Chad A Brautigam

University of Texas Southwestern Medical Center, Dallas, Texas, United States

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Publications (76)454.74 Total impact

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    Methods 04/2015; 76:1-2. DOI:10.1016/j.ymeth.2015.02.001 · 3.22 Impact Factor
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    ABSTRACT: The sexually transmitted disease syphilis is caused by the bacterial spirochete Treponema pallidum. This microorganism is genetically intractable, accounting for the large number of putative and under-characterized members of the pathogen's proteome. In an effort to ascribe a function(s) to the TP0435 (Tp17) lipoprotein, we engineered a soluble variant of the protein (rTP0435) and determined its crystal structure at a resolution of 2.42 Å. The structure is characterized by an eight-stranded β-barrel protein with a shallow “basin” at one end of the barrel and an α-helix stacked on the opposite end. Furthermore, there is a disulfide-linked dimer of the protein in the asymmetric unit of the crystals. Solution hydrodynamic experiments established that purified rTP0435 is monomeric, but specifically forms the disulfide-stabilized dimer observed in the crystal structure. The data herein, when considered with previous work on TP0435, imply plausible roles for the protein in either ligand binding, treponemal membrane architecture, and/or pathogenesis.
    Protein Science 01/2015; 24(1). DOI:10.1002/pro.2576 · 2.86 Impact Factor
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    ABSTRACT: ABSTRACT To adapt to stresses encountered in stationary phase, Gram-negative bacteria utilize the alternative sigma factor RpoS. However, some species lack RpoS; thus, it is unclear how stationary-phase adaptation is regulated in these organisms. Here we defined the growth-phase-dependent transcriptomes of Haemophilus ducreyi, which lacks an RpoS homolog. Compared to mid-log-phase organisms, cells harvested from the stationary phase upregulated genes encoding several virulence determinants and a homolog of hfq. Insertional inactivation of hfq altered the expression of ~16% of the H. ducreyi genes. Importantly, there were a significant overlap and an inverse correlation in the transcript levels of genes differentially expressed in the hfq inactivation mutant relative to its parent and the genes differentially expressed in stationary phase relative to mid-log phase in the parent. Inactivation of hfq downregulated genes in the flp-tad and lspB-lspA2 operons, which encode several virulence determinants. To comply with FDA guidelines for human inoculation experiments, an unmarked hfq deletion mutant was constructed and was fully attenuated for virulence in humans. Inactivation or deletion of hfq downregulated Flp1 and impaired the ability of H. ducreyi to form microcolonies, downregulated DsrA and rendered H. ducreyi serum susceptible, and downregulated LspB and LspA2, which allow H. ducreyi to resist phagocytosis. We propose that, in the absence of an RpoS homolog, Hfq serves as a major contributor of H. ducreyi stationary-phase and virulence gene regulation. The contribution of Hfq to stationary-phase gene regulation may have broad implications for other organisms that lack an RpoS homolog. IMPORTANCE Pathogenic bacteria encounter a wide range of stresses in their hosts, including nutrient limitation; the ability to sense and respond to such stresses is crucial for bacterial pathogens to successfully establish an infection. Gram-negative bacteria frequently utilize the alternative sigma factor RpoS to adapt to stresses and stationary phase. However, homologs of RpoS are absent in some bacterial pathogens, including Haemophilus ducreyi, which causes chancroid and facilitates the acquisition and transmission of HIV-1. Here, we provide evidence that, in the absence of an RpoS homolog, Hfq serves as a major contributor of stationary-phase gene regulation and that Hfq is required for H. ducreyi to infect humans. To our knowledge, this is the first study describing Hfq as a major contributor of stationary-phase gene regulation in bacteria and the requirement of Hfq for the virulence of a bacterial pathogen in humans.
    mBio 12/2014; 5(1). DOI:10.1128/mBio.01081-13 · 6.88 Impact Factor
  • Thomas H. Scheuermann, Chad A. Brautigam
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    ABSTRACT: Isothermal titration calorimetry (ITC) has become a standard and widely available tool to measure the thermodynamic parameters of macromolecular associations. Modern applications of the method, including global analysis and drug screening, require the acquisition of multiple sets of data; sometimes these data sets number in the hundreds. Therefore, there is a need for quick, precise, and automated means to process the data, particularly at the first step of data analysis, which is commonly the integration of the raw data to yield an interpretable isotherm. Herein, we describe enhancements to an algorithm that previously has been shown to provide an automated, unbiased, and high-precision means to integrate ITC data. These improvements allow for the speedy and precise serial integration of an unlimited number of ITC data sets, and they have been implemented in the freeware program NITPIC, version 1.1.0. We present a comprehensive comparison of the performance of this software against an older version of NITPIC and a current version of Origin, which is commonly used for integration. The new methods recapitulate the excellent performance of the previous versions of NITPIC while speeding it up substantially, and their precision is significantly better than that of Origin. This new version of NITPIC is therefore well suited to the serial integration of many ITC data sets.
