Search results for "Macromolecular structure analysis"
showing 10 items of 19 documents
GW-Bodies and P-Bodies Constitute Two Separate Pools of Sequestered Non-Translating RNAs
2015
Non-translating RNAs that have undergone active translational repression are culled from the cytoplasm into P-bodies for decapping-dependent decay or for sequestration. Organisms that use microRNA-mediated RNA silencing have an additional pathway to remove RNAs from active translation. Consequently, proteins that govern microRNA-mediated silencing, such as GW182/Gw and AGO1, are often associated with the P-bodies of higher eukaryotic organisms. Due to the presence of Gw, these structures have been referred to as GW-bodies. However, several reports have indicated that GW-bodies have different dynamics to P-bodies. Here, we use live imaging to examine GW-body and P-body dynamics in the early …
Evolving Notch polyQ tracts reveal possible solenoid interference elements.
2016
ABSTRACTPolyglutamine (polyQ) tracts in regulatory proteins are extremely polymorphic. As functional elements under selection for length, triplet repeats are prone to DNA replication slippage and indel mutations. Many polyQ tracts are also embedded within intrinsically disordered domains, which are less constrained, fast evolving, and difficult to characterize. To identify structural principles underlying polyQ tracts in disordered regulatory domains, here I analyze deep evolution of metazoan Notch polyQ tracts, which can generate alleles causing developmental and neurogenic defects. I show that Notch features polyQ tract turnover that is restricted to a discrete number of conserved “polyQ …
In silico discovery of substituted pyrido[2,3-d]pyrimidines and pentamidine-like compounds with biological activity in myotonic dystrophy models
2016
Myotonic dystrophy type 1 (DM1) is a rare multisystemic disorder associated with an expansion of CUG repeats in mutant DMPK (dystrophia myotonica protein kinase) transcripts; the main effect of these expansions is the induction of pre-mRNA splicing defects by sequestering muscleblind-like family proteins (e.g. MBNL1). Disruption of the CUG repeats and the MBNL1 protein complex has been established as the best therapeutic approach for DM1, hence two main strategies have been proposed: targeted degradation of mutant DMPK transcripts and the development of CUG-binding molecules that prevent MBNL1 sequestration. Herein, suitable CUG-binding small molecules were selected using in silico approach…
Fasciola spp: Mapping of the MF6 epitope and antigenic analysis of the MF6p/HDM family of heme-binding proteins
2017
MF6p/FhHDM-1 is a small cationic heme-binding protein which is recognized by the monoclonal antibody (mAb) MF6, and abundantly present in parenchymal cells and secreted antigens of Fasciola hepatica. Orthologs of this protein (MF6p/HDMs) also exist in other causal agents of important foodborne trematodiasis, such as Clonorchis sinensis, Opisthorchis viverrini and Paragonimus westermani. Considering that MF6p/FhHDM-1 is relevant for heme homeostasis in Fasciola and was reported to have immunomodulatory properties, this protein is expected to be a useful target for vaccination. Thus, in this study we mapped the epitope recognized by mAb MF6 and evaluated its antigenicity in sheep. The sequenc…
Chimeric proteins tagged with specific 3xHA cassettes may present instability and functional problems
2017
Epitope-tagging of proteins has become a widespread technique for the analysis of protein function, protein interactions and protein localization among others. Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo. Different systems have been developed during years in the yeast Saccharomyces cerevisiae. In the present study, we analysed systematically a set of yeast proteins that were fused to different tags. Analysis of the tagged proteins revealed an unexpected general effect on protein level when some specific tagging module was used. This was due in all cases to a destabilization of the proteins and caused a red…
Structural Mechanism of N-Methyl-D-Aspartate Receptor Type 1 Partial Agonism
2012
N-methyl-D-aspartate (NMDA) receptors belong to a family of ionotropic glutamate receptors that contribute to the signal transmission in the central nervous system. NMDA receptors are heterotetramers that usually consist of two GluN1 and GluN2 monomers. The extracellular ligand-binding domain (LBD) of a monomer is comprised of discontinuous segments that form the functional domains D1 and D2. While the binding of a full agonist glycine to LBD of GluN1 is linked to cleft closure and subsequent ion-channel opening, partial agonists are known to activate the receptor only sub-maximally. Although the crystal structures of the LBD of related GluA2 receptor explain the mechanism for the partial a…
CO rebinding kinetics and molecular dynamics simulations highlight dynamic regulation of internal cavities in human cytoglobin
2013
Abstract: Cytoglobin (Cygb) was recently discovered in the human genome and localized in different tissues. It was suggested to play tissue-specific protective roles, spanning from scavenging of reactive oxygen species in neurons to supplying oxygen to enzymes in fibroblasts. To shed light on the functioning of such versatile machinery, we have studied the processes supporting transport of gaseous heme ligands in Cygb. Carbon monoxide rebinding shows a complex kinetic pattern with several distinct reaction intermediates, reflecting rebinding from temporary docking sites, second order recombination, and formation (and dissociation) of a bis-histidyl heme hexacoordinated reaction intermediate…
Human Hsp60 with Its Mitochondrial Import Signal Occurs in Solution as Heptamers and Tetradecamers Remarkably Stable over a Wide Range of Concentrati…
2014
It has been established that Hsp60 can accumulate in the cytosol in various pathological conditions, including cancer and chronic inflammatory diseases. Part or all of the cytosolic Hsp60 could be naive, namely, bear the mitochondrial import signal (MIS), but neither the structure nor the in solution oligomeric organization of this cytosolic molecule has still been elucidated. Here we present a detailed study of the structure and self-organization of naive cytosolic Hsp60 in solution. Results were obtained by different biophysical methods (light and X ray scattering, single molecule spectroscopy and hydrodynamics) that all together allowed us to assay a wide range of concentrations of Hsp60…
Acetylcholine-binding protein in the hemolymph of the planorbid snail Biomphalaria glabrata is a pentagonal dodecahedron (60 subunits).
2012
Nicotinic acetylcholine receptors (nAChR) play important neurophysiological roles and are of considerable medical relevance. They have been studied extensively, greatly facilitated by the gastropod acetylcholine-binding proteins (AChBP) which represent soluble structural and functional homologues of the ligand-binding domain of nAChR. All these proteins are ring-like pentamers. Here we report that AChBP exists in the hemolymph of the planorbid snail Biomphalaria glabrata (vector of the schistosomiasis parasite) as a regular pentagonal dodecahedron, 22 nm in diameter (12 pentamers, 60 active sites). We sequenced and recombinantly expressed two ∼25 kDa polypeptides (BgAChBP1 and BgAChBP2) wit…
Construction of Chimeric Dual-Chain Avidin by Tandem Fusion of the Related Avidins
2011
BackgroundAvidin is a chicken egg-white protein with high affinity to vitamin H, also known as D-biotin. Many applications in life science research are based on this strong interaction. Avidin is a homotetrameric protein, which promotes its modification to symmetrical entities. Dual-chain avidin, a genetically engineered avidin form, has two circularly permuted chicken avidin monomers that are tandem-fused into one polypeptide chain. This form of avidin enables independent modification of the two domains, including the two biotin-binding pockets; however, decreased yields in protein production, compared to wt avidin, and complicated genetic manipulation of two highly similar DNA sequences i…