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Functional and structural similarity of human DNA primase [4Fe4S] cluster domain constructs.
Holt ME, Salay LE, O'Brien E, Barton JK, Chazin WJ
(2018) PLoS One 13: e0209345
MeSH Terms: Binding Sites, Circular Dichroism, Crystallography, X-Ray, DNA, DNA Primase, Molecular Docking Simulation, Nuclear Magnetic Resonance, Biomolecular, Oxidation-Reduction, Protein Binding, Protein Domains, Protein Structure, Secondary, RNA
Show Abstract · Added March 26, 2019
The regulatory subunit of human DNA primase has a C-terminal domain (p58C) that contains a [4Fe4S] cluster and binds DNA. Previous electrochemical analysis of a p58C construct revealed that its affinity for DNA is sensitive to the redox state of the [4Fe4S] cluster. Concerns about the validity of this conclusion have been raised, based in part on differences in X-ray crystal structures of the p58C272-464 construct used for that study and that of a N-terminally shifted p58C266-456 construct and consequently, an assumption that p58C272-464 has abnormal physical and functional properties. To address this controversy, a new p58C266-464 construct containing all residues was crystallized under the conditions previously used for crystallizing p58C272-464, and the solution structures of both constructs were assessed using circular dichroism and NMR spectroscopy. In the new crystal structure, p58C266-464 exhibits the same elements of secondary structure near the DNA binding site as observed in the crystal structure of p58C272-464. Moreover, in solution, circular dichroism and 15N,1H-heteronuclear single quantum coherence (HSQC) NMR spectra show there are no significant differences in the distribution of secondary structures or in the tertiary structure or the two constructs. To validate that the two constructs have the same functional properties, binding of a primed DNA template was measured using a fluorescence-based DNA binding assay, and the affinities for this substrate were the same (3.4 ± 0.5 μM and 2.7 ± 0.3 μM, respectively). The electrochemical properties of p58C266-464 were also measured and this p58C construct was able to engage in redox switching on DNA with the same efficiency as p58C272-464. Together, these results show that although p58C can be stabilized in different conformations in the crystalline state, in solution there is effectively no difference in the structure and functional properties of p58C constructs of different lengths.
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12 MeSH Terms
Integrated Structural Biology for α-Helical Membrane Protein Structure Determination.
Xia Y, Fischer AW, Teixeira P, Weiner B, Meiler J
(2018) Structure 26: 657-666.e2
MeSH Terms: Algorithms, Binding Sites, Electron Spin Resonance Spectroscopy, Humans, Membrane Proteins, Microscopy, Electron, Models, Molecular, Monte Carlo Method, Nuclear Magnetic Resonance, Biomolecular, Protein Binding, Protein Conformation, alpha-Helical, Protein Folding, Protein Interaction Domains and Motifs, Rhodopsin, Thermodynamics
Show Abstract · Added March 17, 2018
While great progress has been made, only 10% of the nearly 1,000 integral, α-helical, multi-span membrane protein families are represented by at least one experimentally determined structure in the PDB. Previously, we developed the algorithm BCL::MP-Fold, which samples the large conformational space of membrane proteins de novo by assembling predicted secondary structure elements guided by knowledge-based potentials. Here, we present a case study of rhodopsin fold determination by integrating sparse and/or low-resolution restraints from multiple experimental techniques including electron microscopy, electron paramagnetic resonance spectroscopy, and nuclear magnetic resonance spectroscopy. Simultaneous incorporation of orthogonal experimental restraints not only significantly improved the sampling accuracy but also allowed identification of the correct fold, which is demonstrated by a protein size-normalized transmembrane root-mean-square deviation as low as 1.2 Å. The protocol developed in this case study can be used for the determination of unknown membrane protein folds when limited experimental restraints are available.
Copyright © 2018 Elsevier Ltd. All rights reserved.
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15 MeSH Terms
Dodecyl-β-melibioside Detergent Micelles as a Medium for Membrane Proteins.
