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Gorshkova Y.E.,Joint Institute for Nuclear Research | Kuklin A.I.,Joint Institute for Nuclear Research | Gordeliy V.I.,RAS Institute for Nuclear Research | Gordeliy V.I.,Institute of Structural Biology
Journal of Surface Investigation | Year: 2017

Results obtained via small-angle neutron scattering studies of the influence of calcium ions on the structure and phase transitions of phospholipid membranes are presented. The main phase transition temperature of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (1 wt %) multilamellar vesicles is demonstrated to increase by more than 1°C even when the calcium-ion content of the solution is low (0.1 mM). Detailed analysis of the multilamellar vesicles transition between “bound” and “unbound” state indicates the continuous character of the investigated process in both liquid and gel phases. The critical Ca2+ ion concentrations which initiate the destruction of the multilamellar structures and the formation of unilamellar vesicles are found to be ~0.3 mM in the gel and ~0.4–0.5 mM in the liquid-crystal phases during heating and ~0.5 mM in the phases under study upon cooling. © 2017, Pleiades Publishing, Ltd.


News Article | May 16, 2017
Site: www.eurekalert.org

An international study led by Monash University has discovered the molecular mechanism by which the potentially deadly superbug 'Golden Staph' evades antibiotic treatment, providing the first important clues on how to counter superbug antibiotic resistance. 'Superbugs' are bacteria that are resistant to commonly used antibiotics, presenting a global health threat. To tackle this global challenge, researchers from Monash's Biomedicine Discovery Institute (BDI) are collaborating with Israel's Weizmann Institute of Science, and the NTU Institute of Structural Biology in Singapore. Now, the Monash BDI researchers have identified the first important clues on how to 'retool' antibiotics to counter the strategies bacteria enlist to evade the life-saving drugs, with the findings published in the journal mBio. Researchers used the latest generation electron microscopes at the Monash Ramaciotti Centre for Electron Microscopy to image at the molecular level -- for the first time -- the changes that take place in superbugs that have become resistant to the most modern antibiotics. Examining bacterial samples of antibiotic-resistant Staphylococcus aureus or 'Golden Staph' taken from a hospital patient, they compared data of a non-resistant strain with their counterparts overseas. These included Shashi Bhushan from NTU, and Zohar Eyal and Nobel Laureate, Professor Ada Yonath from the Weizmann Institute who won the Nobel Prize for Chemistry in 2009. "Using the combined data we could rationalise how the bacteria escapes drug treatment by a really important hospital antibiotic and describe in molecular detail how it becomes like a superbug," said Monash BDI scientist and lead researcher Dr Matthew Belousoff. "The bacteria mutates or evolves to change the shape of the molecule to which the antibiotic would bind so the drug can no longer fit there," Dr Belousoff said. "Knowing what your enemy is doing is the first step to the next phase of new drug design," he said. "We've developed a technique that others can use that might help us speed up the arms race of antibiotic development." Dr Belousoff said Monash BDI researchers are now using this new tool to investigate other drug-resistant bacteria. The research, involving the expertise of Monash microscopist Dr Mazdak Radjainia and mentoring of Professor Trevor Lithgow, was supported by the Australian National Health and Medical Research Council (NHMRC). Read the full paper titled Structural basis for linezolid binding site rearrangement in the Staphylococcus aureus ribosome Committed to making the discoveries that will relieve the future burden of disease, the newly established Monash Biomedicine Discovery Institute at Monash University brings together more than 120 internationally-renowned research teams. Our researchers are supported by world-class technology and infrastructure, and partner with industry, clinicians and researchers internationally to enhance lives through discovery. The World Health Organisation has warned that new antibiotics were urgently needed to counter the growing threat of superbugs. It predicted that deaths from antimicrobial resistance could rise to 10 million by 2050, surpassing the total deaths caused by cancer and diabetes combined. If antibiotics lose their effectiveness, surgery and even treatment of wounds could become life-threatening.


