1,180 research outputs found
The effects of threonine phosphorylation on the stability and dynamics of the central molecular switch region of 18.5-kDa myelin basic protein.
No full textThe classic isoforms of myelin basic protein (MBP) are essential for the formation and maintenance of myelin in the central nervous system of higher vertebrates. The protein is involved in all facets of the development, compaction, and stabilization of the multilamellar myelin sheath, and also interacts with cytoskeletal and signaling proteins. The predominant 18.5-kDa isoform of MBP is an intrinsically-disordered protein that is a candidate auto-antigen in the human demyelinating disease multiple sclerosis. A highly-conserved central segment within classic MBP consists of a proline-rich region (murine 18.5-kDa sequence -T92-P93-R94-T95-P96-P97-P98-S99-) containing a putative SH3-ligand, adjacent to a region that forms an amphipathic α-helix (P82-I90) upon interaction with membranes, or under membrane-mimetic conditions. The T92 and T95 residues within the proline-rich region can be post-translationally modified through phosphorylation by mitogen-activated protein (MAP) kinases. Here, we have investigated the structure of the α-helical and proline-rich regions in dilute aqueous buffer, and have evaluated the effects of phosphorylation at T92 and T95 on the stability and dynamics of the α-helical region, by utilizing four 36-residue peptides (S72-S107) with differing phosphorylation status. Nuclear magnetic resonance spectroscopy reveals that both the α-helical as well as the proline-rich regions are disordered in aqueous buffer, whereas they are both structured in a lipid environment (cf., Ahmed et al., Biochemistry 51, 7475-9487, 2012). Thermodynamic analysis of trifluoroethanol-titration curves monitored by circular dichroism spectroscopy reveals that phosphorylation, especially at residue T92, impedes formation of the amphipathic α-helix. This conclusion is supported by molecular dynamics simulations, which further illustrate that phosphorylation reduces the folding reversibility of the α-helix upon temperature perturbation and affect the global structure of the peptides through altered electrostatic interactions. The results support the hypothesis that the central conserved segment of MBP constitutes a molecular switch in which the conformation and/or intermolecular interactions are mediated by phosphorylation/dephosphorylation at T92 and T95
Solution Nuclear Magnetic Resonance Structure and Molecular Dynamics Simulations of a Murine 18.5 kDa Myelin Basic Protein Segment (S72-S107) in Association with Dodecylphosphocholine Micelles.
No full textpeer reviewedThe 18.5 kDa myelin basic protein (MBP), the most abundant splice isoform in adult mammalian myelin, is a multifunctional, intrinsically disordered protein involved in the development and compaction of the myelin sheath in the central nervous system. A highly conserved central segment comprises a membrane-anchoring amphipathic alpha-helix followed by a proline-rich segment that represents a ligand for SH3 domain-containing proteins. Here, we have determined using solution nuclear magnetic resonance spectroscopy the structure of a 36-residue peptide fragment of MBP (murine 18.5 kDa residues S72-S107, denoted the alpha2-peptide) comprising these two structural motifs, in association with dodecylphosphocholine (DPC) micelles. The structure was calculated using CS-ROSETTA (version 1.01) because the nuclear Overhauser effect restraints were insufficient for this protein. The experimental studies were complemented by molecular dynamics simulations of a corresponding 24-residue peptide fragment (murine 18.5 kDa residues E80-G103, denoted the MD-peptide), also in association with a DPC micelle in silico. The experimental and theoretical results agreed well with one another, despite the independence of the starting structures and analyses, both showing membrane association via the amphipathic alpha-helix, and a sharp bend in the vicinity of the Pro93 residue (murine 18.5 kDa sequence numbering). Overall, the conformations elucidated here show how the SH3 ligand is presented to the cytoplasm for interaction with SH3 domain-containing proteins such as Fyn and contribute to our understanding of myelin architecture at the molecular level
Bridging time and length scales: from macroscopic flux to molecular mechanism of antibiotics diffusion through porins
No full textOur aim in this study was to provide an atomic description of ampicillin translocation through OmpF, the major outer membrane channel in Escherichia coli and main entry point for beta-lactam antibiotics. By applying metadynamics simulations, we also obtained the energy barriers along the diffusion pathway We then studied the effect of mutations that affect the charge and size at the channel constriction zone, and found that in comparison to the wild-type, much lower energy barriers are required for translocation The expected higher translocation rates were confirmed on the macroscopic scale by liposome-swelling assays. A microscopic view on the millisecond timescale was obtained by analysis of temperature-dependent ion current fluctuations in the presence of ampicillin and provide the enthalpic part of the energy barrier. By studying antibiotic translocation over various timescales and length scales, we were able to discern its molecular mechanism and rate-limiting interactions, and draw biologically relevant conclusions that may help in the design of drugs with enhanced permeation rates
