Leu and Ile can influence the expression of ion channel activity, which will be crucial for the de novo designed functional peptides.

Mutation of a highly conserved isoleucine residue in loop 2 of several β-coronavirus macrodomains indicates that enhanced ADP-ribose binding is detrimental to infection.

All coronaviruses (CoVs) encode for a conserved macrodomain (Mac1) located in nonstructural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that binds and hydrolyzes mono-ADP-ribose from target proteins. Previous work has shown that Mac1 is important for virus replication and pathogenesis. Within Mac1, there are several regions that are highly conserved across CoVs, including the GIF (glycine-isoleucine-phenylalanine) motif. To determine how the biochemical activities of these residues impact CoV replication, the isoleucine and the phenylalanine residues were mutated to alanine (I-A/F-A) in both recombinant Mac1 proteins and recombinant CoVs, including murine hepatitis virus (MHV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The F-A mutant proteins had ADP-ribose binding and/or hydrolysis defects that led to attenuated replication and pathogenesis in cell culture and mice. In contrast, the I-A mutations had normal enzyme activity and enhanced ADP-ribose binding. Despite increased ADP-ribose binding, I-A mutant MERS-CoV and SARS-CoV-2 were highly attenuated in both cell culture and mice, indicating that this isoleucine residue acts as a gate that controls ADP-ribose binding for efficient virus replication. These results highlight the function of this highly conserved residue and provide unique insight into how macrodomains control ADP-ribose binding and hydrolysis to promote viral replication.

Backbone and methyl side-chain resonance assignments of the Fab fragment of adalimumab

Adalimumab is a therapeutic monoclonal antibody developed to target human TNF an important mediator of immune-mediated inflammatory diseases such as rheumatoid arthritis, amongst others. The 48 kDa Fab fragment of adalimumab was produced in Escherichia coli using a single chain approach to allow complete isotopic incorporation of deuterium, carbon-13 and nitrogen-15 along with the protonated isoleucine-d, valine and leucine methyl groups. Here we report the near complete resonance assignment of the polypeptide backbone and the methyl groups of isoleucine, leucine and valine residues.

Backbone and methyl side-chain resonance assignments of the single chain Fab fragment of trastuzumab

Trastuzumab is a therapeutic monoclonal antibody developed to target human epidermal growth factor receptor 2 (HER2) present at higher levels in early cancers. Here we report the near complete resonance assignment of trastuzumab-scFab fragment backbone and the methyl groups of isoleucine, leucine and valine residues, as well as their stereo-assignments. The antibody fragment was produced using a single chain approach in Escherichia coli.

Complete characterization of NADH dehydrogenase subunit 1 gene in human hydatid cysts

يمتلك طفيلي الاكياس العدرية سلالاتين متشابهتين (G1 و G3) ونظرا للدور البارز الذي تلعبه عائلة جينات نازعات الهيدروجين في التميز بين هاتين السلالاتين، لذا هدفت الدراسة الحالية الى اجراء التوصيف الجزيئي الكامل لاحد ابرز انواع هذه العائلة وهو الجين NAD1 (من شفرة البدء الى الي شفرة التوقف) للاكياس العدرية المعزولة جراحيا من اكباد المرضى (ولمحافظتي البصرة وميسان) في مستشفى الصدر التعليمي في محافظة البصرة، العراق. عزلت الرؤيسات الاولية من الاكياس تحت ظروف معقمة واستخدمت في استخلاص الحامض النووي منقوص الاوكسجين. اذ ضخم التتابع الكامل للجين في عينات الدراسة وانجز التتابع (السكونس) لهما باستخدام بادئات متخصصه. اظهرت نتائج تحليل السكونس المتكرر Consensus sequence امتلاك كل من عينات البصرة و ميسان 1038 زوجا من القواعد وتقع شفرة البدء GTG في الموقع 87-89 وشفرة التوقف TAA في الموقع 978-980. كما اظهرت استدلالات الاحماض الامينية الى وجود 297 حامض اميني لبروتينات عينات البصرة وميسان. فضلا عن ذلك بينت تحاليل المعلومات الحياتية احتواء هذه البروتينات على الحامض الاميني Isoleucine في الموقع 188 (I188) وهو موجود فقط في سلالة الاغنام G1. وان النموذج التركيبي لبروتينات عينات البصرة وميسان اظهر تشابها كبيرا و يقع I188 في الحلزون 10 (α10). بينت الدراسة ولاول مرة على مستوى العراق التوصيف الكامل للجين NAD1 والعائد الى سلالة الاغنام G1 المسببه لمرض الاكياس العدرية البشري.

