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Reagent cluster anions for multiple gas-phase covalent modifications of peptide and protein cations.
Prentice BM, Stutzman JR, McLuckey SA
(2013) J Am Soc Mass Spectrom 24: 1045-52
MeSH Terms: Acetates, Amino Acid Sequence, Animals, Cations, Cattle, Gases, Indicators and Reagents, Mass Spectrometry, Molecular Sequence Data, Peptide Fragments, Peptides, Proteins, Schiff Bases, Succinimides, Ubiquitin
Show Abstract · Added August 17, 2016
Multiple gas phase ion/ion covalent modifications of peptide and protein ions are demonstrated using cluster-type reagent anions of N-hydroxysulfosuccinimide acetate (sulfo-NHS acetate) and 2-formyl-benzenesulfonic acid (FBMSA). These reagents are used to selectively modify unprotonated primary amine functionalities of peptides and proteins. Multiple reactive reagent molecules can be present in a single cluster ion, which allows for multiple covalent modifications to be achieved in a single ion/ion encounter and at the 'cost' of only a single analyte charge. Multiple derivatizations are demonstrated when the number of available reactive sites on the analyte cation exceeds the number of reagent molecules in the anionic cluster (e.g., data shown here for reactions between the polypeptide [K10 + 3H](3+) and the reagent cluster [5R(5Na) - Na](-)). This type of gas-phase ion chemistry is also applicable to whole protein ions. Here, ubiquitin was successfully modified using an FBMSA cluster anion which, upon collisional activation, produced fragment ions with various numbers of modifications. Data for the pentamer cluster are included as illustrative of the results obtained for the clusters comprised of two to six reagent molecules.
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15 MeSH Terms
Insights into congenital stationary night blindness based on the structure of G90D rhodopsin.
Singhal A, Ostermaier MK, Vishnivetskiy SA, Panneels V, Homan KT, Tesmer JJ, Veprintsev D, Deupi X, Gurevich VV, Schertler GF, Standfuss J
(2013) EMBO Rep 14: 520-6
MeSH Terms: Arrestin, Crystallography, X-Ray, Eye Diseases, Hereditary, Genetic Diseases, X-Linked, HEK293 Cells, Humans, Models, Molecular, Mutation, Missense, Myopia, Night Blindness, Protein Binding, Protein Stability, Protein Structure, Secondary, Protein Structure, Tertiary, Rhodopsin, Schiff Bases, Structural Homology, Protein, Transition Temperature
Show Abstract · Added February 12, 2015
We present active-state structures of the G protein-coupled receptor (GPCRs) rhodopsin carrying the disease-causing mutation G90D. Mutations of G90 cause either retinitis pigmentosa (RP) or congenital stationary night blindness (CSNB), a milder, non-progressive form of RP. Our analysis shows that the CSNB-causing G90D mutation introduces a salt bridge with K296. The mutant thus interferes with the E113Q-K296 activation switch and the covalent binding of the inverse agonist 11-cis-retinal, two interactions that are crucial for the deactivation of rhodopsin. Other mutations, including G90V causing RP, cannot promote similar interactions. We discuss our findings in context of a model in which CSNB is caused by constitutive activation of the visual signalling cascade.
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18 MeSH Terms
The mouse ortholog of NEIL3 is a functional DNA glycosylase in vitro and in vivo.
Liu M, Bandaru V, Bond JP, Jaruga P, Zhao X, Christov PP, Burrows CJ, Rizzo CJ, Dizdaroglu M, Wallace SS
(2010) Proc Natl Acad Sci U S A 107: 4925-30
MeSH Terms: Amino Acid Sequence, Animals, DNA, DNA Damage, DNA Glycosylases, Endodeoxyribonucleases, Escherichia coli, Gamma Rays, Guanidines, Guanosine, Hydantoins, Kinetics, Mice, Molecular Sequence Data, Mutation, Pyrimidines, Schiff Bases, Sequence Alignment, Sequence Homology, Amino Acid, Spiro Compounds, Substrate Specificity, Valine
Show Abstract · Added January 7, 2016
To protect cells from oxidative DNA damage and mutagenesis, organisms possess multiple glycosylases to recognize the damaged bases and to initiate the Base Excision Repair pathway. Three DNA glycosylases have been identified in mammals that are homologous to the Escherichia coli Fpg and Nei proteins, Neil1, Neil2, and Neil3. Neil1 and Neil2 in human and mouse have been well characterized while the properties of the Neil3 protein remain to be elucidated. In this study, we report the characterization of Mus musculus (house mouse) Neil3 (MmuNeil3) as an active DNA glycosylase both in vitro and in vivo. In duplex DNA, MmuNeil3 recognizes the oxidized purines, spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino- 5-formamidopyrimidine (FapyA), but not 8-oxo-7,8-dihydroguanine (8-oxoG). Interestingly, MmuNeil3 prefers lesions in single-stranded DNA and in bubble structures. In contrast to other members of the family that use the N-terminal proline as the nucleophile, MmuNeil3 forms a Schiff base intermediate via its N-terminal valine. We expressed the glycosylase domain of MmuNeil3 (MmuNeil3Delta324) in an Escherichia coli triple mutant lacking Fpg, Nei, and MutY glycosylase activities and showed that MmuNeil3 greatly reduced both the spontaneous mutation frequency and the level of FapyG in the DNA, suggesting that Neil3 plays a role in repairing FapyG in vivo.