    Methods 12/2014; 76. DOI:10.1016/j.ymeth.2014.11.024 · 3.22 Impact Factor
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    ABSTRACT: The spindle checkpoint ensures accurate chromosome segregation by monitoring kinetochore-microtubule attachment. Unattached or tensionless kinetochores activate the checkpoint and enhance the production of the mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20. MCC is a critical checkpoint inhibitor of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase required for anaphase onset. The N-terminal region of BubR1 binds to both Cdc20 and Mad2, thus nucleating MCC formation. The middle region of human BubR1 (BubR1M) also interacts with Cdc20, but the nature and function of this interaction are not understood. Here we identify two critical motifs within BubR1M that contribute to Cdc20 binding and APC/C inhibition: a destruction box (D box) and a phenylalanine-containing motif termed the Phe box. A BubR1 mutant lacking these motifs is defective in MCC maintenance in mitotic human cells, but is capable of supporting spindle-checkpoint function. Thus, the BubR1M-Cdc20 interaction indirectly contributes to MCC homeostasis. Its apparent dispensability in the spindle checkpoint might be due to functional duality or redundant, competing mechanisms. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.
  • Chad A Brautigam
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    ABSTRACT: As isothermal titration calorimetry (ITC) gains popularity for the characterization of enthalpies and equilibrium association constants of simple 1:1 biomolecular interactions, its use for more complex systems is growing. The method is increasingly used to study interactions in which a single binding partner (molecule "A") interacts with multiple copies of a second partner ("B"); thus examinations of ABB and ABBB interactions are not uncommon. The structure of ITC data (commonly formatted as isotherms) has a strong bearing on the ability of the researcher to extract the necessary parameters from them. Usually, only 10-30 injections are recorded in a single ITC experiment. Even if replicates are performed, the data must support the extraction of up to twelve parameters from an ABBB system conducted in triplicate. Further, the refinement of some of the parameters is largely driven by only a subset of the data. The ability of ITC data to guide the deterministic estimation of these parameters may therefore be questioned. This work assesses the ability of both empirical and simulated ITC data of ABB and ABBB systems to support the simultaneous estimation of the desired thermodynamic parameters. The results demonstrate that multiphasic isotherms tend to (but do not always) support the estimation of multiple parameters. On the other hand, uniphasic data obtained from multi-site binding systems are more problematic. In all cases, a thorough exploration of how precisely the estimated parameters are specified by the data is justified. Copyright © 2014. Published by Elsevier Inc.
    Methods 12/2014; 76. DOI:10.1016/j.ymeth.2014.11.018 · 3.22 Impact Factor
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    ABSTRACT: There is a paucity of data concerning gene products that could contribute to the ability of Moraxella catarrhalis to colonize the human nasopharynx. Inactivation of a gene (mesR) encoding a predicted response regulator of a two-component signal transduction system in M. catarrhalis yielded a mutant unable to grow in liquid media. This mesR mutant also exhibited increased sensitivity to certain stressors including polymyxin B, SDS, and hydrogen peroxide. Inactivation of the gene (mesS) encoding the predicted cognate sensor (histidine) kinase yielded a mutant with the same inability to grow in liquid media as the mesR mutant. DNA microarray and real-time RT-PCR analyses indicated that several genes previously shown to be involved in the ability of M. catarrhalis to persist in the chinchilla nasopharynx were up-regulated in the mesR mutant. Two other ORFs up-regulated in the mesR mutant were shown to encode small proteins (Lip1 and Lip2) that had amino acid sequence homology to bacterial adhesins and structural homology to bacterial lysozyme inhibitors. Inactivation of both lip1 and lip2 did not affect the ability of M. catarrhalis O35E to attach to a human bronchial epithelial cell line in vitro. Purified recombinant Lip1 and Lip2 fusion proteins were each shown to inhibit human lysozyme activity in vitro and in saliva. A lip1 lip2 deletion mutant was more sensitive than the wild-type parent strain to killing by human lysozyme in the presence of human apo-lactoferrin. This is the first report of lysozyme inhibitors produced by M. catarrhalis.