News Article | May 17, 2017
Site: www.chromatographytechniques.com

An international study led by Monash University has discovered the molecular mechanism by which the potentially deadly superbug 'Golden Staph' evades antibiotic treatment, providing the first important clues on how to counter superbug antibiotic resistance. 'Superbugs' are bacteria that are resistant to commonly used antibiotics, presenting a global health threat. To tackle this global challenge, researchers from Monash's Biomedicine Discovery Institute (BDI) are collaborating with Israel's Weizmann Institute of Science, and the NTU Institute of Structural Biology in Singapore. Now, the Monash BDI researchers have identified the first important clues on how to 'retool' antibiotics to counter the strategies bacteria enlist to evade the life-saving drugs, with the findings published in the journal mBio. Researchers used the latest generation electron microscopes at the Monash Ramaciotti Centre for Electron Microscopy to image at the molecular level -- for the first time -- the changes that take place in superbugs that have become resistant to the most modern antibiotics. Examining bacterial samples of antibiotic-resistant Staphylococcus aureus or 'Golden Staph' taken from a hospital patient, they compared data of a non-resistant strain with their counterparts overseas. These included Shashi Bhushan from NTU, and Zohar Eyal and Nobel Laureate, Professor Ada Yonath from the Weizmann Institute who won the Nobel Prize for Chemistry in 2009. "Using the combined data we could rationalise how the bacteria escapes drug treatment by a really important hospital antibiotic and describe in molecular detail how it becomes like a superbug," said Monash BDI scientist and lead researcher Dr Matthew Belousoff. "The bacteria mutates or evolves to change the shape of the molecule to which the antibiotic would bind so the drug can no longer fit there," Belousoff said. "Knowing what your enemy is doing is the first step to the next phase of new drug design," he said. "We've developed a technique that others can use that might help us speed up the arms race of antibiotic development."


News Article | May 16, 2017
Site: www.sciencedaily.com

An international study led by Monash University has discovered the molecular mechanism by which the potentially deadly superbug 'Golden Staph' evades antibiotic treatment, providing the first important clues on how to counter superbug antibiotic resistance. 'Superbugs' are bacteria that are resistant to commonly used antibiotics, presenting a global health threat. To tackle this global challenge, researchers from Monash's Biomedicine Discovery Institute (BDI) are collaborating with Israel's Weizmann Institute of Science, and the NTU Institute of Structural Biology in Singapore. Now, the Monash BDI researchers have identified the first important clues on how to 'retool' antibiotics to counter the strategies bacteria enlist to evade the life-saving drugs, with the findings published in the journal mBio. Researchers used the latest generation electron microscopes at the Monash Ramaciotti Centre for Electron Microscopy to image at the molecular level -- for the first time -- the changes that take place in superbugs that have become resistant to the most modern antibiotics. Examining bacterial samples of antibiotic-resistant Staphylococcus aureus or 'Golden Staph' taken from a hospital patient, they compared data of a non-resistant strain with their counterparts overseas. These included Shashi Bhushan from NTU, and Zohar Eyal and Nobel Laureate, Professor Ada Yonath from the Weizmann Institute who won the Nobel Prize for Chemistry in 2009. "Using the combined data we could rationalise how the bacteria escapes drug treatment by a really important hospital antibiotic and describe in molecular detail how it becomes like a superbug," said Monash BDI scientist and lead researcher Dr Matthew Belousoff. "The bacteria mutates or evolves to change the shape of the molecule to which the antibiotic would bind so the drug can no longer fit there," Dr Belousoff said. "Knowing what your enemy is doing is the first step to the next phase of new drug design," he said. "We've developed a technique that others can use that might help us speed up the arms race of antibiotic development."