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22 MeSH Terms
Covalent binding of isoketals to ethanolamine phospholipids.
Bernoud-Hubac N, Fay LB, Armarnath V, Guichardant M, Bacot S, Davies SS, Roberts LJ, Lagarde M
(2004) Free Radic Biol Med 37: 1604-11
MeSH Terms: Aldehydes, Arachidonic Acid, Chromatography, Liquid, Cross-Linking Reagents, Free Radicals, Mass Spectrometry, Phosphatidylethanolamines, Schiff Bases
Show Abstract · Added December 10, 2013
Free radicals have been strongly implicated in the pathogenesis of many human diseases. We previously identified the formation of highly reactive gamma-ketoaldehydes, isoketals, in vivo as products of free radical-induced peroxidation of arachidonic acid. Isoketals react with lysine residues on proteins at a rate that far exceeds that of 4-hydroxynonenal and demonstrate a unique proclivity to crosslink proteins. Hydroxynonenal has been shown to react with aminophospholipids, particularly phosphatidylethanolamine. We explored whether isoketals also react with phosphatidylethanolamine. Using liquid chromatography/electrospray mass spectrometry, we found that isoketals form pyrrole and Schiff base adducts with phosphatidylethanolamine. In addition, the ability of isoketals to covalently modify phosphatidylethanolamine is greater than that of 4-hydroxynonenal. These studies identify in vitro novel isoketal adducts. This provides the basis to explore the formation of isoketal-aminophospholipid adducts in vivo and the biological consequences of the formation of these adducts.
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8 MeSH Terms
Characterization of bis(levuglandinyl) urea derivatives as products of the reaction between prostaglandin H2 and arginine.
Zagol-Ikapitte I, Bernoud-Hubac N, Amarnath V, Roberts LJ, Boutaud O, Oates JA
(2004) Biochemistry 43: 5503-10
MeSH Terms: Aldehydes, Amyloid beta-Peptides, Arginine, Guanidine, Histones, Humans, Lysine, Ornithine, Peptide Fragments, Prostaglandin H2, Prostaglandins E, Schiff Bases, Serum Albumin, Spectrometry, Mass, Electrospray Ionization, Urea
Show Abstract · Added December 10, 2013
Levuglandins are gamma-keto aldehydes formed by rearrangement of prostaglandin (PG) H(2) in aqueous solution. Levuglandins are highly reactive with primary amines. We had previously characterized adducts formed after reaction of levuglandin E(2) (LGE(2)) or PGH(2) with lysine. In this study, we assessed whether reaction of PGH(2) with arginine yielded covalent adducts. Using N(alpha)-acetylarginine and both PGH(2) and synthetic LGE(2), we discovered a novel series of levuglandinyl adducts derived from reaction of two levuglandin moieties with the guanidino group of arginine. Subsequent spontaneous hydrolysis of the adducted amino acid yields bis(levuglandinyl) urea and the corresponding ornithine residue. Using liquid chromatography tandem mass spectrometry, we characterized the molecular structure of these novel adducts and demonstrated their formation after coincubation of PGH(2) with synthetic peptides and proteins. The soluble characteristic of these molecules provides a potential strategy for development of biological markers of lipid modification of proteins following cyclooxygenase activity or lipid peroxidation.
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3 Members
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15 MeSH Terms
Modification of proteins by isoketal-containing oxidized phospholipids.
