Data on the calcium-induced mobility shift of myristoylated and non-myristoylated forms of neurocalcin delta.
Ontology highlight
ABSTRACT:
SUBMITTER: Viviano J
PROVIDER: S-EPMC4802544 | biostudies-other | 2016 Jun
REPOSITORIES: biostudies-other
Publications
Data on the calcium-induced mobility shift of myristoylated and non-myristoylated forms of neurocalcin delta.
Viviano Jeffrey J Krishnan Anuradha A Wu Hao H Venkataraman Venkat V
Data in brief 20160311
This data article presents the differences observed between the myristoylated and non-myristoylated forms of the neuronal calcium sensor protein, neurocalcin delta (NCALD). Analysis of the myristoylated and non-myristoylated versions of the protein by mass spectrometry provided difference in mass values consistent with addition of myristoyl group. In the presence of calcium, mobility retardation was observed upon electrophoresis of the protein in native gels. The retardation was dose-dependent a ...[more]
Similar Datasets
Project description:The increasing resistance of fungi to antibiotics is a severe challenge in public health, and newly effective drugs are required. Promising potential medications are lipopeptides, linear antimicrobial peptides (AMPs) conjugated to a lipid tail, usually at the N-terminus. In this paper, we investigated the in vitro and in vivo antifungal activity of three short myristoylated and non-myristoylated peptides derived from a mutant of the AMP Chionodracine. We determined their interaction with anionic and zwitterionic membrane-mimicking vesicles and their structure during this interaction. We then investigated their cytotoxic and hemolytic activity against mammalian cells. Lipidated peptides showed a broad spectrum of activity against a relevant panel of pathogen fungi belonging to Candida spp., including the multidrug-resistant C. auris. The antifungal activity was also observed vs. biofilms of C. albicans, C. tropicalis, and C. auris. Finally, a pilot efficacy study was conducted on the in vivo model consisting of Galleria mellonella larvae. Treatment with the most-promising myristoylated peptide was effective in counteracting the infection from C. auris and C. albicans and the death of the larvae. Therefore, this myristoylated peptide is a potential candidate to develop antifungal agents against human fungal pathogens.
| S-EPMC8875392 | biostudies-literature
Project description:In eukaryotic Na(+)/Ca(2+) exchangers (NCX) the Ca(2+) binding CBD1 and CBD2 domains form a two-domain regulatory tandem (CBD12). An allosteric Ca(2+) sensor (Ca3-Ca4 sites) is located on CBD1, whereas CBD2 contains a splice-variant segment. Recently, a Ca(2+)-driven interdomain switch has been described, albeit how it couples Ca(2+) binding with signal propagation remains unclear. To resolve the dynamic features of Ca(2+)-induced conformational transitions we analyze here distinct splice variants and mutants of isolated CBD12 at varying temperatures by using small angle x-ray scattering (SAXS) and equilibrium (45)Ca(2+) binding assays. The ensemble optimization method SAXS analysis demonstrates that the apo and Mg(2+)-bound forms of CBD12 are highly flexible, whereas Ca(2+) binding to the Ca3-Ca4 sites results in a population shift of conformational landscape to more rigidified states. Population shift occurs even under conditions in which no effect of Ca(2+) is observed on the globally derived Dmax (maximal interatomic distance), although under comparable conditions a normal [Ca(2+)]-dependent allosteric regulation occurs. Low affinity sites (Ca1-Ca2) of CBD1 do not contribute to Ca(2+)-induced population shift, but the occupancy of these sites by 1 mM Mg(2+) shifts the Ca(2+) affinity (Kd) at the neighboring Ca3-Ca4 sites from ? 50 nM to ? 200 nM and thus, keeps the primary Ca(2+) sensor (Ca3-Ca4 sites) within a physiological range. Thus, Ca(2+) binding to the Ca3-Ca4 sites results in a population shift, where more constraint conformational states become highly populated at dynamic equilibrium in the absence of global conformational transitions in CBD alignment.
| S-EPMC3743486 | biostudies-literature
Project description:We have developed a protocol to produce large quantities of high purity myristoylated and non-myristoylated neuronal calcium sensor 1 (NCS-1) protein. NCS-1 is a member of the neuronal calcium sensor (NCS) family and plays an important role in modulating G-protein signaling and exocytosis pathways in cells. Many of these functions are calcium-dependent and require NCS-1 to be modified with an N-terminal myristoyl moiety. In our system, a C-terminally 6x His-tagged variant of NCS-1 was co-expressed with yeast N-myristoyltransferase (NMT) in ZYP-5052 auto-induction media supplemented with sodium myristate (100-200 microM). With optimized growth conditions and a high capacity metal affinity purification scheme, >50mg of homogenous myristoylated NCS-1 is obtained from 1L of culture in a single step. The properties of the C-terminally tagged NCS-1 variants are indistinguishable from those reported for untagged NCS-1. Using this system, we have also isolated and characterized mutant NCS-1 proteins that have attenuated (NCS-1 E120Q) and abrogated (NCS-1 DeltaEF) ability to bind calcium. The large quantities of NCS-1 proteins isolated from small culture volumes of auto-inducible media will provide the necessary reagents for further biochemical and structural characterization. The affinity tag at the C-terminus of the protein provides a suitable reagent for easily identifying binding partners of the various NCS-1 constructs. Additionally, this method could be used to produce other recombinant proteins of the NCS family, and may be extended to express and isolate myristoylated variants of other proteins.
