Glutamic Acid Residues Research Articles

A subgroup of light-driven sodium pumps with an additional Schiff base counterion

Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterize a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics.

Targeted mutagenesis of negatively charged amino acids outlining the substrate translocation path within the human organic cation transporter 3

Recently published cryo-EM structures of human organic cation transporters of the SLC22 family revealed seven, sequentially arranged glutamic and aspartic acid residues, which may be relevant for interactions with positively charged substrates. We analyzed the functional consequences of removing those negative charges by creating D155N, E232Q, D382N, E390Q, E451Q, E459Q, and D478N mutants of OCT3.E232Q, E459Q, and D478N resulted in a lack of localization in the outer cell membrane and no relevant uptake activity. However, D155N and E451Q showed a substrate-specific loss of transport activity, whereas E390Q had no remaining activity despite correct membrane localization. In contrast, D382N showed almost wild-type-like uptake. D155 is located at the entrance to the substrate binding pocket and could, therefore be involved in guiding cationic substrates towards the inside of the binding pocket. For E390, we confirm its critical function for transporter function as it was recently shown for the corresponding position in OCT1. Interestingly, E451 seems to be located at the bottom of the binding pocket in the outward-open confirmation of the transporter. Substrate-specific loss of transport activity of the E451Q variant suggests an essential role in the transport cycle of specific substances as part of an opportunistic binding site.In general, our study highlights the impact of the cryo-EM structures in guiding mutagenesis studies to understand the molecular level of transporter-ligand interactions, and it also confirms the importance of testing multiple substrates in mutagenesis studies of polyspecific OCTs.

Tryptophan-Centric Bioinformatics Identifies New Lasso Peptide Modifications.

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by a macrolactam linkage between the N-terminus and the side chain of an internal aspartic acid or glutamic acid residue. Instead of adopting a branched-cyclic conformation, lasso peptides are "threaded", with the C-terminal tail passing through the macrocycle to present a kinetically trapped rotaxane conformation. The availability of enhanced bioinformatics methods has led to a significant increase in the number of secondary modifications found on lasso peptides. To uncover new ancillary modifications in a targeted manner, a bioinformatic strategy was developed to discover lasso peptides with modifications to tryptophan. This effort identified numerous putative lasso peptide biosynthetic gene clusters with core regions of the precursor peptides enriched in tryptophan. Parsing of these tryptophan (Trp)-rich biosynthetic gene clusters uncovered several putative ancillary modifying enzymes, including halogenases and dimethylallyltransferases expected to act upon Trp. Characterization of two gene products yielded a lasso peptide with two 5-Cl-Trp modifications (chlorolassin) and another bearing 5-dimethylallyl-Trp and 2,3-didehydro-Tyr modifications (wygwalassin). Bioinformatic analysis of the requisite halogenase and dimethylallyltransferase revealed numerous other putative Trp-modified lasso peptides that remain uncharacterized. We anticipate that the Trp-centric strategy reported herein may be useful in discovering ancillary modifications for other RiPP classes and, more generally, guide the functional prediction of enzymes that act on specific amino acids.

Improving the stability of an unstable lipase by applying different immobilization strategies for the selective hydrolysis of fish oil