    Infection and Immunity 10/2014; 83(1). DOI:10.1128/IAI.02486-14 · 4.16 Impact Factor
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    ABSTRACT: Stu2p/XMAP215 proteins are essential microtubule polymerases that use multiple αβ-tubulin-interacting TOG domains to bind microtubule plus ends and catalyze fast microtubule growth. We report here the structure of the TOG2 domain from Stu2p bound to yeast αβ-tubulin. Like TOG1, TOG2 binds selectively to a fully 'curved' conformation of αβ-tubulin, incompatible with a microtubule lattice. We also show that TOG1-TOG2 binds non-cooperatively to two αβ-tubulins. Preferential interactions between TOGs and fully curved αβ-tubulin that cannot exist elsewhere in the microtubule explain how these polymerases localize to the extreme microtubule end. We propose that these polymerases promote elongation because their linked TOG domains concentrate unpolymerized αβ-tubulin near curved subunits already bound at the microtubule end. This tethering model can explain catalyst-like behavior and also predicts that the polymerase action changes the configuration of the microtubule end.
    eLife Sciences 08/2014; 3:e03069. DOI:10.7554/eLife.03069 · 8.52 Impact Factor
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    ABSTRACT: Accurate measurements of rotor temperature are critical for the interpretation of hydrodynamic parameters in analytical ultracentrifugation. We have recently developed methods for a more accurate determination of the temperature of a spinning rotor utilizing iButton® temperature loggers. Here we report that the temperature measured with the iButton on the counterbalance of a resting rotor, following thermal equilibration under high vacuum, closely corresponded to the temperature of the spinning rotor with a precision better than 0.2 °C. This strategy offers an inexpensive and straightforward approach to monitor the accuracy of the temperature calibration and determine corrective temperature offsets.
    Analytical Biochemistry 08/2014; 458. DOI:10.1016/j.ab.2014.04.029 · 2.31 Impact Factor
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    ABSTRACT: Colonization of the human nasopharynx by Moraxella catarrhalis is presumed to involve attachment of this bacterium to the mucosa. DNA microarray analysis was used to determine whether attachment of M. catarrhalis to human bronchial epithelial (HBE) cells in vitro affected gene expression in this bacterium. Attachment affected expression of at least 454 different genes, with 163 being up-regulated and 291 being down-regulated. Among the up-regulated genes was one (ORF 113) previously annotated as encoding a protein with some similarity to outer membrane protein A (OmpA). The protein encoded by ORF 113 was predicted to have a signal peptidase II cleavage site, and globomycin inhibition experiments confirmed that this protein was indeed a lipoprotein. The ORF 113 protein also contained a predicted peptidoglycan-binding domain in its C-terminal half. The use of mutant and recombinant M. catarrhalis strains confirmed that the ORF 113 protein was present in outer membrane preparations, and this protein was also shown to be at least partially exposed on the bacterial cell surface. A mutant unable to produce the ORF 113 protein showed little or no change in its growth rate in vitro, in its ability to attach to HBE cells in vitro, or in its autoagglutination characteristics, but did exhibit a reduced ability to survive in the chinchilla nasopharynx. This is the first report of a lipoprotein essential to the ability of M. catarrhalis to persist in an animal model.
    Infection and immunity 03/2014; 82(6). DOI:10.1128/IAI.01745-14 · 4.16 Impact Factor
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    ABSTRACT: The alternative sigma factor RpoS in Borrelia burgdorferi plays a central role in modulating host adaptive responses when spirochetes cycle between ticks and mammals. The transcriptional activation of σ54-dependent rpoS requires a Fur homologue designated as BosR. Previously, we found that BosR directly activates rpoS transcription by binding to the rpoS promoter. However, many other DNA-binding features of BosR have remained obscure. In particular, the precise DNA sequence targeted by BosR has not yet been completely elucidated. The prediction of a putative Per box within the rpoS promoter region has further confounded the identification of the BosR binding sequence. Herein, by using electrophoretic mobility shift assays, we demonstrate that the putative Per box predicted in the rpoS promoter region is not involved in the binding of BosR. Rather, a 13-bp palindromic sequence (ATTTAANTTAAAT) with dyad symmetry, which we herein denote as the "BosR box", functions as the core sequence recognized by BosR in the rpoS promoter region of B. burgdorferi. Similar to a Fur box and a Per box, the BosR box likely comprises a 6-1-6 inverted repeat composed of two hexamers (ATTTAA) in a head-to-tail orientation. Selected mutations in the BosR box prevented recombinant BosR from binding to rpoS. In addition, we found that sequences neighboring the BosR box also are required for the formation of BosR-DNA complexes. Identification of the BosR box advances our understanding of how BosR recognizes its DNA target(s), and provides new insight into the mechanistic details behind the unique regulatory function of BosR.