| S-EPMC3770182 | biostudies-literature
Project description:The flagellar calcium-binding protein (FCaBP) of the protozoan Trypanosoma cruzi is targeted to the flagellar membrane where it regulates flagellar function and assembly. As a first step toward understanding the Ca(2+)-induced conformational changes important for membrane-targeting, we report here the x-ray crystal structure of FCaBP in the Ca(2+)-free state determined at 2.2A resolution. The first 17 residues from the N terminus appear unstructured and solvent-exposed. Residues implicated in membrane targeting (Lys-19, Lys-22, and Lys-25) are flanked by an exposed N-terminal helix (residues 26-37), forming a patch of positive charge on the protein surface that may interact electrostatically with flagellar membrane targets. The four EF-hands in FCaBP each adopt a "closed conformation" similar to that seen in Ca(2+)-free calmodulin. The overall fold of FCaBP is closest to that of grancalcin and other members of the penta EF-hand superfamily. Unlike the dimeric penta EF-hand proteins, FCaBP lacks a fifth EF-hand and is monomeric. The unstructured N-terminal region of FCaBP suggests that its covalently attached myristoyl group at the N terminus may be solvent-exposed, in contrast to the highly sequestered myristoyl group seen in recoverin and GCAP1. NMR analysis demonstrates that the myristoyl group attached to FCaBP is indeed solvent-exposed in both the Ca(2+)-free and Ca(2+)-bound states, and myristoylation has no effect on protein structure and folding stability. We propose that exposed acyl groups at the N terminus may anchor FCaBP to the flagellar membrane and that Ca(2+)-induced conformational changes may control its binding to membrane-bound protein targets.
| S-EPMC2516990 | biostudies-literature
Project description:Technetium ((99)Tc) is an abundant, long-lived radioactive fission product whose mobility in the subsurface is largely governed by its oxidation state. Tc immobilization is crucial for radioactive waste management and environmental remediation. Tc(IV) incorporation in spinels has been proposed as a novel method to increase Tc retention in glass waste forms during vitrification. However, experiments under high-temperature and oxic conditions show reoxidation of Tc(IV) to volatile pertechnetate, Tc(VII). Here we examine this problem with ab initio molecular dynamics simulations and propose that, at elevated temperatures, doping with first row transition metal can significantly enhance Tc retention in magnetite in the order Co>Zn>Ni. Experiments with doped spinels at 700?°C provide quantitative confirmation of the theoretical predictions in the same order. This work highlights the power of modern, state-of-the-art simulations to provide essential insights and generate theory-inspired design criteria of complex materials at elevated temperatures.
| S-EPMC4931311 | biostudies-literature
Project description:Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.
| S-EPMC5091922 | biostudies-literature
Project description:BackgroundInsulin is a therapeutic protein that is widely used for the treatment of diabetes. Its biological function was discovered more than 80 years ago and it has since then been characterized extensively. Crystallization of the insulin molecule has always been a key activity since the protein is often administered by subcutaneous injections of crystalline insulin formulations. Over the years, insulin has been crystallized and characterized in a number of crystal systems.ResultsInterestingly, we have now discovered two new crystal forms of human insulin. The crystals were obtained when the two chaotropic agents, urea and thiocyanate were present in the crystallization experiments, and their structures were determined by X-ray crystallography. The crystals belong to the orthorhombic and monoclinic crystal systems, with space groups C2221 and C2 respectively. The orthorhombic crystals were obtained at pH 6.5 and contained three insulin hexamers in R6 conformation in the asymmetric unit whilst the monoclinic C2 crystals were obtained at pH 7.0 and contained one R6 hexamer in the asymmetric unit. Common for the two new crystals is a hexamer-hexamer interaction that has not been found in any of the previous crystal forms of insulin. The contacts involve a tight glutamate-glutamate interaction with a distance of 2.3 A between groups. The short distance suggests a low barrier hydrogen bond. In addition, two tyrosine-tyrosine interactions occupying a known phenol binding pocket contribute to the stabilization of the contacts. Within the crystals, distinct binding sites for urea were found, adding further to the discussion on the role of urea in protein denaturation.ConclusionThe change in space group from C2221 to C2 was primarily caused by an increase in pH. The fewer number of hexamer-hexamer interactions comprising the short hydrogen bond in the C2 space group suggest that pH is the driving force. In addition, the distance between the two glutamates increases from 2.32 A in the C2221 crystals to 2.4 A in the C2 crystals. However, in both cases the low barrier hydrogen bond and the tyrosine-tyrosine interaction should contribute to the stability of the crystals which is crucial when used in pharmaceutical formulations.