AbstractRhizopus oryzae lipase (ROL) is known to present high selectivity in chemical reactions. However, the poor stability of ROL effectively limits its industrial applications. In this study, several immobilization protocols, such as hydrophobic adsorption, covalent immobilization, multi‐point covalent attachment, ionic adsorption/cross‐linking, and ionic interaction, were applied to improve the stability of ROL. Heterogeneous modification of aspartic and glutamic acid residues on the surface of ROL was carried out by 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC) to introduce new amine groups with lower pKb. The highest immobilization yield of 89% was achieved for octyl‐agarose, producing specific activity of 45 U/mg, which is 15 folds higher than the specific activity of the soluble enzyme. Improved stability of ROL was observed, in particular for those derivatives obtained by multi‐point covalent attachment of ROL on glyoxyl‐agarose (Gx‐ROL) and aminated ROL on glyoxyl‐agarose (Gx‐NH2‐ROL) by retaining 28%–36% of their initial activities after 24 h incubation at 60°C. Immobilization also altered the co‐solvent stability profile of the immobilized derivatives producing biocatalysts with varied co‐solvent stabilities. Furthermore, utilization of the immobilized preparations in fish oil hydrolysis revealed the selective release of cis‐5,8,11,14,17‐eicosapentaenoic acid (EPA) and cis‐4,7,10,13,16,19‐docosahexaenoic acid (DHA) in favor of EPA. The highest EPA/DHA selectivity of 33 was observed for the hydrophobically immobilized ROL on octyl‐sepharose.

Poly(Glutamic Acid-Lysine) Hydrogels with Alternating Sequence Resist the Foreign Body Response in Rodents and Non-Human Primates.

The foreign body response (FBR) to implanted biomaterials and biomedical devices can severely impede their functionality and even lead to failure. The discovery of effective anti-FBR materials remains a formidable challenge. Inspire by the enrichment of glutamic acid (E) and lysine (K) residues on human protein surfaces, a class of zwitterionic polypeptide (ZIP) hydrogels with alternating E and K sequences to mitigate the FBR is prepared. When subcutaneously implanted, the ZIP hydrogels caused minimal inflammation after 2 weeks and no obvious collagen capsulation after 6 months in mice. Importantly, these hydrogels effectively resisted the FBR in non-human primate models for at least 2 months. In addition, the enzymatic degradability of the gel can be controlled by adjusting the crosslinking degree or the optical isomerism of amino acid monomers. The long-term FBR resistance and controlled degradability of ZIP hydrogels open up new possibilities for a broad range of biomedical applications.

Cloning human Benchwarmer gene (BNCH) harboring E164K in vector pcDNA3.1 by site-directed mutagenesis method

Benchwarmer (BNCH) gene encodes an orphan transmembrane transporter belonging to the Major Facilitator Superfamily (MFS), facilitating the transport of ions, amino acids, simple sugars and recently lysolipids. The loss of BNCH function caused lethality in several animal models with neurodegeneration and senescence. At the cellular level, dysregulation of BNCH leads to adverse phenotypes of lysosome and also autophagy (i.e. dyshomeostasis, accumulation of carbohydrates and sphingolipids, and enlarged lysosome). However, the molecular function and ligand of BNCH protein remain to be unrevealed. This study aims to create a radical substitution change in human BNCH coding gene to knock out the protein functions. More specifically, lysine (K) was used to replace the glutamic acid residue 164 (E164K) which is conserved in many animals (fly, zebrafish, mouse and human) and this E164K mutation recapitulated BNCH mutant phenotype. In conclusion, BNCH harboring E164K (BNCH*) was successfully produced by site-directed mutagenesis and cloned into pcDNA.3.1 vector. The construct was transformed into E. coli OmniMAX and that provides a valuable cell assay to search for the molecular ligand of BNCH.

Investigation of how gate residues in the main channel affect the catalytic activity of Scytalidium thermophilum catalase.

Catalase is an antioxidant enzyme that breaks down hydrogen peroxide (H2O2) into molecular oxygen and water. In all monofunctional catalases the pathway that H2O2 takes to the catalytic centre is via the `main channel'. However, the structure of this channel differs in large-subunit and small-subunit catalases. In large-subunit catalases the channel is 15 Å longer and consists of two distinct parts, including a hydrophobic lower region near the heme and a hydrophilic upper region where multiple H2O2 routes are possible. Conserved glutamic acid and threonine residues are located near the intersection of these two regions. Mutations of these two residues in the Scytalidium thermophilum catalase had no significant effect on catalase activity. However, the secondary phenol oxidase activity was markedly altered, with kcat and kcat/Km values that were significantly increased in the five variants E484A, E484I, T188D, T188I and T188F. These variants also showed a lower affinity for inhibitors of oxidase activity than the wild-type enzyme and a higher affinity for phenolic substrates. Oxidation of heme b to heme d did not occur in most of the studied variants. Structural changes in solvent-chain integrity and channel architecture were also observed. In summary, modification of the main-channel gate glutamic acid and threonine residues has a greater influence on the secondary activity of the catalase enzyme, and the oxidation of heme b to heme d is predominantly inhibited by their conversion to aliphatic and aromatic residues.

