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RosettaEPR: rotamer library for spin label structure and dynamics.
Alexander NS, Stein RA, Koteiche HA, Kaufmann KW, McHaourab HS, Meiler J
(2013) PLoS One 8: e72851
MeSH Terms: Bacteriophage T4, Electron Spin Resonance Spectroscopy, Mesylates, Models, Molecular, Molecular Dynamics Simulation, Muramidase, Protein Conformation, Proteins, Reproducibility of Results, Software, Spin Labels
Show Abstract · Added January 24, 2015
An increasingly used parameter in structural biology is the measurement of distances between spin labels bound to a protein. One limitation to these measurements is the unknown position of the spin label relative to the protein backbone. To overcome this drawback, we introduce a rotamer library of the methanethiosulfonate spin label (MTSSL) into the protein modeling program Rosetta. Spin label rotamers were derived from conformations observed in crystal structures of spin labeled T4 lysozyme and previously published molecular dynamics simulations. Rosetta's ability to accurately recover spin label conformations and EPR measured distance distributions was evaluated against 19 experimentally determined MTSSL labeled structures of T4 lysozyme and the membrane protein LeuT and 73 distance distributions from T4 lysozyme and the membrane protein MsbA. For a site in the core of T4 lysozyme, the correct spin label conformation (Χ1 and Χ2) is recovered in 99.8% of trials. In surface positions 53% of the trajectories agree with crystallized conformations in Χ1 and Χ2. This level of recovery is on par with Rosetta performance for the 20 natural amino acids. In addition, Rosetta predicts the distance between two spin labels with a mean error of 4.4 Å. The width of the experimental distance distribution, which reflects the flexibility of the two spin labels, is predicted with a mean error of 1.3 Å. RosettaEPR makes full-atom spin label modeling available to a wide scientific community in conjunction with the powerful suite of modeling methods within Rosetta.
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11 MeSH Terms
DEER EPR measurements for membrane protein structures via bifunctional spin labels and lipodisq nanoparticles.
Sahu ID, McCarrick RM, Troxel KR, Zhang R, Smith HJ, Dunagan MM, Swartz MS, Rajan PV, Kroncke BM, Sanders CR, Lorigan GA
(2013) Biochemistry 52: 6627-32
MeSH Terms: Cyclic N-Oxides, Electron Spin Resonance Spectroscopy, Lipid Bilayers, Membrane Proteins, Mesylates, Nanoparticles, Potassium Channels, Voltage-Gated, Spin Labels
Show Abstract · Added November 21, 2018
Pulsed EPR DEER structural studies of membrane proteins in a lipid bilayer have often been hindered by difficulties in extracting accurate distances when compared to those of globular proteins. In this study, we employed a combination of three recently developed methodologies, (1) bifunctional spin labels (BSL), (2) SMA-Lipodisq nanoparticles, and (3) Q band pulsed EPR measurements, to obtain improved signal sensitivity, increased transverse relaxation time, and more accurate and precise distances in DEER measurements on the integral membrane protein KCNE1. The KCNE1 EPR data indicated an ∼2-fold increase in the transverse relaxation time for the SMA-Lipodisq nanoparticles when compared to those of proteoliposomes and narrower distance distributions for the BSL when compared to those of the standard MTSL. The certainty of information content in DEER data obtained for KCNE1 in SMA-Lipodisq nanoparticles is comparable to that in micelles. The combination of techniques will enable researchers to potentially obtain more precise distances in cases where the traditional spin labels and membrane systems yield imprecise distance distributions.
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MeSH Terms
Structural refinement from restrained-ensemble simulations based on EPR/DEER data: application to T4 lysozyme.
Islam SM, Stein RA, McHaourab HS, Roux B
(2013) J Phys Chem B 117: 4740-54
MeSH Terms: Bacteriophage T4, Crystallography, X-Ray, Cyclic N-Oxides, Electron Spin Resonance Spectroscopy, Electrons, Mesylates, Molecular Dynamics Simulation, Muramidase, Protein Structure, Tertiary, Spin Labels
Show Abstract · Added February 19, 2015
DEER (double electron-electron resonance) is a powerful pulsed ESR (electron spin resonance) technique allowing the determination of distance histograms between pairs of nitroxide spin-labels linked to a protein in a native-like solution environment. However, exploiting the huge amount of information provided by ESR/DEER histograms to refine structural models is extremely challenging. In this study, a restrained ensemble (RE) molecular dynamics (MD) simulation methodology is developed to address this issue. In RE simulation, the spin-spin distance distribution histograms calculated from a multiple-copy MD simulation are enforced, via a global ensemble-based energy restraint, to match those obtained from ESR/DEER experiments. The RE simulation is applied to 51 ESR/DEER distance histogram data from spin-labels inserted at 37 different positions in T4 lysozyme (T4L). The rotamer population distribution along the five dihedral angles connecting the nitroxide ring to the protein backbone is determined and shown to be consistent with available information from X-ray crystallography. For the purpose of structural refinement, the concept of a simplified nitroxide dummy spin-label is designed and parametrized on the basis of these all-atom RE simulations with explicit solvent. It is demonstrated that RE simulations with the dummy nitroxide spin-labels imposing the ESR/DEER experimental distance distribution data are able to systematically correct and refine a series of distorted T4L structures, while simple harmonic distance restraints are unsuccessful. This computationally efficient approach allows experimental restraints from DEER experiments to be incorporated into RE simulations for efficient structural refinement.