Scarsdale J.N.,Institute of Structural Biology | Walavalkar N.M.,Virginia Commonwealth University | Buchwald W.A.,Virginia Commonwealth University | Ginder G.D.,University of North Carolina at Chapel Hill | Williams Jr. D.C.,University of North Carolina at Chapel Hill
Journal of Biological Chemistry | Year: 2014

Although highly homologous to other methylcytosine-binding domain (MBD) proteins, MBD3 does not selectively bind methylated DNA, and thus the functional role of MBD3 remains in question. To explore the structural basis of its binding properties and potential function, we characterized the solution structure and binding distribution of the MBD3 MBD on hydroxymethylated, methylated, and unmethylated DNA. The overall fold of this domain is very similar to other MBDs, yet a key loop involved in DNA binding is more disordered than previously observed. Specific recognition of methylated DNA constrains the structure of this loop and results in large chemical shift changes in NMR spectra. Based on these spectral changes, we show that MBD3 preferentially localizes to methylated and, to a lesser degree, unmethylated cytosine-guanosine dinucleotides (CpGs), yet does not distinguish between hydroxymethylated and unmethylated sites. Measuring residual dipolar couplings for the different bound states clearly shows that the MBD3 structure does not change between methylation-specific and nonspecific binding modes. Furthermore, residual dipolar couplings measured for MBD3 bound to methylated DNA can be described by a linear combination of those for the methylation and nonspecific binding modes, confirming the preferential localization to methylated sites. The highly homologous MBD2 protein shows similar but much stronger localization to methylated as well as unmethylated CpGs. Together, these data establish the structural basis for the relative distribution of MBD2 and MBD3 on genomic DNA and their observed occupancy at active and inactive CpG-rich promoters. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.


Strulovich R.,Institute of Structural Biology | Attali B.,Tel Aviv University | Hirsch J.A.,Institute of Structural Biology | Hirsch J.A.,Tel Aviv University
Biochemistry | Year: 2016

The Kv7 (KCNQ) channel family, comprising voltage-gated potassium channels, plays major roles in fine-tuning cellular excitability by reducing firing frequency and controlling repolarization. Kv7 channels have a unique intracellular C-terminal (CT) domain bound constitutively by calmodulin (CaM). This domain plays key functions in channel tetramerization, trafficking, and gating. CaM binds to the proximal CT, comprising helices A and B. Kv7.2 and Kv7.3 are expressed in neural tissues. Together, they form the heterotetrameric M channel. We characterized Kv7.2, Kv7.3, and chimeric Kv7.3 helix A-Kv7.2 helix B (Q3A-Q2B) proximal CT/CaM complexes by solution methods at various Ca2+concentrations and determined them all to have a 1:1 stoichiometry. We then determined the crystal structure of the Q3A-Q2B/CaM complex at high Ca2+ concentration to 2.0 Å resolution. CaM hugs the antiparallel coiled coil of helices A and B, braced together by an additional helix. The structure displays a hybrid apo-Ca2+ CaM conformation even though four Ca2+ ions are bound. Our results pinpoint unique interactions enabling the possible intersubunit pairing of Kv7.3 helix A and Kv7.2 helix B while underlining the potential importance of Kv7.3 helix A's role in stabilizing channel oligomerization. Also, the structure can be used to rationalize various channelopathic mutants. Functional testing of the chimeric channel found it to have a voltage-dependence similar to the M channel, thereby demonstrating helix A's importance in imparting gating properties. © 2016 American Chemical Society.


Gargallo R.,University of Barcelona | Eritja R.,Institute of Structural Biology | Kudrev A.G.,Saint Petersburg State University
Russian Journal of General Chemistry | Year: 2010

UV absorption spectra and circular dichroism spectra of aqueous solutions of cytosin- and thyminecontaining single-stranded Oligodeoxyribonucleotide 5′-CCTTTCCTTTTCCTTTCC-3′(ckit4) were measured at various pH in the range 3.3-8.9. The chemometric analysis of the multiinstrumental data matrix was carrie out. The diagrams of relative contents of complex forms of the DNA molecule absorbing in the studied wavelength range (220-320 nm) were constructed by the ALS-MCR soft simulation procedure without initial postulation of their chemical compositions. The model of equilibrium complex formation describing observed changes in the spectra depending on the solution acidity was developed on the basis of the matrix method. Intrinsic protonation constants of the oligonucleotide ckit4 were calculated. The formation of intramolecular complexes between cytosine C.C+ bases in the studied DNA molecule is of a cooperative nature, and thei subsequent protonation is an anticooperative process. © 2010 Pleiades Publishing, Ltd.


