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The TFE-induced transient native-like structure of the intrinsically disordered [Formula: see text] domain of Escherichia coli RNA polymerase.
Get PDFThe transient folding of domain 4 of an E. coli RNA polymerase [Formula: see text] subunit ([Formula: see text]) induced by an increasing concentration of 2,2,2-trifluoroethanol (TFE) in an aqueous solution was monitored by means of CD and heteronuclear NMR spectroscopy. NMR data, collected at a 30 % TFE, allowed the estimation of the population of a locally folded [Formula: see text] structure (CSI descriptors) and of local backbone dynamics ((15)N relaxation). The spontaneous organization of the helical regions of the initially unfolded protein into a TFE-induced 3D structure was revealed from structural constraints deduced from (15)N- to (13)C-edited NOESY spectra. In accordance with all the applied criteria, three highly populated α-helical regions, separated by much more flexible fragments, form a transient HLHTH motif resembling those found in PDB structures resolved for homologous proteins. All the data taken together demonstrate that TFE induces a transient native-like structure in the intrinsically disordered protein
Global analysis of S-nitrosylation sites in the wild type and APP transgenic mouse brain-clues for synaptic pathology
Get PDFAlzheimer’s disease (AD) is characterized by an early synaptic
loss, which strongly correlates with the severity of
dementia. The pathogenesis and causes of characteristic
AD symptoms are not fully understood. Defects in various
cellular cascades were suggested, including the imbalance
in production of reactive oxygen and nitrogen species.
Alterations in S-nitrosylation of several proteins
were previously demonstrated in various AD animal models
and patients. In this work, using combined biotinswitch
affinity/nano-LC-MS/MS and bioinformatic approaches
we profiled endogenous S-nitrosylation of brain
synaptosomal proteins from wild type and transgenic
mice overexpressing mutated human Amyloid Precursor
Protein (hAPP). Our data suggest involvement of S-nitrosylation
in the regulation of 138 synaptic proteins, including
MAGUK, CamkII, or synaptotagmins. Thirty-eight
proteins were differentially S-nitrosylated in hAPP mice
only. Ninety-five S-nitrosylated peptides were identified
for the first time (40% of total, including 33 peptides exclusively
in hAPP synaptosomes). We verified differential
S-nitrosylation of 10 (26% of all identified) synaptosomal
proteins from hAPP mice, by Western blotting with specific
antibodies. Functional enrichment analysis linked S-nitrosylated
proteins to various cellular pathways, including:
glycolysis, gluconeogenesis, calcium homeostasis, ion, and
vesicle transport, suggesting a basic role of this post-translational
modification in the regulation of synapses. The linkage
of SNO-proteins to axonal guidance and other processes
related to APP metabolism exclusively in the hAPP
brain, implicates S-nitrosylation in the pathogenesis of
Alzheimer’s disease.
Factors influencing compact-extended structure equilibrium in oligomers of Aβ1–40 peptide – an ion mobility mass spectrometry study
Get PDFOligomers formed by amyloid beta peptide (Aβ) are widely believed to be the main neurotoxic agent in Alzheimer’s disease. Studies discovered a broad variety of oligomeric forms, which display different levels of toxicity. Some of these forms may further assemble into mature fibrils, while other might be off-pathway from conversion to fibrils and assemble into alternative forms. To better understand a relationship between the structure and toxicity of Aβ oligomers, systematic characterization and classification of all possible forms is required, facilitating rational design of the beneficial modifiers of their activity. In previous ion-mobility analysis of Aβ1–40 oligomers, we have detected the coexistence of two alternative structural forms (compact and extended) in a pool of low-order Aβ1–40 oligomers. These forms may represent two pathways of the oligomer evolution, either leading to fibrils or to off-pathway oligomers, potential candidates for the neurotoxic species. Here, we have analyzed the impact of incubation time, the presence of selected metal ions and the effect of a series of point mutations on mutual population of alternative forms. We have shown that a salt bridge D23K28 provides stabilization of the compact form whereas G25 is required for the existence of the extended form. We have found that binding of metal ions also stabilizes the compact form. These results improve our understanding of the possible molecular mechanism of the bifurcation of structural evolution of non-monomeric Aβ species into an off-fibril pathway, ultimately leading to the formation of potentially neurotoxic species
To control and to be controlled: understanding the Arabidopsis SLIM1 function in sulfur deficiency through comprehensive investigation of the EIL protein family.