Brame CJ, Boutaud O, Davies SS, Yang T, Oates JA, Roden D, Roberts LJ
(2004) J Biol Chem 279: 13447-51
MeSH Terms: Animals, Carbon Tetrachloride, Esters, Free Radicals, Isoprostanes, Lipid Peroxidation, Male, Membrane Proteins, Myocardium, Oxidative Stress, Phospholipids, Potassium Channels, Rats, Rats, Sprague-Dawley, Schiff Bases
Show Abstract · Added December 10, 2013
Oxidative stress frequently leads to altered function of membrane proteins. Isoketals are highly reactive products of the isoprostane pathway of free radical-induced lipid peroxidation that rapidly form covalent protein adducts and exhibit a remarkable proclivity to form protein cross links in vitro. Examination of isoketal adducts from an animal model of oxidative injury revealed that initial adducts were formed by isoketals esterified in phospholipids, representing a novel oxidative injury-associated modification of proteins by phospholipids. Maturation of adducts involved cleavage from phospholipids and conversion of adducts to a more stable chemical form that can be detected for extended periods. Because initial adducts were formed by phospholipid-esterified isoketals, the functional consequence of isoketal adduction was examined using a model membrane protein (a cardiac K(+) channel). These studies revealed that isoketal adduction profoundly altered protein function, inhibiting potassium current in a dose-dependent manner. These findings indicate that phospholipid-esterified isoketals rapidly adduct membrane proteins and that such modification can alter protein function, suggesting a generalized cellular mechanism for alteration of membrane function as a consequence of oxidative stress.
1 Communities
4 Members
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15 MeSH Terms
Identification of oxidized derivatives of neuroketals.
Bernoud-Hubac N, Roberts LJ
(2002) Biochemistry 41: 11466-71
MeSH Terms: Aldehydes, Humans, Hydroxylation, Ketones, Molecular Structure, Neurodegenerative Diseases, Oxidation-Reduction, Oxidative Stress, Schiff Bases, Spectrometry, Mass, Electrospray Ionization, Structure-Activity Relationship
Show Abstract · Added December 10, 2013
Oxidative stress and protein aggregation have been implicated in the pathogenesis of neurodegenerative diseases. The formation of neuroprostanes, isoprostane-like compounds formed from oxidation of docosahexaenoic acid, which is uniquely enriched in the brain, is increased in Alzheimer's disease. We recently identified the formation of a new class of highly reactive gamma-keto aldehydes, neuroketals, in vivo as products of the neuroprostane pathway. Neuroketals adduct to lysine residues of proteins with remarkable rapidity and induce cross-linking. Because neuroketals have either a 1,4-pentadiene or 1,4,7-octatriene side chain structure, we hypothesized that they could undergo further oxidation to form neuroketals with an additional hydroxyl group. Oxidation of docosahexaenoic acid in vitro yielded a series of compounds that were confirmed to be oxidized neuroketals by mass spectrometric analyses. Analysis of oxidized neuroketal adducts during oxidation of docosahexaenoic acid in the presence of lysine revealed the formation of oxidized Schiff base and hydroxylactam adducts. Oxidized hydroxylactam neuroketal-lysyl protein adducts, analyzed after digestion of proteins to individual amino acids, were not detected in nonoxidized rat brain synaptosomes but were readily detected following oxidation of synaptosomes. These studies indicate that neuroketals can undergo further oxidation, which in turn suggests that measurement of only unoxidized neuroketal adducts likely underestimates the amount of neuroketal adducts present in the brain in disorders of oxidant stress.
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11 MeSH Terms
Crystal structure of the D85S mutant of bacteriorhodopsin: model of an O-like photocycle intermediate.