Unraveling motion in proteins by combining NMR relaxometry and molecular dynamics simulations: A case study on ubiquitin.

Nuclear magnetic resonance (NMR) relaxation experiments shine light onto the dynamics of molecular systems in the picosecond to millisecond timescales. As these methods cannot provide an atomically resolved view of the motion of atoms, functional groups, or domains giving rise to such signals, relaxation techniques have been combined with molecular dynamics (MD) simulations to obtain mechanistic descriptions and gain insights into the functional role of side chain or domain motion. In this work, we present a comparison of five computational methods that permit the joint analysis of MD simulations and NMR relaxation experiments. We discuss their relative strengths and areas of applicability and demonstrate how they may be utilized to interpret the dynamics in MD simulations with the small protein ubiquitin as a test system. We focus on the aliphatic side chains given the rigidity of the backbone of this protein. We find encouraging agreement between experiment, Markov state models built in the χ1/χ2 rotamer space of isoleucine residues, explicit rotamer jump models, and a decomposition of the motion using ROMANCE. These methods allow us to ascribe the dynamics to specific rotamer jumps. Simulations with eight different combinations of force field and water model highlight how the different metrics may be employed to pinpoint force field deficiencies. Furthermore, the presented comparison offers a perspective on the utility of NMR relaxation to serve as validation data for the prediction of kinetics by state-of-the-art biomolecular force fields.

The Greater Celandine: Identification and Characterization of an Antimicrobial Peptide from Chelidonium majus.

Chelidonium majus, known as Greater Celandine, is a latex-bearing plant that has been leveraged for its anticancer and antimicrobial properties. Herein, C. majus aerial tissue is mined for the presence of antimicrobial peptides. A highly abundant cysteine-rich peptide with a length of 25 amino acids, deemed CM-AMP1, is characterized through multiple mass spectrometric approaches. Electron-activated dissociation is leveraged to differentiate between isoleucine and leucine residues and complement conventional collision-induced dissociation to gain full sequence coverage of the full-length peptide. CM-AMP1 shares little sequence similarity with any proteins in publicly available databases, highlighting the novelty of its cysteine landscape and core motif. The presence of three disulfide bonds in the native peptide confers proteolytic stability, and antimicrobial activity is greatly decreased upon the alkylation of the cysteine residues. Synthetic variants of CM-AMP1 are used to confirm the activity of the full-length sequence and the core motif. To assess the biological impact, E. coli was grown in a sublethal concentration of CM-AMP1 and quantitative proteomics was used to identify proteins produced by the bacteria under stress, ultimately suggesting a membrane lytic antimicrobial mechanism of action. This study integrates multiple analytical methods for molecular and biological characterization of a unique antimicrobial peptide identified from C. majus.

Protective effect of tretinoin on cervical cancer growth and proliferation through downregulation of pFAK2 expression.

Cervical cancer, a life-threatening disease, is the seventh most commonly detected cancer among women throughout the world. The present study investigated the effect of tretinoin on cervical cancer growth and metastasis in vitro and in vivo in the mice model. Cell Counting Kit-8, clonogenic survival, and transwell chamber assays were used for determination cells proliferation, colony formation, and invasiveness. Western blotting assay was used for assessment of protein expression whereas AutoDock Vina and Discovery studio software for in silico studies. Tretinoin treatment significantly (p < .05) reduced the proliferation of HT-3 and Caski cells in concentration-based manner. Incubation with tretinoin caused a significant decrease in clonogenic survival of HT-3 and Caski cells compared with the control cultures. The invasive potential of HT-3 cells was decreased to 18%, whereas that of Caski cells to 21% on treatment with 8 μM concentration of tretinoin. In HT-3 cells, tretinoin treatment led to a prominent reduction in p-focal adhesion kinase (FAK), matrix metalloproteinases (MMP)-2, and MMP-9 expression in HT-3 cells. Treatment of the cervical cancer mice model with tretinoin led to a prominent decrease in tumor growth. The metastasis of tumor in model cervical cancer mice group was effectively inhibited in spleen, intestines, and peritoneal cavity. In silico studies showed that tretinoin interacts with alanine, proline, isoleucine, and glycine amino acid residues of FAK protein to block its activation. The 2-dimensional diagram of interaction of tretinoin with FAK protein revealed that tretinoin binds to alanine and glycine amino acids through conventional hydrogen bonding. In summary, tretinoin suppressed the proliferation, colony formation, and invasiveness of cervical cancer cells in vitro. It decreased the expression of activated focal adhesion kinase, MMP-2, and MMP-9 in HT-3 cells in dose-dependent manner. In silico studies showed that tretinoin interacts with alanine and glycine amino acids through conventional hydrogen bonding. In vivo data demonstrated that treatment of the cervical cancer mice model with tretinoin led to a prominent decrease in tumor growth. Therefore, tretinoin can be developed as an effective therapeutic agent for cervical cancer treatment.