Forced intercalation-induced light-up peptides as fluorogenic indicators for the HIV-1 TAR RNA-ligand assay.

Fluorescence indicators capable of binding to human immunodeficiency virus-1 (HIV-1) trans-activation responsive (TAR) RNA are powerful tools for the exploratory studies of the identification of anti-HIV drug candidates. This work presents a new design strategy for fluorogenic indicators with a transactivator of transcription (Tat)-derived peptide based on the forced intercalation of thiazole orange (TO) dyes (FIT). The developed 9-mer FIT peptide (RKKRR-TO-RRR: named FiLuP) features the TO unit integrated onto a Dap (2,3-diaminopropionic acid) residue in the middle of the Tat peptide sequence; the Q (glutamic acid) residue in the Tat peptide (RKKRR-Q-RRR) is replaced with TO as if it were an amino acid surrogate. This facilitates a significant light-up response (450-fold at λem = 541 nm, Φfree = 0.0057, and Φbound = 0.61) upon binding to TAR RNA. The response of FiLuP is highly selective to TAR RNA over other non-cognate RNAs, and FiLuP maintains strong binding affinity (Kd = 1.0 ± 0.6 nM). Significantly, in contrast to previously developed Tat peptide-based FRET probes, FiLuP is able to discriminate between "competitive" and "noncompetitive" inhibitors when used in the fluorescence indicator displacement (FID) assay. The FID assay under stringent screening conditions is also possible, enabling super-strong competitive binders toward TAR RNA to be sieved out.

Structural features of the active centre and quaternary structure of inulinase

Now many researchers started to pay attention to the fact that not only traditional sorbents and ion exchangers, but also a number of natural components contained in plant and animal organisms have the ability to absorb other organic molecules and ions, enter into complex formation reactions with metal ions, and exhibit catalytic properties. Such natural substances with sorption and ion-exchange properties include aminopolysaccharides (chitin, chitosan), nucleic and ribonucleic acids, saponins, and enzymes. The provided list of substances can be classified as high-molecular compounds with sorption, ligand and catalytic properties. This study presents the results of the investigation of the functional groups of the active centre of inulinase and the features of its quaternary structure. It was established that the enzyme has a quaternary structure, represented by two subunits with Mr 76900 Da and 10140 Da, which have catalytic activity. The imidazole radical of histidine, SH groups and carboxyl groups take part in the formation of the enzyme-substrate complex. The purpose of this study was investigation of the functional groups of the active centre and some features of the quaternary structure of inulinase. In this study, we used a preparation of inulinase isolated from Aspergillus awamori Ts2250, purified by ion exchange chromatography on columns with diethylaminoethylcellulose (DEAE): [-O-(CH2)2-N(C2H5)2]. The homogeneity of the drug was confirmed by gel electrophoresis [24-25]. The catalytic activity of inulinase was determined on the substrate inulin (Spofa, Czech Republic) spectrophotometrically using resorcinol at λ = 540 nm, and the molecular weight was determined by gel chromatography on Sephadex G-200. The presence of electrophilic groups COO- was determined using the Dixon method [28], and also by IR spectroscopy on a Vertex-70 device (Bruker, Germany) in the frequency range 4000-400 cm-1. The active centre of the enzyme includes γ and δ-carboxyl groups of aspartic and glutamic acid residues, respectively. Hydrogen ion H+ splits off from the carboxyl group of the glutamine residue of the enzyme and binds to oxygen connecting rings A and B of the substrate. As a result, the oxygen bond with the ring is broken, and the carbon located in position I of ring A forms a carbonium ion, which is stabilized by COO- group of the aspartic acid residue of the enzyme. An ОН- ion delivered by a water molecule interacts with carbonium ion, and H+ ion of water is fixed in the place of H+ ion lost by the glutamic acid residue in inulinase. After this, inulin molecules leave the enzyme, freeing it for subsequent reaction with the substrate. However, this mechanism is not the only one. Histidine imidazole also takes part in the formation of the enzyme-substrate complex. When interacting with inulin, the imidazole group is hydrogen bonded to the oxygen connecting rings A and B of the substrate. There is also an orientation of the COO- ion of the enzyme ion relative to the resulting carbonium ion in ring A. Subsequently, Н+ and ОН- ions from water molecules are fixed in inulinase in place of H+, lost Glu, and OH- is fixed on the carbonium ion of inulin.