    Microbiology 03/2014; 160(Pt 4). DOI:10.1099/mic.0.075655-0 · 2.84 Impact Factor
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    ABSTRACT: The Lyme disease agent Borrelia burgdorferi, which is transmitted via a tick vector, is dependent on its tick and mammalian hosts for a number of essential nutrients. Like other bacterial diderms, it must transport these biochemicals from the extracellular milieu across two membranes, ultimately to the B. burgdorferi cytoplasm. In the current study, we established that a gene cluster comprising genes bb0215 through bb0218 is cotranscribed and is therefore an operon. Sequence analysis of these proteins suggested that they are the components of an ABC-type transporter responsible for translocating phosphate anions from the B. burgdorferi periplasm to the cytoplasm. Biophysical experiments established that the putative ligand-binding protein of this system, BbPstS (BB0215), binds to phosphate in solution. We determined the high-resolution (1.3 Å) crystal structure of the protein in the absence of phosphate, revealing that the protein's fold is similar to other phosphate-binding proteins, and residues that are implicated in phosphate binding in other such proteins are conserved in BbPstS. Taken together, the gene products of bb0215-0218 function as a phosphate transporter for B. burgdorferi.
    Protein Science 02/2014; 23(2). DOI:10.1002/pro.2406 · 2.86 Impact Factor
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    ABSTRACT: The presence of DNA in the cytoplasm is a danger signal that triggers immune and inflammatory responses. Cytosolic DNA binds to and activates cyclic GMP-AMP (cGAMP) synthase (cGAS), which produces the second messenger cGAMP. cGAMP binds to the adaptor protein STING and activates a signaling cascade that leads to the production of type I interferons and other cytokines. Here, we report the crystal structures of human cGAS in its apo form, representing its autoinhibited conformation as well as in its cGAMP- and sulfate-bound forms. These structures reveal switch-like conformational changes of an activation loop that result in the rearrangement of the catalytic site. The structure of DNA-bound cGAS reveals a complex composed of dimeric cGAS bound to two molecules of DNA. Functional analyses of cGAS mutants demonstrate that both the protein-protein interface and the two DNA binding surfaces are critical for cGAS activation. These results provide insights into the mechanism of DNA sensing by cGAS.
    Cell Reports 01/2014; DOI:10.1016/j.celrep.2014.01.003 · 7.21 Impact Factor
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    ABSTRACT: Bacterial transporter proteins are involved in the translocation of many essential nutrients and metabolites. However, many of these key bacterial transport systems remain to be identified, including those involved in the transport of riboflavin (vitamin B2). Pathogenic spirochetes lack riboflavin biosynthetic pathways, implying reliance on obtaining riboflavin from their hosts. Using structural and functional characterizations of possible ligand-binding components, we have identified an ABC-type riboflavin transport system within pathogenic spirochetes. The putative lipoprotein ligand-binding components of these systems from three different spirochetes were cloned, hyperexpressed in Escherichia coli, and purified to homogeneity. Solutions of all three of the purified recombinant proteins were bright yellow. UV-visible spectra demonstrated that these proteins were likely flavoproteins; electrospray ionization mass spectrometry and thin-layer chromatography confirmed that they contained riboflavin. A 1.3-Å crystal structure of the protein (TP0298) encoded by Treponema pallidum, the syphilis spirochete, demonstrated that the protein’s fold is similar to the ligand-binding components of ABC-type transporters. The structure also revealed other salient details of the riboflavin binding site. Comparative bioinformatics analyses of spirochetal genomes, coupled with experimental validation, facilitated the discovery of this new ABC-type riboflavin transport system(s). We denote the ligand-binding component as riboflavin uptake transporter A (RfuA). Taken together, it appears that pathogenic spirochetes have evolved an ABC-type transport system (RfuABCD) for survival in their host environments, particularly that of the human host.