Screening for antibiotics with enhanced permeation properties: proof of concept using molecular simulations
No full textTowards screening for antibiotics with enhanced permeation properties through bacterial porins
No full textGram-negative bacteria are protected by an outer membrane barrier, and to reach their periplasmic target, penicillins have to diffuse through outer membrane porins such as OmpF. Here we propose a structure-dynamics-based strategy for improving such antibiotic uptake. Using a variety of experiments (high-resolution single channel recording, Minimum Inhibitory Concentration (MIC), liposome swelling assay) and accelerated molecular simulations, we decipher the subtle balance of interactions governing ampicillin diffusion through the porin OmpF. This suggests mutagenesis of a hot spot residue of OmpF for which additional simulations reveal drastic changes in the molecular and energetic pathway of ampicillin's diffusion. Inverting the problem, we predict and describe how benzylpenicillin diffuses with a lower effective energy barrier by interacting differently with OmpF. The thorough comparison between the theoretical predictions and the three independent experiments, which were set up to measure the kinetics of transport and biological activity, gives insights on how to combine such different investigation techniques with the aim of providing complementary validation. Our study illustrates the importance of microscopic interactions at the constriction region of the biological channel to control the antibiotic flux through it. We conclude by providing a complete inventory of the channel and antibiotic hot spots and discuss the implications in terms of antibacterial screening and design
Kiśïniòvcura Rroma. Studia pala lenqëri història thaj aćara
No full textThe Kishiniovtsy Gypsy group consists of around 3000 people today and hasn’t been described in literature. Ancestors of the group were serfs in Bessarabia. They migrated in Ukraine after abolition of the serfdom in the Russian Empire (the 1861 year’s reform). Now they live in Ukraine and Russia. Until the very roam prohibition in October, 1956, the economic basis of the life of Kishiniovtsy consisted of the men’s domestic thievery and the women’s fortunetelling. By now the ethnic group has trade earnings. Kishiniovtsy didn’t avoid the massive deportations in Stalin’s period. Many were arrested in June, 1933 in Moscow and were deported to Siberia. Unlike most of Gypsy groups, Kishiniovtsy didn’t take an active part in the World War II. While the Russka Roma and the Ukrainian and South-Russian Servi joined the army and were even decorated, Kishiniovtsy always tried to avoid the mobilization. There are many facts about Nazi-genocide in the article. Kishiniovtsy are orthodox Christians. The Moldavian folklore is now forgotten by them. In everyday life the Kishiniovtsy usually sing the songs of Russka Roma. N. Bessonov described their nomadic life and clothing. He built genealogy of some families. Also there is information about customs and justice. Gypsy court in Kishiniovtsy’s dialect is called źudekàta (after Romanian judecată). As for the place of Kishiniovtsy in the Gypsy ethnic group’s hierarchy, they have strong positions. Kishiniovtsy retain their traditions speak their Romany dialect very good and are materially successful. Their problem is the reputation of “dangerous people”. In many aspects such point of view has lost its acuteness (though in conflicts Kishiniovtsy just like Krymy can still protect themselves). In the newest conditions some young people managed to finish school, training colleges and even enter a university, creating a new educated class. Today the legal education is one of the most prestigious. This Gypsy group have good perspectives in modern Russia.O endaj e Rromenqëro Kiśïniovconqëro adadives si grùpa andar pretele 3000 Ŵene thaj palem si bipinŴardo and-e studienqëri literatùra. Lenqëre purane manuśa säs krepostni and-e Besaràbia. Kana lias agor e krepostněnqëro phandlipen and-e Rusiaqëro prathagaripen (pal-e refòrma and-o b. 1861), durile te Ŵan and-e Ukraìna. Akana Ŵiven and-e Ukraìna thaj Rùsia. O avtòro mothovel so akala Rroma, Ŵi k-ob. 1956, Ŵivènas andar-o ćoripen thaj lenqëre rromnǎ zumavènas (dorakǎrènas). Akana kinen thaj bikinen. And-o Baredivaj 1933 nesave lenθär säs deportuime andar-e Mòskva Ŵi k-e Sibèria. Kana pharilǎs o 2-to Sundalìtko Mariben, o Rroma Kiśïniòvcura na marde pen aj vi naśènas katar-e mobilizàcia, Ŵikaj javëra Rroma (sär o „Rùska Rroma thaj o Sèrvi andar-e Ukraìna) säs but aktìvo mamuj-i nazìsmo, phandile e armiaça (xaladimaça) aj vi xuterde xaladìtka dekoràcie. O artìklo den informàcie pal-o Samudaripen. Rroma Kiśïniòvcura si dasa (ortodòksǎ). Bisterde aba o moldavìtko folklòro thaj akana gilaben (bagen) e Ruska Rromenqëre gilǎ. O Besonòvo mothovel pala lenqëro phirutno Ŵivipen aj lodipen, pala lenqëre gada aj nesave irrenqëre genealògie thaj vi lenqëre aćara (tradìcie) aj lenqëri kris rromani, savi lenθe akhardǒl „źudekàta” (andar-e rumunìcko ćhib - judecată). And-e Rromenqëre hieràrxia, o than e Rromenqëro Kiśïniovconqëro si but opre. ŵivde si lenqëre tradìcie (aćara) thaj but śukär rakëren e rromani ćhib. And-e ekonòmia śukär keren love thaj naj len javër problèma, sadaj sol en dikhen javëra manuśa sar „bilaćhe” (traśorne). AZkana fededër dikhen len o javëra, thaj o krimitìka Rroma butivar javen te den len vast kana si konflìkto (ćingar). Terne Rroma Kiśïniòvcura agordine śkòle thaj universitète, thaj kerde jekh nevi klàsa siklǎrdi. Maśkär lenθe si so siklǒn juridika. Akaja rromani endaj si la laćhe perspektìve and-i adadivesutuni Rùsia.Natalia Gancar