Iron-Mediated Electrolysis for Selective Oxidative Decarboxylation of C-Terminal Acids Using Metal-Binding Residue

With the increasing concern of the environmental impact of solid-phase peptide synthesis (SPPS), semi-recombinant processes, in which recombinantly expressed peptides are selectively modified, are of growing interest. The strategies employed for these modifications must overcome the challenges associated with recombinant peptides: selectivity across many functional groups and solubility of the full-length peptide. Electrochemistry is a viable solution to these challenges as it is a mild and tunable process which can operate in both organic and aqueous systems. Herein, a model tetramer with a redox-active residue, tyrosine, is selectively decarboxylated at the C-terminus in an iron-mediated electrolysis. Chemoselectivity is achieved by exploiting the metal-binding property of a C-terminal glutamic acid residue. In the presence of the iron mediator, the tyrosine oxidation pathway is suppressed while the oxidative decarboxylation pathway is favored. Mechanistic controls demonstrate that the glutamic acid, iron, and applied current are all necessary to impart the desired chemoselectivity.

Glutamate affects self-assembly, protein corona, and anti-4 T1 tumor effects of melittin/vitamin E-succinic acid-(glutamate)n nanoparticles

Melittin (M) has attracted increasing attention for its significant antitumor effects and various immunomodulatory effects. However, various obstacles such as the short plasma half-life and adverse reactions restrict its application. This study aimed to systematically investigate the self-assembly mechanism, components of the protein corona, targeting behavior, and anti-4 T1 tumor effect of vitamin E-succinic acid-(glutamate)n /melittin nanoparticles with varying amounts of glutamic acid. Here, we present a new vitamin E-succinic acid-(glutamate)5 (E5), vitamin E-succinic acid-(glutamate)10 (E10) or vitamin E-succinic acid-(glutamate)15 (E15), and their co-assembly system with positively charged melittin in water. The molecular dynamics simulations demonstrated that the electrostatic energy and van der Waals force in the system decreased significantly with the increase in the amount of glutamic acid. The melittin and E15 system exhibited the optimal stability for nanoparticle self-assembly. When nanoparticles derived from different self-assembly systems were co-incubated with plasma from patients with breast cancer, the protein corona showed heterogeneity. In vivo imaging demonstrated that an increase in the number of glutamic acid residues enhanced circulation duration and tumor-targeting effects. Both in vitro and in vivo antitumor evaluation indicated a significant increase in the antitumor effect with the addition of glutamic acid. According to our research findings, the number of glutamic acid residues plays a crucial role in the targeted delivery of melittin for immunomodulation and inhibition of 4 T1 breast cancer. Due to the self-assembly capabilities of vitamin E-succinic acid-(glutamate)n in water, these nanoparticles carry significant potential for delivering cationic peptides such as melittin.

Mutational Analysis of the Nitrogenase Carbon Monoxide Protective Protein CowN Reveals That a Conserved C-Terminal Glutamic Acid Residue Is Necessary for Its Activity.