    mBio 12/2013; 4(1). DOI:10.1128/mBio.00615-12 · 6.88 Impact Factor
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    ABSTRACT: eLife digest A key question in neurobiology is how the brain becomes wired up. How do axons—the ‘wires’ along which neural signals flow—know in which direction to grow to reach their intended targets? A family of signalling proteins called semaphorins contribute to this process by acting as stop signals for axons that are heading in the wrong direction. The actions of semaphorins are mediated by receptors known as plexins, which are found on the membranes of axons. Plexins contain an extracellular domain that binds semaphorin, and a large domain inside the cell that can turn semaphorin binding into cellular responses. When a semaphorin protein binds to the extracellular domain of a plexin receptor, the domain inside the cell joins with the intracellular domain of a neighbouring receptor to form a dimer. This activates the intracellular domain, which turns on its ability to inactivate a molecule called Rap. The end result is that the axon stops growing and changes direction, but the molecular mechanisms through which these events occur are not well understood. Now, Wang, Pascoe et al. have worked out the structure of the dimers formed by the intracellular plexin domains, both alone and in complex with Rap. The structures reveal how the dimer drives a shape change of the intracellular domain to enable it to bind Rap, and show that Rap itself adopts a novel conformation upon binding to plexin. This conformational change in Rap catalyses the breakdown of a signalling molecule called GTP, which inactivates Rap and triggers an intracellular signalling cascade that causes the axon to collapse and change direction. Lastly, Wang, Pascoe et al. have shown that the highly specific nature of these interactions depends on particular amino-acid residues in both Rap and the plexin receptor. Further work is now required to determine whether this pattern of activation represents a general mechanism for signalling by plexin receptors, and for the inhibition of Rap. DOI: http://dx.doi.org/10.7554/eLife.01279.002
    eLife Sciences 10/2013; 2:e01279. DOI:10.7554/eLife.01279 · 8.52 Impact Factor
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    ABSTRACT: Expression of the lspB-lspA2 operon encoding a virulence-related two-partner secretion system in Haemophilus ducreyi 35000HP is directly regulated by the CpxRA regulatory system [Labandeira-Rey M, Mock JR, Hansen E. Regulation of expression of the Haemophilus ducreyi LspB and LspA2 proteins by CpxR. Infect. Immun. 77: 3402-3411 (2009)]. In the present study, we show that this secretion system is also regulated by the small nucleoid-associated protein Fis. Inactivation of the H. ducreyi fis gene resulted in a reduction in expression of both the H. ducreyi LspB and LspA2 proteins. DNA microarray experiments showed that a H. ducreyi fis deletion mutant exhibited altered expression of genes encoding other important H. ducreyi virulence factors including DsrA and Flp1, suggesting a possible global role for Fis in control of virulence in this obligate human pathogen. While the H. ducreyi Fis protein has a high degree of sequence and structural similarity to the Fis proteins of other bacteria, its temporal pattern of expression was very different from that of enterobacterial Fis proteins. The use of a lacZ-based transcriptional reporter provided evidence which indicated that the H. ducreyi Fis homolog is a positive regulator of gyrB, a gene that is negatively regulated by Fis in enteric bacteria. Taken together, the Fis protein expression data and the observed regulatory effects of Fis in H. ducreyi suggest that this small DNA binding protein has a regulatory role in H. ducreyi which may differ in substantial ways from that of other Fis proteins.
    Infection and immunity 08/2013; DOI:10.1128/IAI.00714-13 · 4.16 Impact Factor
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    ABSTRACT: The tumor-suppressive Hippo pathway controls tissue homeostasis through balancing cell proliferation and apoptosis. Activation of the kinases Mst1 and Mst2 (Mst1/2) is a key upstream event in this pathway and remains poorly understood. Mst1/2 and their critical regulators RASSFs contain Salvador/RASSF1A/Hippo (SARAH) domains that can homo- and heterodimerize. Here, we report the crystal structures of human Mst2 alone and bound to RASSF5. Mst2 undergoes activation through transautophosphorylation at its activation loop, which requires SARAH-mediated homodimerization. RASSF5 disrupts Mst2 homodimer and blocks Mst2 autoactivation. Binding of RASSF5 to already activated Mst2, however, does not inhibit its kinase activity. Thus, RASSF5 can act as an inhibitor or a potential positive regulator of Mst2, depending on whether it binds to Mst2 before or after activation-loop phosphorylation. We propose that these temporally sensitive functions of RASSFs enable the Hippo pathway to respond to and integrate diverse cellular signals.
