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Results: 21 to 30 of 452

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The homology model of PMP22 suggests mutations resulting in peripheral neuropathy disrupt transmembrane helix packing.
Mittendorf KF, Kroncke BM, Meiler J, Sanders CR
(2014) Biochemistry 53: 6139-41
MeSH Terms: Amino Acid Sequence, Claudins, Crystallography, X-Ray, Models, Molecular, Molecular Sequence Data, Mutation, Myelin Proteins, Peripheral Nervous System Diseases, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, Sequence Homology, Amino Acid
Show Abstract · Added January 20, 2015
Peripheral myelin protein 22 (PMP22) is a tetraspan membrane protein strongly expressed in myelinating Schwann cells of the peripheral nervous system. Myriad missense mutations in PMP22 result in varying degrees of peripheral neuropathy. We used Rosetta 3.5 to generate a homology model of PMP22 based on the recently published crystal structure of claudin-15. The model suggests that several mutations known to result in neuropathy act by disrupting transmembrane helix packing interactions. Our model also supports suggestions from previous studies that the first transmembrane helix is not tightly associated with the rest of the helical bundle.
1 Communities
3 Members
0 Resources
12 MeSH Terms
Structural investigation of the transmembrane domain of KCNE1 in proteoliposomes.
Sahu ID, Kroncke BM, Zhang R, Dunagan MM, Smith HJ, Craig A, McCarrick RM, Sanders CR, Lorigan GA
(2014) Biochemistry 53: 6392-401
MeSH Terms: Amino Acid Substitution, Humans, Lipid Bilayers, Liposomes, Molecular Dynamics Simulation, Phosphatidylcholines, Phosphatidylglycerols, Potassium Channels, Voltage-Gated, Protein Structure, Secondary, Protein Structure, Tertiary, Thermodynamics
Show Abstract · Added November 21, 2018
KCNE1 is a single-transmembrane protein of the KCNE family that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in KCNE1 have been linked to diseases such as long QT syndrome (LQTS), atrial fibrillation, sudden infant death syndrome, and deafness. The transmembrane domain (TMD) of KCNE1 plays a key role in mediating the physical association with KCNQ1 and in subsequent modulation of channel gating kinetics and conductance. However, the mechanisms associated with these roles for the TMD remain poorly understood, highlighting a need for experimental structural studies. A previous solution NMR study of KCNE1 in LMPG micelles revealed a curved transmembrane domain, a structural feature proposed to be critical to KCNE1 function. However, this curvature potentially reflects an artifact of working in detergent micelles. Double electron electron resonance (DEER) measurements were conducted on KCNE1 in LMPG micelles, POPC/POPG proteoliposomes, and POPC/POPG lipodisq nanoparticles to directly compare the structure of the TMD in a variety of different membrane environments. Experimentally derived DEER distances coupled with simulated annealing molecular dynamic simulations were used to probe the bilayer structure of the TMD of KCNE1. The results indicate that the structure is helical in proteoliposomes and is slightly curved, which is consistent with the previously determined solution NMR structure in micelles. The evident resilience of the curvature in the KCNE1 TMD leads us to hypothesize that the curvature is likely to be maintained upon binding of the protein to the KCNQ1 channel.
0 Communities
1 Members
0 Resources
MeSH Terms
Kinetic analysis of lauric acid hydroxylation by human cytochrome P450 4A11.