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10 MeSH Terms
Effect on ex vivo platelet aggregation and in vivo cyclic flow with Na+/H+ exchange inhibition: Gumina, NHE-1 inhibition and platelet aggregation.
Gumina RJ, Newman PJ, Gross GJ
(2011) J Thromb Thrombolysis 31: 431-5
MeSH Terms: Animals, Benzamides, Blood Flow Velocity, Blood Platelets, Disease Models, Animal, Dogs, Femoral Artery, Humans, Mesylates, Platelet Aggregation, Pyrroles, Sodium-Hydrogen Exchangers
Show Abstract · Added February 21, 2015
BIIB 513 and EMD 85131 are selective inhibitors of the Na+/H+ exchanger-1 (NHE-1) that are benzoylguanidine derivatives of the clinically employed diuretic amiloride. Prior studies have suggested a role for NHE-1 activity in platelet activation and aggregation using amiloride or its non- benzoylguanidines derivatives. However, the concentrations employed in these prior studies were at levels known to exert effects on other ion transport systems besides the NHE-1. Therefore, the purpose of this study was to examine the effects of more selective NHE-1 inhibitors, BIIB 513 and EMD 85131, on platelet aggregation and in vivo cyclic flow following arterial injury. BIIB 513 and EMD 85131 effects on ex vivo canine and human platelet aggregation in response to various agents was monitored via platelet aggregation. For analysis of in vivo thrombus formation, a femoral artery crush injury model was employed and a flow meter was used to monitor the effect of BIIB 513 on cyclic blood flow. Treatment of either canine or human platelets with up to 1 mM of BIIB 513 had no effect on aggregation induced by platelet activating factor (PAF), thrombin receptor activator peptide (TRAP), or adenosine diphosphate (ADP). Additionally, the structurally related compound EMD 85131 at up to 1 mM failed to inhibit TRAP induced platelet aggregation. In vivo administration of up to 9 mg/kg of BIIB 513 intravenously failed to affect cyclic flow in a canine model of femoral artery injury. These data demonstrate that the specific and selective NHE-1 inhibitors BIIB 513 or EMD 85131 have no effect on ex vivo platelet aggregation or in vivo cyclic flow following arterial injury.
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The ClC-3 Cl-/H+ antiporter becomes uncoupled at low extracellular pH.
Matsuda JJ, Filali MS, Collins MM, Volk KA, Lamb FS
(2010) J Biol Chem 285: 2569-79
MeSH Terms: Acids, Animals, Aorta, Cell Compartmentation, Cell Line, Chloride Channels, Chlorides, Glutamic Acid, Green Fluorescent Proteins, Humans, Hydrogen, Hydrogen-Ion Concentration, Kidney, Membrane Potentials, Mesylates, Mice, Mice, Mutant Strains, Muscle, Smooth, Vascular, Mutagenesis, Patch-Clamp Techniques, RNA, Small Interfering, Sulfhydryl Reagents
Show Abstract · Added February 22, 2016
Adenovirus expressing ClC-3 (Ad-ClC-3) induces Cl(-)/H(+) antiport current (I(ClC-3)) in HEK293 cells. The outward rectification and time dependence of I(ClC-3) closely resemble an endogenous HEK293 cell acid-activated Cl(-) current (ICl(acid)) seen at extracellular pH
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22 MeSH Terms
A new multi-gram synthetic route to labeling precursors for the D(2/3) PET agent 18F-fallypride.
Kim K, Miller NR, Sulikowski GA, Lindsley CW
(2008) Bioorg Med Chem Lett 18: 4467-9
MeSH Terms: Benzamides, Chemistry, Pharmaceutical, Chlorine, Dopamine D2 Receptor Antagonists, Drug Design, Mesylates, Models, Chemical, Positron-Emission Tomography, Pyrrolidines, Radiopharmaceuticals, Receptors, Dopamine D2, Receptors, Dopamine D3
Show Abstract · Added March 5, 2014
This Letter describes a new multi-gram synthetic protocol for the preparation of the classic tosylate labeling precursor for the D(2/3) PET agent [(18)F]fallypride. In the course of our studies, we also discovered two novel labeling precusors, the previously undescribed mesylate and chloro congeners of fallypride.