    Structure 08/2013; DOI:10.1016/j.str.2013.07.008 · 6.79 Impact Factor
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    ABSTRACT: Cohesin, along with positive regulators, establishes sister-chromatid cohesion by forming a ring to circle chromatin. The wings apart-like protein (Wapl) is a key negative regulator of cohesin and forms a complex with precocious dissociation of sisters protein 5 (Pds5) to promote cohesin release from chromatin. Here we report the crystal structure and functional characterization of human Wapl. Wapl contains a flexible, variable N-terminal region (Wapl-N) and a conserved C-terminal domain (Wapl-C) consisting of eight HEAT (Huntingtin, Elongation factor 3, A subunit, and target of rapamycin) repeats. Wapl-C folds into an elongated structure with two lobes. Structure-based mutagenesis maps the functional surface of Wapl-C to two distinct patches (I and II) on the N lobe and a localized patch (III) on the C lobe. Mutating critical patch I residues weaken Wapl binding to cohesin and diminish sister-chromatid resolution and cohesin release from mitotic chromosomes in human cells and Xenopus egg extracts. Surprisingly, patch III on the C lobe does not contribute to Wapl binding to cohesin or its known regulators. Although patch I mutations reduce Wapl binding to intact cohesin, they do not affect Wapl-Pds5 binding to the cohesin subcomplex of sister chromatid cohesion protein 1 (Scc1) and stromal antigen 2 (SA2) in vitro, which is instead mediated by Wapl-N. Thus, Wapl-N forms extensive interactions with Pds5 and Scc1-SA2. Wapl-C interacts with other cohesin subunits and possibly unknown effectors to trigger cohesin release from chromatin.
    Proceedings of the National Academy of Sciences 06/2013; DOI:10.1073/pnas.1304594110 · 9.81 Impact Factor
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    ABSTRACT: Sedimentation velocity (SV) is a method based on first-principles that provides a precise hydrodynamic characterization of macromolecules in solution. Due to recent improvements in data analysis, the accuracy of experimental SV data emerges as a limiting factor in its interpretation. Our goal was to unravel the sources of experimental error and develop improved calibration procedures. We implemented the use of a Thermochron iButton® temperature logger to directly measure the temperature of a spinning rotor, and detected deviations that can translate into an error of as much as 10% in the sedimentation coefficient. We further designed a precision mask with equidistant markers to correct for instrumental errors in the radial calibration, which were observed to span a range of 8.6%. The need for an independent time calibration emerged with use of the current data acquisition software (Zhao et al., doi 10.1016/j.ab.2013.02.011) and we now show that smaller but significant time errors of up to 2% also occur with earlier versions. After application of these calibration corrections, the sedimentation coefficients obtained from eleven instruments displayed a significantly reduced standard deviation of ∼ 0.7%. This study demonstrates the need for external calibration procedures and regular control experiments with a sedimentation coefficient standard.
    Analytical Biochemistry 05/2013; DOI:10.1016/j.ab.2013.05.011 · 2.31 Impact Factor

Publication Stats

2k Citations
454.74 Total Impact Points

Institutions

  • 2004–2015
    • University of Texas Southwestern Medical Center
      • • Department of Biophysics
      • • Department of Biochemistry
      • • Department of Microbiology
      Dallas, Texas, United States
  • 2004–2014
    • University of Texas at Dallas
      • Biochemistry
      Richardson, Texas, United States
  • 2007
    • Johns Hopkins University
      • Department of Pharmacology and Molecular Sciences
      Baltimore, Maryland, United States
  • 2006
    • Indiana University-Purdue University Indianapolis
      • Department of Microbiology and Immunology
      Indianapolis, Indiana, United States
  • 1998–2000
    • Yale University
      • Department of Molecular Biophysics and Biochemistry
      New Haven, CT, United States
    • University of New Haven
      New Haven, Connecticut, United States
  • 1999
    • University of Chicago
      • Department of Chemistry
      Chicago, Illinois, United States