Hutchison JM, Lu Z, Li GC, Travis B, Mittal R, Deatherage CL, Sanders CR
(2017) Biochemistry 56: 5481-5484
MeSH Terms: Amyloid beta-Protein Precursor, Detergents, Diacylglycerol Kinase, Disaccharides, Dynamic Light Scattering, Enzyme Stability, Escherichia coli Proteins, Glucosides, Glycolipids, Hot Temperature, Humans, Micelles, Myelin Proteins, Nuclear Magnetic Resonance, Biomolecular, Particle Size, Peptide Fragments, Protein Interaction Domains and Motifs, Protein Stability, Receptor, Notch1
Show Abstract · Added November 21, 2018
There remains a need for new non-ionic detergents that are suitable for use in biochemical and biophysical studies of membrane proteins. Here we explore the properties of n-dodecyl-β-melibioside (β-DDMB) micelles as a medium for membrane proteins. Melibiose is d-galactose-α(1→6)-d-glucose. Light scattering showed the β-DDMB micelle to be roughly 30 kDa smaller than micelles formed by the commonly used n-dodecyl-β-maltoside (β-DDM). β-DDMB stabilized diacylglycerol kinase (DAGK) against thermal inactivation. Moreover, activity assays conducted using aliquots of DAGK purified into β-DDMB yielded activities that were 40% higher than those of DAGK purified into β-DDM. β-DDMB yielded similar or better TROSY-HSQC NMR spectra for two single-pass membrane proteins and the tetraspan membrane protein peripheral myelin protein 22. β-DDMB appears be a useful addition to the toolbox of non-ionic detergents available for membrane protein research.
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Analysis of DNA binding by human factor xeroderma pigmentosum complementation group A (XPA) provides insight into its interactions with nucleotide excision repair substrates.
Sugitani N, Voehler MW, Roh MS, Topolska-Woś AM, Chazin WJ
(2017) J Biol Chem 292: 16847-16857
MeSH Terms: Amino Acid Substitution, DNA Repair, DNA Repair Enzymes, DNA, Single-Stranded, Humans, Mutation, Missense, Nuclear Magnetic Resonance, Biomolecular, Protein Binding, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Structural Homology, Protein, Xeroderma Pigmentosum, Xeroderma Pigmentosum Group A Protein
Show Abstract · Added March 24, 2018
Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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13 MeSH Terms
Carbon and amide detect backbone assignment methods of a novel repeat protein from the staphylocoagulase in S. aureus.
Voehler M, Ashoka MA, Meiler J, Bock PE
(2017) Biomol NMR Assign 11: 243-249
MeSH Terms: Amides, Carbon, Coagulase, Nuclear Magnetic Resonance, Biomolecular, Staphylococcus aureus
Show Abstract · Added March 17, 2018
The C-terminal repeat domain of staphylocoagulase that is secreted by the S. aureus is believed to play an important role interacting with fibrinogen and promotes blood clotting. To study this interaction by NMR, full assignment of each amide residue in the HSQC spectrum was required. Despite of the short sequence of the repeat construct, the HSQC spectrum contained a substantial amount of overlapped and exchange broadened resonances, indicating little secondary or tertiary structure. This caused severe problems while using the conventional, amide based NMR method for the backbone assignment. With the growing interest in small apparently disordered proteins, these issues are being faced more frequently. An alternative strategy to improve the backbone assignment capability involved carbon direct detection methods. Circumventing the amide proton detection offers a larger signal dispersion and more uniform signal intensity. For peptides with higher concentrations and in combination with the cold carbon channels of new cryoprobes, higher fields, and sufficiently long relaxation times, the disadvantage of the lower sensitivity of the C nucleus can be overcome. Another advantage of this method is the assignment of the proline backbone residues. Complete assignment with the carbon-detected strategy was achieved with a set of only two 3D, one 2D, and a HNCO measurement, which was necessary to translate the information to the HSQC spectrum.