Sachyani D.,Institute of Structural Biology | Dvir M.,Tel Aviv University | Strulovich R.,Institute of Structural Biology | Tria G.,German Electron Synchrotron | And 7 more authors.
Structure | Year: 2014

Kv7 channels tune neuronal and cardiomyocyte excitability. In addition to the channel membrane domain, they also have a unique intracellular C-terminal (CT) domain, bound constitutively to calmodulin (CaM). This CT domain regulates gating and tetramerization. We investigated the structure of the membrane proximal CT module in complex with CaM by X-ray crystallography. The results show how the CaM intimately hugs a two-helical bundle, explaining many channelopathic mutations. Structure-based mutagenesis of this module in the context of concatemeric tetramer channels and functional analysis along with in vitro data lead us to propose that one CaM binds to one individual protomer, without crosslinking subunits and that this configuration is required for proper channel expression and function. Molecular modeling of the CT/CaM complex in conjunction with small-angle X-ray scattering suggests that the membrane proximal region, having a rigid lever arm, is a critical gating regulator. © 2014 Elsevier Ltd.


Dames S.A.,TU Munich | Dames S.A.,Helmholtz Center Munich | Dames S.A.,Institute of Structural Biology
Journal of Peptide Science | Year: 2015

The nematocyst walls of Hydra are formed by proteins containing small cysteine-rich domains (CRDs) of ~25 amino acids. The first CRD of nematocyst outer all antigen (NW1) and the C-terminal CRD of minicollagen-1 (Mcol1C) contain six cysteines at identical sequence positions, however adopt different disulfide bonded structures. NW1 shows the disulfide connectivities C2-C14/C6-C19/C10-C18 and Mcol1C C2-C18/C6-C14/C10-C19. To analyze if both show structural preferences in the open, non-disulfide bonded form, which explain the formation of either disulfide connectivity pattern, molecular dynamics (MD) simulations at different temperatures were performed. NW1 maintained in the 100-ns MD simulations at 283 K a rather compact fold that is stabilized by specific hydrogen bonds. The Mcol1C structure fluctuated overall more, however stayed most of the time also rather compact. The analysis of the backbone Φ/ψ angles indicated different turn propensities for NW1 and Mcol1C, which mostly can be explained based on published data about the influence of different amino acid side chains on the local backbone conformation. Whereas a folded precursor mechanism may be considered for NW1, Mcol1C may fold according to the quasi-stochastic folding model involving disulfide bond reshuffling and conformational changes, locking the native disulfide conformations. The study further demonstrates the power of MD simulations to detect local structural preferences in rather dynamic systems such as the open, non-disulfide bonded forms of NW1 and Mcol1C, which complement published information from NMR backbone residual dipolar couplings. Because the backbone structural preferences encoded by the amino acid sequence embedding the cysteines influence which disulfide connectivities are formed, the data are generally interesting for a better understanding of oxidative folding and the design of disulfide stabilized therapeutics. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.


Holton S.J.,German Electron Synchrotron | Anandhakrishnan M.,German Electron Synchrotron | Geerlof A.,German Electron Synchrotron | Geerlof A.,Institute of Structural Biology | Wilmanns M.,German Electron Synchrotron
Journal of Structural Biology | Year: 2013

Hydroxyacid dehydrogenases, responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids in lactic acid producing bacteria, have a range of biotechnology applications including antibiotic synthesis, flavor development in dairy products and the production of valuable synthons. The genome of Lactobacillus delbrueckii ssp. bulgaricus, a member of the heterogeneous group of lactic acid bacteria, encodes multiple hydroxyacid dehydrogenases whose structural and functional properties remain poorly characterized. Here, we report the apo and coenzyme NAD+ complexed crystal structures of the L. bulgaricus D-isomer specific 2-hydroxyacid dehydrogenase, D2-HDH. Comparison with closely related members of the NAD-dependent dehydrogenase family reveals that whilst the D2-HDH core fold is structurally conserved, the substrate-binding site has a number of non-canonical features that may influence substrate selection and thus dictate the physiological function of the enzyme. © 2012 Elsevier Inc.

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