Molecular Dynamics and Docking Simulations of Homologous RsmE Methyltransferases Hints at a General Mechanism for Substrate Release upon Uridine Methylation on 16S rRNA

In this study, molecular dynamics (MD) and docking simulations were carried out on the crystal structure of Neisseria Gonorrhoeae RsmE aiming at free energy of binding estimation (ΔGbinding) of the methyl transfer substrate S-adenosylmethionine (SAM), as well as its homocysteine precursor S-adenosylhomocysteine (SAH). The mechanistic insight gained was generalized in view of existing homology to two other crystal structures of RsmE from Escherichia coli and Aquifex aeolicus. As a proof of concept, the crystal poses of SAM and SAH were reproduced reflecting a more general pattern of molecular interaction for bacterial RsmEs. Our results suggest that a distinct set of conserved residues on loop segments between β12, α6, and Met169 are interacting with SAM and SAH across these bacterial methyltransferases. Comparing molecular movements over time (MD trajectories) between Neisseria gonorrhoeae RsmE alone or in the presence of SAH revealed a hitherto unknown gatekeeper mechanism by two isoleucine residues, Ile171 and Ile219. The proposed gating allows switching from an open to a closed state, mimicking a double latch lock. Additionally, two key residues, Arg221 and Thr222, were identified to assist the exit mechanism of SAH, which could not be observed in the crystal structures. To the best of our knowledge, this study describes for the first time a general catalytic mechanism of bacterial RsmE on theoretical ground.

Leader peptide removal in lasso peptide biosynthesis based on penultimate isoleucine residue.

Lasso peptides are ribosomally synthesized peptides that undergo post-translational modifications including leader peptide removal by B (or the segregated B1 and B2) proteins and core peptide macrolactamization by C proteins to form a unique lariat topology. A conserved threonine residue at the penultimate position of leader peptide is hitherto found in lasso peptide precursors and shown to be a critical recognition element for effective enzymatic processing. We identified a lasso peptide biosynthetic gene cluster (bsf) from Bradymonas sediminis FA350, a Gram-negative and facultatively prey-dependent bacterium that belongs to a novel bacterial order Bradymonadales in the class Deltaproteobacteria. The kinase BsfK specifically catalyzes the phosphorylation of the precursor peptide BsfA on the Ser3 residue. BsfB1 performs dual functions to accelerate the post-translational phosphorylation and assist BsfB2 in leader peptide removal. Most importantly, the penultimate residue of leader peptide is an isoleucine rather than the conserved threonine and this isoleucine has a marked impact on the phosphorylation of Ser3 as well as leader peptide removal, implying that BsfB1 and BsfB2 exhibit a new substrate selectivity for leader peptide binding and excision. This is the first experimentally validated penultimate isoleucine residue in a lasso peptide precursor to our knowledge. In silico analysis reveals that the leader peptide Ile/Val(-2) residue is rare but not uncommon in phosphorylated lasso peptides, as this residue is also discovered in Acidobacteriaceae and Sphingomonadales in addition to Bradymonadales.