Kim D, Cha GS, Nagy LD, Yun CH, Guengerich FP
(2014) Biochemistry 53: 6161-72
MeSH Terms: Algorithms, Binding, Competitive, Biocatalysis, Cytochrome P-450 CYP4A, Cytochrome P-450 Enzyme System, Cytochromes b5, Deuterium, Electron Transport, Ferric Compounds, Ferrous Compounds, Humans, Hydroxylation, Kinetics, Lauric Acids, Models, Chemical, Models, Molecular, Oxidation-Reduction, Protein Binding, Protein Structure, Tertiary, Substrate Specificity, Tritium
Show Abstract · Added January 20, 2015
Cytochrome P450 (P450) 4A11 is the only functionally active subfamily 4A P450 in humans. P450 4A11 catalyzes mainly ω-hydroxylation of fatty acids in liver and kidney; this process is not a major degradative pathway, but at least one product, 20-hydroxyeicosatetraenoic acid, has important signaling properties. We studied catalysis by P450 4A11 and the issue of rate-limiting steps using lauric acid ω-hydroxylation, a prototypic substrate for this enzyme. Some individual reaction steps were studied using pre-steady-state kinetic approaches. Substrate and product binding and release were much faster than overall rates of catalysis. Reduction of ferric P450 4A11 (to ferrous) was rapid and not rate-limiting. Deuterium kinetic isotope effect (KIE) experiments yielded low but reproducible values (1.2-2) for 12-hydroxylation with 12-(2)H-substituted lauric acid. However, considerable "metabolic switching" to 11-hydroxylation was observed with [12-(2)H3]lauric acid. Analysis of switching results [Jones, J. P., et al. (1986) J. Am. Chem. Soc. 108, 7074-7078] and the use of tritium KIE analysis with [12-(3)H]lauric acid [Northrop, D. B. (1987) Methods Enzymol. 87, 607-625] both indicated a high intrinsic KIE (>10). Cytochrome b5 (b5) stimulated steady-state lauric acid ω-hydroxylation ∼2-fold; the apoprotein was ineffective, indicating that electron transfer is involved in the b5 enhancement. The rate of b5 reoxidation was increased in the presence of ferrous P450 mixed with O2. Collectively, the results indicate that both the transfer of an electron to the ferrous·O2 complex and C-H bond-breaking limit the rate of P450 4A11 ω-oxidation.
0 Communities
1 Members
0 Resources
21 MeSH Terms
Structural analysis of replication protein A recruitment of the DNA damage response protein SMARCAL1.
Feldkamp MD, Mason AC, Eichman BF, Chazin WJ
(2014) Biochemistry 53: 3052-61
MeSH Terms: Amino Acid Sequence, Crystallography, X-Ray, DNA Helicases, Models, Molecular, Replication Protein A, Sequence Alignment
Show Abstract · Added May 19, 2014
SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like1 (SMARCAL1) is a recently identified DNA damage response protein involved in remodeling stalled replication forks. The eukaryotic single-strand DNA binding protein replication protein A (RPA) recruits SMARCAL1 to stalled forks in vivo and facilitates regression of forks containing leading strand gaps. Both activities are mediated by a direct interaction between an RPA binding motif (RBM) at the N-terminus of SMARCAL1 and the C-terminal winged-helix domain of the RPA 32 kDa subunit (RPA32C). Here we report a biophysical and structural characterization of the SMARCAL1-RPA interaction. Isothermal titration calorimetry and circular dichroism spectroscopy revealed that RPA32C binds SMARCAL1-RBM with a Kd of 2.5 μM and induces a disorder-to-helix transition. The crystal structure of RPA32C was refined to 1.4 Å resolution, and the SMARCAL1-RBM binding site was mapped on the structure on the basis of nuclear magnetic resonance chemical shift perturbations. Conservation of the interaction surface to other RBM-containing proteins allowed construction of a model for the RPA32C/SMARCAL1-RBM complex. The implications of our results are discussed with respect to the recruitment of SMARCAL1 and other DNA damage response and repair proteins to stalled replication forks.
1 Communities
2 Members
0 Resources
6 MeSH Terms
Levuglandin forms adducts with histone h4 in a cyclooxygenase-2-dependent manner, altering its interaction with DNA.
Carrier EJ, Zagol-Ikapitte I, Amarnath V, Boutaud O, Oates JA
(2014) Biochemistry 53: 2436-41
MeSH Terms: Chromatography, Liquid, Cyclooxygenase 2, DNA, Histones, Mass Spectrometry, Prostaglandin D2, Prostaglandins E, Solubility
Show Abstract · Added January 22, 2015
Inflammation and subsequent cyclooxygenase-2 (COX-2) activity has long been linked with the development of cancer, although little is known about any epigenetic effects of COX-2. A product of COX-2 activation, levuglandin (LG) quickly forms covalent bonds with nearby primary amines, such as those in lysine, which leads to LG-protein adducts. Here, we demonstrate that COX-2 activity causes LG-histone adducts in cultured cells and liver tissue, detectable through LC-MS, with the highest incidence in histone H4. Adduction is blocked by a γ-ketoaldehyde scavenger, which has no effect on COX-2 activity as measured by PGE2 production. Formation of the LG-histone adduct is associated with an increased histone solubility in NaCl, indicating destabilization of the nucleosome structure; this is also reversed with scavenger treatment. These data demonstrate that COX-2 activity can cause histone adduction and loosening of the nucleosome complex, which could lead to altered transcription and contribute to carcinogenesis.