Get PDFSLIM1, a member of the EIN3-like (EIL) family of transcription factors in Arabidopsis, is the regulator of many sulfur-deficiency responsive genes. Among the five other proteins of the family, three regulate ethylene responses and two have unassigned functions. Contrary to the well-defined ethylene signaling, the pathway leading from sensing sulfate status to the activation of its acquisition via SLIM1 is completely unknown. SLIM1 binds to the 20 nt-long specific UPE-box sequence; however, it also recognizes the shorter TEIL sequence, unique for the whole EIL family. SLIM1 takes part in the upregulation and downregulation of various sulfur metabolism genes, but also it controls the degradation of glucosinolates under sulfur deficient conditions. Besides facilitating the increased flux through the sulfate assimilation pathway, SLIM1 induces microRNA395, specifically targeting ATP sulfurylases and a low-affinity sulfate transporter, SULTR2;1, thus affecting sulfate translocation to the shoot. Here, we briefly review the identification, structural characteristics and molecular function of SLIM1 from the perspective of the whole EIL protein family
Patterns of structural dynamics in RACK1 protein retained throughout evolution a hydrogen-deuterium exchange study of three orthologs.
Get PDFRACK1 is a member of the WD repeat family of proteins and is involved in multiple fundamental cellular processes. An intriguing feature of RACK1 is its ability to interact with at least 80 different protein partners. Thus, the structural features enabling such interactomic flexibility are of great interest. Several previous studies of the crystal structures of RACK1 orthologs described its detailed architecture and confirmed predictions that RACK1 adopts a seven-bladed β-propeller fold. However, this did not explain its ability to bind to multiple partners. We performed hydrogen-deuterium (H-D) exchange mass spectrometry on three orthologs of RACK1 (human, yeast, and plant) to obtain insights into the dynamic properties of RACK1 in solution. All three variants retained similar patterns of deuterium uptake, with some pronounced differences that can be attributed to RACK1's divergent biological functions. In all cases, the most rigid structural elements were confined to B-C turns and, to some extent, strands B and C, while the remaining regions retained much flexibility. We also compared the average rate constants for H-D exchange in different regions of RACK1 and found that amide protons in some regions exchanged at least 1000-fold faster than in others. We conclude that its evolutionarily retained structural architecture might have allowed RACK1 to accommodate multiple molecular partners. This was exemplified by our additional analysis of yeast RACK1 dimer, which showed stabilization, as well as destabilization, of several interface regions upon dimer formation
Atomic resolution structure of a protein prepared by non-enzymatic His-tag removal. Crystallographic and NMR study of GmSPI-2 inhibitor.
Get PDFPurification of suitable quantity of homogenous protein is very often the bottleneck in protein structural studies. Overexpression of a desired gene and attachment of enzymatically cleavable affinity tags to the protein of interest made a breakthrough in this field. Here we describe the structure of Galleria mellonella silk proteinase inhibitor 2 (GmSPI-2) determined both by X-ray diffraction and NMR spectroscopy methods. GmSPI-2 was purified using a new method consisting in non-enzymatic His-tag removal based on a highly specific peptide bond cleavage reaction assisted by Ni(II) ions. The X-ray crystal structure of GmSPI-2 was refined against diffraction data extending to 0.98 Å resolution measured at 100 K using synchrotron radiation. Anisotropic refinement with the removal of stereochemical restraints for the well-ordered parts of the structure converged with R factor of 10.57% and Rfree of 12.91%. The 3D structure of GmSPI-2 protein in solution was solved on the basis of 503 distance constraints, 10 hydrogen bonds and 26 torsion angle restraints. It exhibits good geometry and side-chain packing parameters. The models of the protein structure obtained by X-ray diffraction and NMR spectroscopy are very similar to each other and reveal the same β2αβ fold characteristic for Kazal-family serine proteinase inhibitors
Sulphate reducing activity detected in soil samples from Antarctica, Ecology Glacier forefield, King George Island
Get PDFWe determined sulphate-reducing activities in media inoculated with soils and with kettle lake sediments in order to investigate their potential in geomicrobiological processes in low-temperature, terrestrial maritime Antarctic habitats. Soil and sediment samples were collected in a glacier valley abandoned by Ecology Glacier during the last 30 years: from a new formed kettle lake sediment and forefield soil derived from ground moraine. Inoculated with these samples, liquid Postgate C and minimal media supplemented with various carbon sources as electron donors were incubated for 8 weeks at 4°C. High rates of sulphate reduction were observed only in media inoculated with soil. No sulphate reduction was detected in media inoculated with kettle lake sediments. In soil samples culture media calcite and elemental sulphur deposits were observed, demonstrating that sulphate-reducing activity is associated with a potential to mineral formation in cold environments. Cells observed on scanning microscopy (SEM) micrographs of post-culture-soil deposits could be responsible for sulphate-reducing activity
Simvastatin Treatment Highlights a New Role for the Isoprenoid/Cholesterol Biosynthetic Pathway in the Modulation of Emotional Reactivity and Cognitive Performance in Rats.