Nitrogenase is the only enzyme that catalyzes the reduction of nitrogen gas into ammonia. Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Many nitrogen fixing bacteria protect nitrogenase from CO inhibition using the protective protein CowN. This work demonstrates that a conserved glutamic acid residue near the C-terminus of Gluconacetobacter diazotrophicus CowN is necessary for its function. Mutation of the glutamic acid residue abolishes both CowN's protection against CO inhibition and the ability of CowN to bind to nitrogenase. In contrast, a conserved C-terminal cysteine residue is not important for CO protection by CowN. Overall, this work uncovers structural features in CowN that are required for its function and provides new insights into its nitrogenase binding and CO protection mechanism.

Bacteroides fragilis ubiquitin homologue drives intraspecies bacterial competition in the gut microbiome.

Interbacterial antagonism and associated defensive strategies are both essential during bacterial competition. The human gut symbiont Bacteroides fragilis secretes a ubiquitin homologue (BfUbb) that is toxic to a subset of B. fragilis strains in vitro. In the present study, we demonstrate that BfUbb lyses certain B. fragilis strains by non-covalently binding and inactivating an essential peptidyl-prolyl isomerase (PPIase). BfUbb-sensitivity profiling of B. fragilis strains revealed a key tyrosine residue (Tyr119) in the PPIase and strains that encode a glutamic acid residue at Tyr119 are resistant to BfUbb. Crystal structural analysis and functional studies of BfUbb and the BfUbb-PPIase complex uncover a unique disulfide bond at the carboxy terminus of BfUbb to mediate the interaction with Tyr119 of the PPIase. In vitro coculture assays and mouse studies show that BfUbb confers a competitive advantage for encoding strains and this is further supported by human gut metagenome analyses. Our findings reveal a previously undescribed mechanism of bacterial intraspecies competition.

Programming co-assembled peptide nanofiber morphology via anionic amino acid type: Insights from molecular dynamics simulations.

Co-assembling peptides can be crafted into supramolecular biomaterials for use in biotechnological applications, such as cell culture scaffolds, drug delivery, biosensors, and tissue engineering. Peptide co-assembly refers to the spontaneous organization of two different peptides into a supramolecular architecture. Here we use molecular dynamics simulations to quantify the effect of anionic amino acid type on co-assembly dynamics and nanofiber structure in binary CATCH(+/-) peptide systems. CATCH peptide sequences follow a general pattern: CQCFCFCFCQC, where all C's are either a positively charged or a negatively charged amino acid. Specifically, we investigate the effect of substituting aspartic acid residues for the glutamic acid residues in the established CATCH(6E-) molecule, while keeping CATCH(6K+) unchanged. Our results show that structures consisting of CATCH(6K+) and CATCH(6D-) form flatter β-sheets, have stronger interactions between charged residues on opposing β-sheet faces, and have slower co-assembly kinetics than structures consisting of CATCH(6K+) and CATCH(6E-). Knowledge of the effect of sidechain type on assembly dynamics and fibrillar structure can help guide the development of advanced biomaterials and grant insight into sequence-to-structure relationships.

Pharmacokinetics of Glutapyrone and its Impact on Expression of the Genes Involved in DNA Repair and Free Radical Production in Intact and Diabetic Rats

Abstract Modification of expression of several genes encoding enzymes involved in radical production and DNA repair by a 1,4-DHP derivative glutapyrone was studied in intact rats and animals with streptozotocin diabetes mellitus. Glutapyrone stimulated iNos mRNA production in both kidneys and blood. The effect was stronger in kidneys of diabetic animals, however in blood the expression was down-regulated. The compound increased the Parp 1 gene expression in kidneys of both intact and diabetic animals; in blood the effect was adverse. Expression of XDh gene was significantly increased by glutapyrone in kidneys of intact and diabetic animals. Administration of the compound to intact animals triggered significant increase of DNA damage in white blood cells assayed by comet assay; in diabetic animals no effect was produced. To explain discrepancies with the formerly described effects of glutapyrone on cultured cells, metabolism of the compound was studied. Glutapyrone is either oxidised or the residue of glutamic acid is removed, glutapyrone turns into AV-153, and the latter is metabolised to smaller compounds. Formation of AV-153, a DNA binder and genotoxic compound in high concentrations, can explain DNA damage in white blood cells and stimulation of DNA repair.