Phirutne rromnǎ: lenqëre hakaja, dotǎ aj xod-aktualizàcia
No full textTill now, a state of nomadic Romani women has been described only from a position of an external observer. The author supposes that narrative sources should be used more actively. To actualize this concept he studies recollections of nomadic life, focusing on Romani women’s traditional ways of earning, such as fortune-telling and begging, and their main value – a family. The article shows how Romani women used to risk for husbands and children. They had to take the initiative and to show mother with every day to feed themselves and their relatives. At the same time, married Romani women lived at the mercy of patriarchy. That created the basic paradox of their lives. Gypsy fortune tellers had real economical independence. Outside of families they looked as free women, but inside they uncomplainingly received whipping. The author want to show subjects that are hardly shown in special literature at the moment. Particularly, he writes about the fact that Romani women have actively taken part in a fight against Nazism during the World War II. Another little known subject is women’s literacy. Though most nomadic Romani women couldn’t read, but indeed exclusions existed. It is interesting, too, such a fact as Romani women could get a high social status with an elderly age, so that men take into account their opinion in traditional court. Sometimes, as it can be seen from examples, families were named for female ancestors. The author studies also psychological possibilities used by Gypsy wives to affect their husbands’ decisions.Natalia Gancar
The Biophysics Of Antibiotics Translocation Through OmpF Revealed By Computer Simulations
No full textIn Gram-negative bacteria, the outer membrane porin F (OmpF) constitute the preferred entry point of antibiotics. Since bacteria can resist antibiotics by altering the expression or structures of OmpF, it is of fundamental importance to investigate on the permeation mechanisms at a molecular level. A key feature in the structure of OmpF is the presence of a constriction region, characterized by both a spatial (with dimensions as low as 7x11Å) and an electrostatic (a transversal field formed by negative and positive residues facing each others) restriction.
To study the translocation process at a molecular scale, we performed molecular dynamic simulations combined with the metadynamic algorithm. This recently designed algorithm overcomes the time scale problem by accelerating properly defined reaction coordinates. We compared the following modeling methodologies: (i) OmpF as monomers or trimers, (ii) membranes as surrounding detergent molecules or lipid bilayers, (iii) antibiotics of different structural and chemical properties (penicillins, fluorokinolones, cephalosporines).
We evaluated how site mutations on OmpF alter electrostatic or spatial restriction at the constriction region and affect antibiotics binding and transport. We reconstructed the free energy surface of each antibiotic translocation and compared their preferred path, orientation, affinity sites. We find that translocation is governed by specific (polar, hydrophobic) interactions. This leads us to discuss the applicability of analytical models in this transport. Our results, such as energy barriers for translocations, compared well with the translocation rates obtained by experimental collaborators using electrophysiology and MIC measurements. Furthermore, our methodology suggested new measurements, such as testing novel OmpF variants, low-temperature measurements and liposome swelling assays.
This study demonstrates how theory and experiments combined can reveal the mechanism and the molecular basis of OmpF permeation. This work will benefits to the design of antibiotics with improved transport properties
Rekonstrùkcia thaj brakhipen e rromane ʒivipnasqëre aj folkloraqëre
No full textAuthor is a painter. Together with the members of the Romen Theatre in Moscow he carries out a project of theatrical reconstructions of the traditional scenes and situations of the gypsy camp life (dances among others). The acts are prepared according to detailed historical studies. They are subsequently photographed and filmed to form an archival collection to be used by researchers in the future
Crystal chemistry of selenates with mineral-like structures: VIII. Butlerite chains in the structure of K(UO2)(SeO4)(OH)(H2O)
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