Rouhani S, Cartailler JP, Facciotti MT, Walian P, Needleman R, Lanyi JK, Glaeser RM, Luecke H
(2001) J Mol Biol 313: 615-28
MeSH Terms: Amino Acid Substitution, Bacterial Proteins, Bacteriorhodopsins, Binding Sites, Crystallography, X-Ray, Cytoplasm, Halobacterium, Hydrogen Bonding, Isomerism, Models, Molecular, Protein Structure, Secondary, Retinaldehyde, Schiff Bases
Show Abstract · Added April 15, 2010
Crystal structures are reported for the D85S and D85S/F219L mutants of the light-driven proton/hydroxyl-pump bacteriorhodopsin. These mutants crystallize in the orthorhombic C222(1) spacegroup, and provide the first demonstration that monoolein-based cubic lipid phase crystallization can support the growth of well-diffracting crystals in non-hexagonal spacegroups. Both structures exhibit similar and substantial differences relative to wild-type bacteriorhodopsin, suggesting that they represent inherent features resulting from neutralization of the Schiff base counterion Asp85. We argue that these structures provide a model for the last photocycle intermediate (O) of bacteriorhodopsin, in which Asp85 is protonated, the proton release group is deprotonated, and the retinal has reisomerized to all-trans. Unlike for the M and N photointermediates, where structural changes occur mainly on the cytoplasmic side, here the large-scale changes are confined to the extracellular side. As in the M intermediate, the side-chain of Arg82 is in a downward configuration, and in addition, a pi-cloud hydrogen bond forms between Trp189 NE1 and Trp138. On the cytoplasmic side, there is increased hydration near the surface, suggesting how Asp96 might communicate with the bulk during the rise of the O intermediate.
Copyright 2001 Academic Press.
1 Communities
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13 MeSH Terms
Coupling photoisomerization of retinal to directional transport in bacteriorhodopsin.
Luecke H, Schobert B, Cartailler JP, Richter HT, Rosengarth A, Needleman R, Lanyi JK
(2000) J Mol Biol 300: 1237-55
MeSH Terms: Amino Acid Substitution, Bacteriorhodopsins, Crystallography, X-Ray, Cytoplasm, Hydrogen Bonding, Ion Transport, Isomerism, Light, Membrane Proteins, Models, Molecular, Molecular Sequence Data, Mutation, Protein Structure, Secondary, Protons, Retinaldehyde, Schiff Bases, Static Electricity, Structure-Activity Relationship, Water
Show Abstract · Added April 15, 2010
In order to understand how isomerization of the retinal drives unidirectional transmembrane ion transport in bacteriorhodopsin, we determined the atomic structures of the BR state and M photointermediate of the E204Q mutant, to 1.7 and 1.8 A resolution, respectively. Comparison of this M, in which proton release to the extracellular surface is blocked, with the previously determined M in the D96N mutant indicates that the changes in the extracellular region are initiated by changes in the electrostatic interactions of the retinal Schiff base with Asp85 and Asp212, but those on the cytoplasmic side originate from steric conflict of the 13-methyl retinal group with Trp182 and distortion of the pi-bulge of helix G. The structural changes suggest that protonation of Asp85 initiates a cascade of atomic displacements in the extracellular region that cause release of a proton to the surface. The progressive relaxation of the strained 13-cis retinal chain with deprotonated Schiff base, in turn, initiates atomic displacements in the cytoplasmic region that cause the intercalation of a hydrogen-bonded water molecule between Thr46 and Asp96. This accounts for the lowering of the pK(a) of Asp96, which then reprotonates the Schiff base via a newly formed chain of water molecules that is extending toward the Schiff base.
Copyright 2000 Academic Press.
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19 MeSH Terms
Structural changes in bacteriorhodopsin during ion transport at 2 angstrom resolution.
Luecke H, Schobert B, Richter HT, Cartailler JP, Lanyi JK
(1999) Science 286: 255-61
MeSH Terms: Bacteriorhodopsins, Binding Sites, Crystallography, X-Ray, Cytoplasm, Hydrogen Bonding, Hydrogen-Ion Concentration, Ion Transport, Isomerism, Light, Models, Molecular, Photolysis, Photons, Point Mutation, Protein Conformation, Protein Structure, Secondary, Proton Pumps, Protons, Retinaldehyde, Schiff Bases, Thermodynamics, Water
Show Abstract · Added May 7, 2010
Crystal structures of the Asp96 to Asn mutant of the light-driven proton pump bacteriorhodopsin and its M photointermediate produced by illumination at ambient temperature have been determined to 1.8 and 2.0 angstroms resolution, respectively. The trapped photoproduct corresponds to the late M state in the transport cycle-that is, after proton transfer to Asp85 and release of a proton to the extracellular membrane surface, but before reprotonation of the deprotonated retinal Schiff base. Its density map describes displacements of side chains near the retinal induced by its photoisomerization to 13-cis,15-anti and an extensive rearrangement of the three-dimensional network of hydrogen-bonded residues and bound water that accounts for the changed pKa values (where Ka is the acid constant) of the Schiff base and Asp85. The structural changes detected suggest the means for conserving energy at the active site and for ensuring the directionality of proton translocation.
1 Communities
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21 MeSH Terms