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5 MeSH Terms
Analysis of Functional Dynamics of Modular Multidomain Proteins by SAXS and NMR.
Thompson MK, Ehlinger AC, Chazin WJ
(2017) Methods Enzymol 592: 49-76
MeSH Terms: DNA Primase, Humans, Multiprotein Complexes, Nuclear Magnetic Resonance, Biomolecular, Protein Conformation, Protein Domains, Replication Protein A, Scattering, Small Angle, X-Ray Diffraction
Show Abstract · Added March 24, 2018
Multiprotein machines drive virtually all primary cellular processes. Modular multidomain proteins are widely distributed within these dynamic complexes because they provide the flexibility needed to remodel structure as well as rapidly assemble and disassemble components of the machinery. Understanding the functional dynamics of modular multidomain proteins is a major challenge confronting structural biology today because their structure is not fixed in time. Small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy have proven particularly useful for the analysis of the structural dynamics of modular multidomain proteins because they provide highly complementary information for characterizing the architectural landscape accessible to these proteins. SAXS provides a global snapshot of all architectural space sampled by a molecule in solution. Furthermore, SAXS is sensitive to conformational changes, organization and oligomeric states of protein assemblies, and the existence of flexibility between globular domains in multiprotein complexes. The power of NMR to characterize dynamics provides uniquely complementary information to the global snapshot of the architectural ensemble provided by SAXS because it can directly measure domain motion. In particular, NMR parameters can be used to define the diffusion of domains within modular multidomain proteins, connecting the amplitude of interdomain motion to the architectural ensemble derived from SAXS. Our laboratory has been studying the roles of modular multidomain proteins involved in human DNA replication using SAXS and NMR. Here, we present the procedure for acquiring and analyzing SAXS and NMR data, using DNA primase and replication protein A as examples.
© 2017 Elsevier Inc. All rights reserved.
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9 MeSH Terms
Structural and biochemical differences between the Notch and the amyloid precursor protein transmembrane domains.
Deatherage CL, Lu Z, Kroncke BM, Ma S, Smith JA, Voehler MW, McFeeters RL, Sanders CR
(2017) Sci Adv 3: e1602794
MeSH Terms: Amyloid beta-Protein Precursor, Humans, Models, Molecular, Nuclear Magnetic Resonance, Biomolecular, Protein Domains, Receptors, Notch
Show Abstract · Added May 3, 2017
γ-Secretase cleavage of the Notch receptor transmembrane domain is a critical signaling event for various cellular processes. Efforts to develop inhibitors of γ-secretase cleavage of the amyloid-β precursor C99 protein as potential Alzheimer's disease therapeutics have been confounded by toxicity resulting from the inhibition of normal cleavage of Notch. We present biochemical and structural data for the combined transmembrane and juxtamembrane Notch domains (Notch-TMD) that illuminate Notch signaling and that can be compared and contrasted with the corresponding traits of C99. The Notch-TMD and C99 have very different conformations, adapt differently to changes in model membrane hydrophobic span, and exhibit different cholesterol-binding properties. These differences may be exploited in the design of agents that inhibit cleavage of C99 while allowing Notch cleavage.
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6 MeSH Terms
A pH-Mediated Topological Switch within the N-Terminal Domain of Human Caveolin-3.