Vitamin K and Calcium Chelation in Vascular Health.

The observation that the extent of artery calcification correlates with the degree of atherosclerosis was the background for the alternative treatment of cardiovascular disease with chelator ethylenediamine tetraacetate (EDTA). Recent studies have indicated that such chelation treatment has only marginal impact on the course of vascular disease. In contrast, endogenous calcium chelation with removal of calcium from the cardiovascular system paralleled by improved bone mineralization exerted, i.e., by matrix Gla protein (MGP) and osteocalcin, appears to significantly delay the development of cardiovascular diseases. After post-translational vitamin-K-dependent carboxylation of glutamic acid residues, MGP and other vitamin-K-dependent proteins (VKDPs) can chelate calcium through vicinal carboxyl groups. Dietary vitamin K is mainly provided in the form of phylloquinone from green leafy vegetables and as menaquinones from fermented foods. Here, we provide a review of clinical studies, addressing the role of vitamin K in cardiovascular diseases, and an overview of vitamin K kinetics and biological actions, including vitamin-K-dependent carboxylation and calcium chelation, as compared with the action of the exogenous (therapeutic) chelator EDTA. Consumption of vitamin-K-rich foods and/or use of vitamin K supplements appear to be a better preventive strategy than EDTA chelation for maintaining vascular health.

Prevalence of vitamin D deficiency in children with sickle cell anemia: a systematic review

Introduction: Sickle cell anemia is a highly prevalent genetic disease in the Brazilian population, mainly present in black ethnic groups from slave migration, as well as from the miscegenation process. It is a disease of clinical expression of the homozygosity of the hemoglobin S gene, which may be a genetic and/or hereditary disease, caused by the replacement of the normal residue of glutamic acid by the amino acid valine in the sixth position of the polypeptide chains of the beta-globin protein, generating biochemical alteration in the hemoglobin S molecules, which polymerize inside the erythrocyte and transform into a sickle cell. The possible lack of vitamin D may be linked to disease processes, as well as to the individual's painful crises. Objectives: To analyze whether or not there was a prevalence of vitamin D deficiency in children with sickle cell anemia, through a systematic review. Methodology: A systematic review was carried out by searching for original articles in the scientific databases: Pubmed, Science Direct, Lilacs and Scielo in the languages: English and Portuguese. Results: It was found that out of 10 articles, 8 showed the prevalence of vitamin D deficiency (< 20 ng/ml) in children with sickle cell anemia. Conclusion: The prevalence of vitamin D deficiency was high in patients with the disease, so that its supplementation may serve as a means of assisting in the treatment to improve the condition and prevent deficiency. However, more targeted studies with this association and/or intervention are needed to have more concrete results.

Abstract 16468: A Microtubule-Connexin 43 Regulatory Link Suppresses Cardiac Fibrosis and Arrhythmias in Duchenne Muscular Dystrophy Mice

Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular degenerative disorder that leads to premature death in the teens because of multiple dysfunctions in the heart and skeletal muscle. DMD is caused by the absence of dystrophin, a sarcolemma protein encoded by the dmd gene, located on the X chromosome. 1:5,000 boys are diagnosed with DMD annually, and there is no cure. Clinically, steroid-based treatments reduce inflammation and thus, palliate skeletal muscle pathology. With improvements in the skeletal muscle, DMD-associated cardiomyopathy has become the leading cause of mortality because nearly all patients develop cardiac disease by age 20. Connexin 43 (Cx43) is a gap junction protein and is responsible for the electrical propagation of signals in the heart; it is upregulated and undergoes pathological remodeling, away from the intercalated disc in the heart of DMD mice (mdx) and patients. Hypo-phosphorylation of a specific triplet of serines in Cx43 had been found in mdx and DMD hearts. To study a potential rescue role for Cx43 phosphorylation in DMD, the triplet of serine residues was replaced by glutamic acid or alanine residues to create a phospho-mimic (Cx43-S3E) or a phospho-dead (Cx43-S3A) version of Cx43 respectively. We found that Cx43-S3E but not Cx43-S3A prevents Cx43 remodeling and development of fibrotic cardiomyopathy, arrhythmias, and heart failure in mdx mice. Remarkably, inhibition of hyperdensified, dystrophic microtubule (MT) polymerization by treatment with colchicine (Colch) enhances Cx43 phosphorylation and reduces Cx43 remodeling in dystrophic hearts, suggesting the presence of a MT-Cx43 regulatory connection. This connection could play an important role in the development of dystrophic cardiomyopathy. Our studies explore the electrophysiological properties of Cx43-S3E in DMD cardiomyocytes, and investigate a requirement for Cx43-S3A in the Colch-dependent improvement of the MT dynamics, uncovering a novel link between phospho-Cx43 and MT organization. Furthermore, the results suggest a novel target for the treatment of DMD-cardiomyopathy.

Disorder and amino acid composition in proteins: their potential role in the adaptation of extracellular pilins to the acidic media, where Acidithiobacillus thiooxidans grows.

There are few biophysical studies or structural characterizations of the type IV pilin system of extremophile bacteria, such as the acidophilic Acidithiobacillus thiooxidans. We set out to analyze their pili-comprising proteins, pilins, because these extracellular proteins are in constant interaction with protons of the acidic medium in which At. thiooxidans grows. We used the web server Operon Mapper to analyze and identify the cluster codified by the minor pilin of At. thiooxidans. In addition, we carried an in-silico characterization of such pilins using the VL-XT algorithm of PONDR® server. Our results showed that structural disorder prevails more in pilins of At. thiooxidans than in non-acidophilic bacteria. Further computational characterization showed that the pilins of At. thiooxidans are significantly enriched in hydroxy (serine and threonine) and amide (glutamine and asparagine) residues, and significantly reduced in charged residues (aspartic acid, glutamic acid, arginine and lysine). Similar results were obtained when comparing pilins from other Acidithiobacillus and other acidophilic bacteria from another genus versus neutrophilic bacteria, suggesting that these properties are intrinsic to pilins from acidic environments, most likely by maintaining solubility and stability in harsh conditions. These results give guidelines for the application of extracellular proteins of acidophiles in protein engineering.

Effect of mutations on the folding and stability of γD-crystallin protein

Interprotein interactions between the partially unfolded states of γD-crystallin (γD-crys) protein are known to cause cataracts. Therefore, understanding the unfolding pathways of native γD-crys is extremely crucial to delineate their aggregation mechanism. In this study, we have performed extensive all-atom Molecular Dynamics simulations with explicit solvent to understand the role of the critical residues that drive the stability of the motifs and domains of γD-crys in its wild type and mutant forms. Our findings show that while the individual motifs of wild type are not stable in the native form, the individual domains remain structurally stable at 425K. This enhanced stability of the domain was attributed to the hydrophobic interactions between the motifs. Single and double point mutations of the domains with negatively charged aspartic and glutamic acid amino acid residues (I3E, W42D, W42E, I3D/W42D, I3E/W42E, and L92D/W157D) decreases the structural stability, leading to unfolding of individual domains of γD-crys. We believe that our study sheds light on the weakest links of γD-crys, along with the role of interactions stabilizing the domains. Further, this study bolsters and provides a better understanding of the domain swapping mechanism of aggregation of γD-crys. Communicated by Ramaswamy H. Sarma