| S-EPMC2241603 | biostudies-literature
Project description:The implementation of governmental Non-Pharmaceutical Interventions (NPIs) has been the primary means of controlling the spread of the COVID-19 disease. One of the intended effects of these NPIs has been to reduce population mobility. Due to the huge costs of implementing these NPIs, it is essential to have a good understanding of their efficacy. Using aggregated mobility data per country, released by Apple and Google we investigated the proportional contribution of NPIs to the magnitude and rate of mobility changes at a multi-national level. NPIs with the greatest impact on the magnitude of mobility change were lockdown measures; declaring a state of emergency; closure of businesses and public services and school closures. NPIs with the greatest effect on the rate of mobility change were implementation of lockdown measures and limitation of public gatherings. As confirmed by chi-square and cluster analysis, separately recorded NPIs like school closure and closure of businesses and public services were closely correlated with each other, both in timing and occurrence. This suggests that the observed significant NPI effects are mixed with and amplified by their correlated NPI measures. We observed direct and similar effects of NPIs on both Apple and Google mobility data. In addition, although Apple and Google data were obtained by different methods they were strongly correlated indicating that they are reflecting overall mobility on a country level. The availability of this data provides an opportunity for governments to build timely, uniform and cost-effective mechanisms to monitor COVID-19 or future pandemic countermeasures.
| S-EPMC8119480 | biostudies-literature
Project description:Here, we demonstrate that aptamers tethered to gold nanoparticles enable direct visualization of protein-oligonucleotide interactions during gel electrophoresis. This technique is used to confirm that an aptamer previously identified as binding to C-reactive protein (CRP) only binds to the monomeric form of CRP. While native, pentameric CRP (pCRP) is used in clinical assays to predict cardiovascular disease (CVD) risk, it is the monomeric isoform that is more strongly associated with pro-inflammatory and pro-atherogenic effects. To visualize this selectivity, the CRP-aptamer was conjugated to streptavidin-coated gold nanoparticles and the mobility of the free oligonucleotide-nanoparticle conjugate (ON-NP) and the protein/ON-NP complex bands were visualized and recorded during electrophoresis using a simple digital camera. At a concentration of 6??g/mL, monomeric CRP showed a significant decrease in the observed ON-NP mobility, whereas no change in mobility was observed with pCRP up to 18??g/mL. Advantages of this nanoparticle-based electrophoretic mobility shift assay (NP-EMSA) over the traditional EMSA include real-time detection of protein-oligonucleotide interactions, the avoidance of harmful radioisotopes, and elimination of the need for expensive gel imagers. The availability of both the NP-EMSA technique and an mCRP-specific probe will allow for improved clinical diagnostic to more accurately predict future CVD risk.
| S-EPMC3307786 | biostudies-literature
Project description:The metabolic requirements change during cell proliferation and differentiation. Upon antigen-stimulation, effector T cells switch from adenosine-triphospate (ATP)-production by oxidative phosphorylation in the mitochondria to glycolysis. In the gut it was shown that short chain fatty acids (SCFA), fermentation products of the microbiota in colon, ameliorate inflammatory reactions by supporting the differentiation of regulatory T cells. SCFA are a major energy source, but they are also anabolic metabolites, histone-deacetylase-inhibitors and activators of G protein receptors. Recently, it was reported that a topical application of the SCFA butyrate promotes regulatory T cells in the skin. Here we ask if the SCFA butyrate, propionate and acetate affect the energy metabolism and inflammatory potential of dendritic epidermal T cells (DETC), the innate resident skin γδ T cell population. Using the Seahorse™ technology, we measured glycolysis and oxidative phosphorylation (OXPHOS) in a murine DETC cell line, 7-17, upon TCR-stimulation by CD3/CD28 crosslinking, with or without SCFA addition. TCR engagement resulted in a change of the ratio glycolysis/OXPHOS. A similar metabolic shift has been described for activated CD4 T cells. Addition of 5 mM SCFA, in particular butyrate, antagonized the effect. Stimulated DETC secrete cytokines, e.g. the pro-inflammatory cytokine interferon-gamma (IFNγ), and thereby regulate skin homeostasis. Addition of butyrate and propionate to the cultures at non-toxic concentrations decreased secretion of IFNγ by DETC and increased the expression of the immunoregulatory surface receptor CD69. We hypothesize that SCFA can dampen the inflammatory activity of DETC.
| S-EPMC7105938 | biostudies-literature