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12 MeSH Terms
Serotonin and cocaine-sensitive inactivation of human serotonin transporters by methanethiosulfonates targeted to transmembrane domain I.
Henry LK, Adkins EM, Han Q, Blakely RD
(2003) J Biol Chem 278: 37052-63
MeSH Terms: Cocaine, HeLa Cells, Humans, Immunoblotting, Mesylates, Receptors, Serotonin, Serotonin, Structure-Activity Relationship
Show Abstract · Added July 10, 2013
To explore aqueous accessibility and functional contributions of transmembrane domain (TM) 1 in human serotonin transporter (hSERT) proteins, we utilized the largely methanethiosulfonate (MTS) insensitive hSERT C109A mutant and mutated individual residues of hSERT TM1 to Cys followed by tests of MTS inactivation of 5-hydroxytryptamine (5-HT) transport. Residues in TM1 cytoplasmic to Gly-94 were largely unaffected by Cys substitution, whereas the mutation of residues extracellular to Ile-93 variably diminished transport activity. TM1 Cys substitutions displayed differential sensitivity to MTS reagents, with residues more cytoplasmic to Asp-98 being largely insensitive to MTS inactivation. Aminoethylmethanethiosulfonate (MTSEA), [2-(trimethylammonium) ethyl]methanethiosulfonate bromide (MTSET), and sodium (2-sulfonatoethyl)-methanethiosulfonate (MTSES) similarly and profoundly inactivated 5-HT transport by SERT mutants D98C, G100C, W103C, and Y107C. MTSEA uniquely inactivated transport activity of S91C, G94C, Y95C but increased activity at I108C. MTSEA and MTSET, but not MTSES, inactivated transport function at N101C. Notably, 5-HT provided partial to complete protection from MTSET inactivation for D98C, G100C, N101C, and Y107C. Equivalent blockade of MTSET inactivation at N101C was observed with 5-HT at both room temperature and at 4 degrees C, inconsistent with major conformational changes leading to protection. Notably, cocaine also protected MTSET inactivation of G100C and N101C, although MTS incubations with N101C that eliminate 5-HT transport do not preclude cocaine analog binding nor its inhibition by 5-HT. 5-HT modestly enhanced the inactivation by MTSET at I93C and Y95C, whereas cocaine significantly enhanced MTSET sensitivity at Y107C and I108C. In summary, our studies reveal physical differences in TM1 accessibility to externally applied MTS reagents and reveal sites supporting substrate and antagonist modulation of MTS inactivation. Moreover, we identify a limit to accessibility for membrane-impermeant MTS reagents that may reflect aspects of an occluded permeation pathway.
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8 MeSH Terms
Na(+)/H(+) exchange inhibition-induced cardioprotection in dogs: effects on neutrophils versus cardiomyocytes.
Gumina RJ, Auchampach J, Wang R, Buerger E, Eickmeier C, Moore J, Daemmgen J, Gross GJ
(2000) Am J Physiol Heart Circ Physiol 279: H1563-70
MeSH Terms: Animals, CD18 Antigens, Collateral Circulation, Coronary Circulation, Dogs, Gases, Heart, Hemodynamics, Mesylates, Myocardial Infarction, Myocardial Ischemia, Myocardial Reperfusion Injury, Myocardium, Neutrophils, Peroxidase, Sodium-Hydrogen Exchangers
Show Abstract · Added February 21, 2015
Numerous studies have examined the effect of Na(+)/H(+) exchanger (NHE) inhibition on the myocardium; however, the effect of NHE-1 inhibition on neutrophil function has not been adequately examined. An in vivo canine model of myocardial ischemia-reperfusion injury in which 60 min of left anterior descending coronary artery occlusion followed by 3 h of reperfusion was used to examine the effect of NHE-1 inhibition on infarct size (IS) and neutrophil function. BIIB-513, a selective inhibitor of NHE-1, was infused before ischemia. IS was expressed as a percentage of area at risk (IS/AAR). NHE-1 inhibition significantly reduced IS/AAR and reduced neutrophil accumulation in the ischemic myocardium. NHE-1 inhibition attenuated both phorbol 12-myristate 13-acetate- and platelet-activating factor-induced neutrophil respiratory burst but not CD18 upregulation. Furthermore, NHE-1 inhibition directly protected cardiomyocytes against metabolic inhibition-induced lactate dehydrogenase release and hypercontracture. This study provides evidence that the cardioprotection induced by NHE-1 inhibition is likely due to specific protection of cardiomyocytes and attenuation of neutrophil activity.