0 Communities
2 Members
0 Resources
8 MeSH Terms
5-methylcytosine recognition by Arabidopsis thaliana DNA glycosylases DEMETER and DML3.
Brooks SC, Fischer RL, Huh JH, Eichman BF
(2014) Biochemistry 53: 2525-32
MeSH Terms: 5-Methylcytosine, Arabidopsis, Arabidopsis Proteins, Base Sequence, DNA Glycosylases, DNA Primers, Models, Molecular, Nuclear Proteins, Polymerase Chain Reaction, Substrate Specificity
Show Abstract · Added May 27, 2014
Methylation of cytosine to 5-methylcytosine (5mC) is important for gene expression, gene imprinting, X-chromosome inactivation, and transposon silencing. Active demethylation in animals is believed to proceed by DNA glycosylase removal of deaminated or oxidized 5mC. In plants, 5mC is removed from the genome directly by the DEMETER (DME) family of DNA glycosylases. Arabidopsis thaliana DME excises 5mC to activate expression of maternally imprinted genes. Although the related Repressor of Silencing 1 (ROS1) enzyme has been characterized, the molecular basis for 5mC recognition by DME has not been investigated. Here, we present a structure-function analysis of DME and the related DME-like 3 (DML3) glycosylases for 5mC and its oxidized derivatives. Relative to 5mC, DME and DML3 exhibited robust activity toward 5-hydroxymethylcytosine, limited activity for 5-carboxylcytosine, and no activity for 5-formylcytosine. We used homology modeling and mutational analysis of base excision and DNA binding to identify residues important for recognition of 5mC within the context of DNA and inside the enzyme active site. Our results indicate that the 5mC binding pocket is composed of residues from discrete domains and is responsible for discrimination against 5mC derivatives, and suggest that DME, ROS1, and DML3 utilize subtly different mechanisms to probe the DNA duplex for cytosine modifications.
1 Communities
1 Members
0 Resources
10 MeSH Terms
Fiber diffraction of the prion-forming domain HET-s(218-289) shows dehydration-induced deformation of a complex amyloid structure.
Wan W, Stubbs G
(2014) Biochemistry 53: 2366-70
MeSH Terms: Amyloid, Models, Molecular, Molecular Structure, Prions, Water, X-Ray Diffraction
Show Abstract · Added February 15, 2016
Amyloids are filamentous protein aggregates that can be formed by many different proteins and are associated with both disease and biological functions. The pathogenicities or biological functions of amyloids are determined by their particular molecular structures, making accurate structural models a requirement for understanding their biological effects. One potential factor that can affect amyloid structures is hydration. Previous studies of simple stacked β-sheet amyloids have suggested that dehydration does not impact structure, but other studies indicated dehydration-related structural changes of a putative water-filled nanotube. Our results show that dehydration significantly affects the molecular structure of the fungal prion-forming domain HET-s(218-289), which forms a β-solenoid with no internal solvent-accessible regions. The dehydration-related structural deformation of HET-s(218-289) indicates that water can play a significant role in complex amyloid structures, even when no obvious water-accessible cavities are present.
0 Communities
1 Members
0 Resources
6 MeSH Terms
Purification and structural study of the voltage-sensor domain of the human KCNQ1 potassium ion channel.