Kim JH, Schlebach JP, Lu Z, Peng D, Reasoner KC, Sanders CR
(2016) Biophys J 110: 2475-2485
MeSH Terms: Amino Acid Sequence, Caveolin 3, Circular Dichroism, Humans, Hydrogen-Ion Concentration, Membranes, Artificial, Micelles, Models, Molecular, Mutation, Nuclear Magnetic Resonance, Biomolecular, Phosphatidylglycerols, Protein Structure, Secondary, Solubility, Solutions
Show Abstract · Added November 21, 2018
Caveolins mediate the formation of caveolae, which are small omega-shaped membrane invaginations involved in a variety of cellular processes. There are three caveolin isoforms, the third of which (Cav3) is expressed in smooth and skeletal muscles. Mutations in Cav3 cause a variety of human muscular diseases. In this work, we characterize the secondary structure, dynamics, and topology of the monomeric form of the full-length lipidated protein. Cav3 consists of a series of membrane-embedded or surface-associated helical elements connected by extramembrane connecting loops or disordered domains. Our results also reveal that the N-terminal domain undergoes a large scale pH-mediated topological rearrangement between soluble and membrane-anchored forms. Considering that roughly one-third of pathogenic mutations in Cav3 influence charged residues located in this domain, we hypothesize that this transition is likely to be relevant to the molecular basis of Cav3-linked diseases. These results provide insight into the structure of Cav3 and set the stage for mechanistic investigations of the effects of pathogenic mutations.
Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.
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Absolute Configuration of (-)-Centratherin, a Sesquiterpenoid Lactone, Defined by Means of Chiroptical Spectroscopy.
Junior FM, Covington CL, de Albuquerque AC, Lobo JF, Borges RM, de Amorim MB, Polavarapu PL
(2015) J Nat Prod 78: 2617-23
MeSH Terms: Circular Dichroism, Lactones, Models, Chemical, Molecular Structure, Nuclear Magnetic Resonance, Biomolecular, Optical Rotatory Dispersion, Sesquiterpenes, Stereoisomerism
Show Abstract · Added February 15, 2016
(-)-Centratherin is a bioactive sesquiterpenoid lactone, whose absolute configuration (AC) was not established, but has been proposed based on those of germacrane precursors. To verify this proposal, the experimental electronic circular dichroism (ECD), electronic dissymmetry factor (EDF), optical rotatory dispersion (ORD), vibrational circular dichroism (VCD), and vibrational dissymmetry factor (VDF) spectra of (-)-centratherin have been analyzed with the corresponding density functional theoretical predictions. These analyses suggest the AC of naturally occurring (-)-centratherin to be (6R,7R,8S,10R,2'Z).
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8 MeSH Terms
Promotion of BRCA2-Dependent Homologous Recombination by DSS1 via RPA Targeting and DNA Mimicry.
Zhao W, Vaithiyalingam S, San Filippo J, Maranon DG, Jimenez-Sainz J, Fontenay GV, Kwon Y, Leung SG, Lu L, Jensen RB, Chazin WJ, Wiese C, Sung P
(2015) Mol Cell 59: 176-87
MeSH Terms: Amino Acid Substitution, BRCA2 Protein, Breast Neoplasms, Cell Line, Female, HeLa Cells, Homologous Recombination, Humans, Models, Biological, Molecular Mimicry, Mutagenesis, Site-Directed, Nuclear Magnetic Resonance, Biomolecular, Proteasome Endopeptidase Complex, Protein Subunits, Rad51 Recombinase, Recombinant Proteins, Replication Protein A
Show Abstract · Added February 5, 2016
The tumor suppressor BRCA2 is thought to facilitate the handoff of ssDNA from replication protein A (RPA) to the RAD51 recombinase during DNA break and replication fork repair by homologous recombination. However, we find that RPA-RAD51 exchange requires the BRCA2 partner DSS1. Biochemical, structural, and in vivo analyses reveal that DSS1 allows the BRCA2-DSS1 complex to physically and functionally interact with RPA. Mechanistically, DSS1 acts as a DNA mimic to attenuate the affinity of RPA for ssDNA. A mutation in the solvent-exposed acidic domain of DSS1 compromises the efficacy of RPA-RAD51 exchange. Thus, by targeting RPA and mimicking DNA, DSS1 functions with BRCA2 in a two-component homologous recombination mediator complex in genome maintenance and tumor suppression. Our findings may provide a paradigm for understanding the roles of DSS1 in other biological processes.
Copyright © 2015 Elsevier Inc. All rights reserved.
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17 MeSH Terms