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16 MeSH Terms
Inhibition of the Na(+)/H(+) exchanger confers greater cardioprotection against 90 minutes of myocardial ischemia than ischemic preconditioning in dogs.
Gumina RJ, Buerger E, Eickmeier C, Moore J, Daemmgen J, Gross GJ
(1999) Circulation 100: 2519-26; discussion 2469-72
MeSH Terms: Amiloride, Animals, Dogs, Drug Evaluation, Preclinical, Guanidines, Hemodynamics, Hydrogen, Ion Transport, Ischemic Preconditioning, Myocardial, Mesylates, Muscle Proteins, Myocardial Infarction, Myocardial Reperfusion Injury, Sodium, Sodium-Hydrogen Exchangers, Sulfones
Show Abstract · Added February 21, 2015
BACKGROUND - This study compared the efficacy of ischemic preconditioning (IPC) and sodium-hydrogen exchanger (NHE)-1 inhibition to reduce infarct size (IS) induced by a 90-minute ischemic insult and examined the interaction between NHE-1 inhibition and IPC.
METHODS AND RESULTS - In a canine infarct model, either IPC, produced by 1 or four 5-minute coronary artery occlusions, or the specific NHE-1 inhibitor BIIB 513, 0.75 or 3.0 mg/kg, was administered 15 minutes before either a 60- or 90-minute coronary artery occlusion followed by 3 hours of reperfusion. IS was determined by TTC staining and expressed as a percentage of the area at risk (IS/AAR). Although both IPC and BIIB 513 at 0.75 mg/kg produced comparable and significant reductions in IS/AAR in the 60-minute occlusion model, insignificant reductions in IS/AAR were observed in the 90-minute occlusion model. However, BIIB 513 at 3.0 mg/kg markedly reduced IS in both models (P<0.05). Next, to examine the interaction between NHE-1 blockade and IPC, BIIB 0.75 mg/kg was administered either before IPC or during the washout phase of IPC before 90 minutes of coronary artery occlusion. Both combinations resulted in a greater-than-additive reduction in IS/AAR (P<0.05).
CONCLUSIONS - These data demonstrate that although IPC and NHE-1 inhibition provide comparable protection against 60 minutes of myocardial ischemia, NHE-1 inhibition is more efficacious than IPC at protecting against a 90-minute ischemic insult. Furthermore, the combination of NHE-1 inhibition and IPC produces a greater-than-additive reduction in IS/AAR, suggesting either that NHE activity limits the efficacy of IPC or that different mechanisms are involved in the cardioprotective effect of IPC and NHE-1 inhibition.
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The bis(naphthalimide) DMP-840 causes cytotoxicity by its action against eukaryotic topoisomerase II.
Nitiss JL, Zhou J, Rose A, Hsiung Y, Gale KC, Osheroff N
(1998) Biochemistry 37: 3078-85
MeSH Terms: Adenine, Alleles, Antineoplastic Agents, DNA Topoisomerases, Type II, Drug Resistance, Microbial, Humans, Imides, Isoquinolines, Mesylates, Naphthalimides, Organophosphonates, Saccharomyces cerevisiae
Show Abstract · Added March 5, 2014
DMP 840 ((R,R)-2,2'-[1,2-ethanediylbis[imino(1-methyl-2, 1-ethanediyl)]-bis(5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione] dimethanesulfonate) is a novel bis(naphthalimide) that has shown promising antitumor activity in a variety of preclinical model systems. The compound binds to DNA with high affinity and intercalates, but the mechanism of cell killing has not been elucidated. We have used yeast strains to test whether DMP-840 is active against either topoisomerase I or II. We found that temperature-sensitive top2 mutants resistant to etoposide or amsacrine also confer resistance to DMP-840. In addition, cells overexpressing yeast topoisomerase II were hypersensitive to the drug. By contrast, top1 deletions rendered cells hypersensitive to the drug. These results strongly suggest that DMP-840 acts against eukaryotic topoisomerase II and kills cells by converting the enzyme into a cellular poison. We verified that DMP-840 is active against eukaryotic topoisomerase II by demonstrating that the drug stimulates formation of a cleavage complex with purified yeast topoisomerase II in vitro. We also demonstrated that the drug is active against human topoisomerase II by showing that expression of human topoisomerase II restored sensitivity of resistant yeast cells to DMP-840. We have also directly demonstrated that DMP-840 acts as a poison against purified human topoisomerase II alpha. Taken together, these results indicate that DMP-840 acts like other intercalating topoisomerase II poisons; it kills eukaryotic cells by stabilizing the cleavage complex of topoisomerase II with DNA.
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12 MeSH Terms