Peng D, Kim JH, Kroncke BM, Law CL, Xia Y, Droege KD, Van Horn WD, Vanoye CG, Sanders CR
(2014) Biochemistry 53: 2032-42
MeSH Terms: Amino Acid Sequence, Deuterium Exchange Measurement, Humans, KCNQ1 Potassium Channel, Molecular Sequence Data, Protein Structure, Tertiary, Structure-Activity Relationship
Show Abstract · Added November 21, 2018
KCNQ1 (also known as KV7.1 or KVLQT1) is a voltage-gated potassium channel modulated by members of the KCNE protein family. Among multiple functions, KCNQ1 plays a critical role in the cardiac action potential. This channel is also subject to inherited mutations that cause certain cardiac arrhythmias and deafness. In this study, we report the overexpression, purification, and preliminary structural characterization of the voltage-sensor domain (VSD) of human KCNQ1 (Q1-VSD). Q1-VSD was expressed in Escherichia coli and purified into lyso-palmitoylphosphatidylglycerol micelles, conditions under which this tetraspan membrane protein yields excellent nuclear magnetic resonance (NMR) spectra. NMR studies reveal that Q1-VSD shares a common overall topology with other channel VSDs, with an S0 helix followed by transmembrane helices S1-S4. The exact sequential locations of the helical spans do, however, show significant variations from those of the homologous segments of previously characterized VSDs. The S4 segment of Q1-VSD was seen to be α-helical (with no 310 component) and underwent rapid backbone amide H-D exchange over most of its length. These results lay the foundation for more advanced structural studies and can be used to generate testable hypotheses for future structure-function experiments.
0 Communities
2 Members
0 Resources
MeSH Terms
Mechanism of quinolone action and resistance.
Aldred KJ, Kerns RJ, Osheroff N
(2014) Biochemistry 53: 1565-74
MeSH Terms: Animals, Anti-Bacterial Agents, Bacteria, Bacterial Infections, Bacterial Proteins, Drug Resistance, Bacterial, Humans, Quinolones
Show Abstract · Added March 13, 2014
Quinolones are one of the most commonly prescribed classes of antibacterials in the world and are used to treat a variety of bacterial infections in humans. Because of the wide use (and overuse) of these drugs, the number of quinolone-resistant bacterial strains has been growing steadily since the 1990s. As is the case with other antibacterial agents, the rise in quinolone resistance threatens the clinical utility of this important drug class. Quinolones act by converting their targets, gyrase and topoisomerase IV, into toxic enzymes that fragment the bacterial chromosome. This review describes the development of the quinolones as antibacterials, the structure and function of gyrase and topoisomerase IV, and the mechanistic basis for quinolone action against their enzyme targets. It will then discuss the following three mechanisms that decrease the sensitivity of bacterial cells to quinolones. Target-mediated resistance is the most common and clinically significant form of resistance. It is caused by specific mutations in gyrase and topoisomerase IV that weaken interactions between quinolones and these enzymes. Plasmid-mediated resistance results from extrachromosomal elements that encode proteins that disrupt quinolone-enzyme interactions, alter drug metabolism, or increase quinolone efflux. Chromosome-mediated resistance results from the underexpression of porins or the overexpression of cellular efflux pumps, both of which decrease cellular concentrations of quinolones. Finally, this review will discuss recent advancements in our understanding of how quinolones interact with gyrase and topoisomerase IV and how mutations in these enzymes cause resistance. These last findings suggest approaches to designing new drugs that display improved activity against resistant strains.
0 Communities
1 Members
0 Resources
8 MeSH Terms
Differential localization of G protein βγ subunits.
Betke KM, Rose KL, Friedman DB, Baucum AJ, Hyde K, Schey KL, Hamm HE
(2014) Biochemistry 53: 2329-43
MeSH Terms: Amino Acid Sequence, Animals, Chromatography, Liquid, GTP-Binding Protein beta Subunits, GTP-Binding Protein gamma Subunits, Male, Mice, Mice, Inbred C57BL, Molecular Sequence Data, Protein Isoforms, Signal Transduction, Subcellular Fractions, Synaptosomes, Tandem Mass Spectrometry
Show Abstract · Added May 27, 2014
G protein βγ subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both Gβ and Gγ isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics approach known as multiple-reaction monitoring to analyze localization patterns of Gβ and Gγ isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular Gβ and Gγ subunits were found to exhibit distinct regional and subcellular localization patterns throughout the brain. Significant differences in subcellular localization between pre- and postsynaptic fractions were observed within the striatum for most Gβ and Gγ isoforms, while others exhibited completely unique expression patterns in all four brain regions examined. Such differences are a prerequisite for understanding roles of individual subunits in regulating specific signaling pathways throughout the central nervous system.
0 Communities
2 Members
0 Resources
14 MeSH Terms