Get PDFThe aim of the present work was to shed light on the role played by the isoprenoid/cholesterol biosynthetic pathway in the modulation of emotional reactivity and memory consolidation in rodents through the inhibition of the key and rate-limiting enzyme 3-hydroxy 3-methylglutaryl Coenzyme A reductase (HMGR) both in vivo and in vitro with simvastatin. Three-month-old male Wistar rats treated for 21 days with simvastatin or vehicle were tested in the social interaction, elevated plus-maze, and inhibitory avoidance tasks; after behavioral testing, the amygdala, hippocampus, prefrontal cortex, dorsal and ventral striatum were dissected out for biochemical assays. In order to delve deeper into the molecular mechanisms underlying the observed effects, primary rat hippocampal neurons were used. Our results show that HMGR inhibition by simvastatin induces anxiogenic-like effects in the social interaction but not in the elevated plus maze test, and improves memory consolidation in the inhibitory avoidance task. These effects are accompanied by imbalances in the activity of specific prenylated proteins, Rab3 and RhoA, involved in neurotransmitter release and synaptic plasticity, respectively. Taken together, the present findings indicate that the isoprenoid/cholesterol biosynthetic pathway is critically involved in the physiological modulation of both emotional and cognitive processes in rodents.Neuropsychopharmacology accepted article preview online, 10 October 2013. doi:10.1038/npp.2013.284
Di-Tyrosine Cross-link Decreases the Collisional Cross-Section of Aβ Peptide Dimers and Trimers in the Gas Phase – an Ion Mobility Study
Get PDFOligomeric forms of Aβ peptide are most likely the main synaptotoxic and neurotoxic agent in Alzheimer’s disease. Toxicity of various Aβ oligomeric forms has been confirmed in vivo and also in vitro. However, in vitro preparations were found to be orders of magnitude less toxic than oligomers obtained from in vivo sources. This difference can be explained by the presence of a covalent cross-link, which would stabilize the oligomer. In the present work, we have characterized the structural properties of Aβ dimers and trimers stabilized by di- and tri-tyrosine cross-links. Using ion mobility mass spectrometry we have compared the collisional cross-section of non-cross-linked and cross-linked species. We have found that the presence of cross-links does not generate new unique forms but rather shifts the equilibrium towards more compact oligomer types that can also be detected for non-cross-linked peptide. In consequence, more extended forms, probable precursors of off-pathway oligomeric species, become relatively destabilized in cross-linked oligomers and the pathway of oligomer evolution becomes redirected towards fibrillar structures
Crystal structure of the 5hmC specific endonuclease PvuRts1I
Get PDFPvuRts1I is a prototype for a larger family of restriction endonucleases that cleave DNA containing 5-hydroxymethylcytosine (5hmC) or 5-glucosylhydroxymethylcytosine (5ghmC), but not 5-methylcytosine (5mC) or cytosine. Here, we report a crystal structure of the enzyme at 2.35 Å resolution. Although the protein has been crystallized in the absence of DNA, the structure is very informative. It shows that PvuRts1I consists of an N-terminal, atypical PD-(D/E)XK catalytic domain and a C-terminal SRA domain that might accommodate a flipped 5hmC or 5ghmC base. Changes to predicted catalytic residues of the PD-(D/E)XK domain or to the putative pocket for a flipped base abolish catalytic activity. Surprisingly, fluorescence changes indicative of base flipping are not observed when PvuRts1I is added to DNA substrates containing pyrrolocytosine in place of 5hmC (5ghmC). Despite this caveat, the structure suggests a model for PvuRts1I activity and presents opportunities for protein engineering to alter the enzyme properties for biotechnological applications