A mutagenesis study of autoantigen optimization for potential T1D vaccine design

A previously reported autoreactive antigen, termed the X-idiotype, isolated from a unique cell population in Type 1 diabetes (T1D) patients, was found to stimulate their CD4+ T cells. This antigen was previously determined to bind more favorably than insulin and its mimic (insulin superagonist) to HLA-DQ8, supporting its strong role in CD4+ T cell activation. In this work, we probed HLA-X-idiotype-TCR binding and designed enhanced-reactive pHLA-TCR antigens using an in silico mutagenesis approach which we functionally validated by cell proliferation assays and flow cytometry. From a combination of single, double, and swap mutations, we identified antigen-binding sites p4 and p6 as potential mutation sites for HLA binding affinity enhancement. Site p6 is revealed to favor smaller but more hydrophobic residues than the native tyrosine, such as valine (Y6V) and isoleucine (Y6I), indicating a steric mechanism in binding affinity improvement. Meanwhile, site p4 methionine mutation to hydrophobic residues isoleucine (M4I) or leucine (M4L) modestly increases HLA binding affinity. Select p6 mutations to cysteine (Y6C) or isoleucine (Y6I) exhibit favorable TCR binding affinities, while a swap p5-p6 tyrosine-valine double mutant (V5Y_Y6V) and a p6-p7 glutamine-glutamine double mutant (Y6Q_Y7Q) exhibit enhanced HLA binding affinity but weakened TCR affinity. This work holds relevance to potential T1D antigen-based vaccine design and optimization.

Molecular Characterization of Dehydrin in Azraq Saltbush among Related Atriplex Species

Atriplex spp. (saltbush) is known to survive extremely harsh environmental stresses such as salinity and drought. It mitigates such conditions based on specialized physiological and biochemical characteristics. Dehydrin genes (DHNs) are considered major players in this adaptation. In this study, a novel DHN gene from Azrak (Jordan) saltbush was characterized along with other Atriplex species from diverse habitats. Intronless DHN-expressed sequence tags (495-761 bp) were successfully cloned and sequenced. Saltbush dehydrins contain one S-segment followed by three K-segments: an arrangement called SK3-type. Two substantial insertions were detected including three copies of the K2-segemnet in A. canescens. New motif variants other than the six-serine standard were evident in the S-segment. AhaDHN1 (A. halimus) has a cysteine residue (SSCSSS), while AgaDHN1 (A. gardneri var. utahensis) has an isoleucine residue (SISSSS). In contrast to the conserved K1-segment, both the K2- and K3-segment showed several substitutions, particularly in AnuDHN1 (A. nummularia). In addition, a parsimony phylogenetic tree based on homologs from related genera was constructed. The phylogenetic tree resolved DHNs for all of the investigated Atriplex species in a superclade with an 85% bootstrap value. Nonetheless, the DHN isolated from Azraq saltbush was uniquely subclustred with a related genera Halimione portulacoides. The characterized DHNs revealed tremendous diversification among the Atriplex species, which opens a new venue for their functional analysis.

Voltage sensitive ion channels, minimal size of ordered phases and the evolution of metazoa.

The complex electrical, mechanical and optical behavior of a voltage sensitive ion channel (VSIC) requires that the macromolecule possess at least one ordered phase. For an object to support order, it must be of a minimum size to overcome thermal fluctuations. The limiting size of a stable phase, the correlation length, depends on temperature. Converging lines of experimental evidence indicate that the VSIC is a ferroelectric liquid crystal of the blue phase type. Reaching beyond the analogy of a gated pore, the Channel Activation by Electrostatic Repulsion (CAbER) hypothesis (Leuchtag 2022 Biophys J 121(3):92a) postulates that voltage sensitivity arises from polarization due to the induction of dipoles in the branched sidechains of the numerous isoleucine, leucine and valine residues, producing a polar order in the excitable (closed) phase and making it an insulator by compressing and crowding the ion-conducting segments. On threshold depolarization the dipoles collapse into thermal chaos, reducing the macromolecule's dielectric permittivity and thereby increasing the long-range Coulomb repulsions between the conserved positive charges of the four S4 segments, pushing them apart. The consequent relaxation of the S5 and S6 segments allows their core hydrogen bonds to widen and allow permeant ions to enter and percolate stochastically through the membrane. The critical role of VSIC size in the CAbER model has implications for the beginning of multicellularity during the emergence of the Metazoa in the genomic revolution. At about 300 K, the size effect underlies VSIC activation as an order–disorder transition. This property of the size effect implies that the evolution of Metazoa became possible only after the organism's DNA had grown extensive enough to code for ion channels with dimensions larger than the correlation length to sustain an ordered polar, bioferroelectric, phase.