Kinetics of Transferrin and Transferrin-Receptor during Iron Transport through Blood Brain Barrier

NASA Astrophysics Data System (ADS)

Khan, Aminul; Liu, Jin; Dutta, Prashanta

2017-11-01

Transferrin and its receptors play an important role during the uptake and transcytosis of iron by blood brain barrier (BBB) endothelial cells to maintain iron homeostasis in BBB endothelium and brain. In the blood side of BBB, ferric iron binds with the apo-transferrin to form holo-transferrin which enters the endothelial cell via transferrin receptor mediated endocytosis. Depending on the initial concentration of iron inside the cell endocytosed holo-transferrin can either be acidified in the endosome or exocytosed through the basolateral membrane. Acidification of holo-transferrin in the endosome releases ferrous irons which may either be stored and used by the cell or transported into brain side. Exocytosis of the holo-transferrin through basolateral membrane leads to transport of iron bound to transferrin into brain side. In this work, kinetics of internalization, recycling and exocytosis of transferrin and its receptors are modeled by laws of mass action during iron transport in BBB endothelial cell. Kinetic parameters for the model are determined by least square analysis. Our results suggest that the cell's initial iron content determines the extent of the two possible iron transport pathways, which will be presented in this talk Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R01GM122081.

Analysis and prediction of calcium-binding pockets from apo-protein structures exhibiting calcium-induced localized conformational changes

PubMed Central

Wang, Xue; Zhao, Kun; Kirberger, Michael; Wong, Hing; Chen, Guantao; Yang, Jenny J

2010-01-01

Calcium binding in proteins exhibits a wide range of polygonal geometries that relate directly to an equally diverse set of biological functions. The binding process stabilizes protein structures and typically results in local conformational change and/or global restructuring of the backbone. Previously, we established the MUG program, which utilized multiple geometries in the Ca2+-binding pockets of holoproteins to identify such pockets, ignoring possible Ca2+-induced conformational change. In this article, we first report our progress in the analysis of Ca2+-induced conformational changes followed by improved prediction of Ca2+-binding sites in the large group of Ca2+-binding proteins that exhibit only localized conformational changes. The MUGSR algorithm was devised to incorporate side chain torsional rotation as a predictor. The output from MUGSR presents groups of residues where each group, typically containing two to five residues, is a potential binding pocket. MUGSR was applied to both X-ray apo structures and NMR holo structures, which did not use calcium distance constraints in structure calculations. Predicted pockets were validated by comparison with homologous holo structures. Defining a “correct hit” as a group of residues containing at least two true ligand residues, the sensitivity was at least 90%; whereas for a “correct hit” defined as a group of residues containing at least three true ligand residues, the sensitivity was at least 78%. These data suggest that Ca2+-binding pockets are at least partially prepositioned to chelate the ion in the apo form of the protein. PMID:20512971

Opposing Intermolecular Tuning of Ca2+ Affinity for Calmodulin by Neurogranin and CaMKII Peptides.

PubMed

Zhang, Pengzhi; Tripathi, Swarnendu; Trinh, Hoa; Cheung, Margaret S

2017-03-28

We investigated the impact of bound calmodulin (CaM)-target compound structure on the affinity of calcium (Ca 2+ ) by integrating coarse-grained models and all-atomistic simulations with nonequilibrium physics. We focused on binding between CaM and two specific targets, Ca 2+ /CaM-dependent protein kinase II (CaMKII) and neurogranin (Ng), as they both regulate CaM-dependent Ca 2+ signaling pathways in neurons. It was shown experimentally that Ca 2+ /CaM (holoCaM) binds to the CaMKII peptide with overwhelmingly higher affinity than Ca 2+ -free CaM (apoCaM); the binding of CaMKII peptide to CaM in return increases the Ca 2+ affinity for CaM. However, this reciprocal relation was not observed in the Ng peptide (Ng 13-49 ), which binds to apoCaM or holoCaM with binding affinities of the same order of magnitude. Unlike the holoCaM-CaMKII peptide, whose structure can be determined by crystallography, the structural description of the apoCaM-Ng 13-49 is unknown due to low binding affinity, therefore we computationally generated an ensemble of apoCaM-Ng 13-49 structures by matching the changes in the chemical shifts of CaM upon Ng 13-49 binding from nuclear magnetic resonance experiments. Next, we computed the changes in Ca 2+ affinity for CaM with and without binding targets in atomistic models using Jarzynski's equality. We discovered the molecular underpinnings of lowered affinity of Ca 2+ for CaM in the presence of Ng 13-49 by showing that the N-terminal acidic region of Ng peptide pries open the β-sheet structure between the Ca 2+ binding loops particularly at C-domain of CaM, enabling Ca 2+ release. In contrast, CaMKII peptide increases Ca 2+ affinity for the C-domain of CaM by stabilizing the two Ca 2+ binding loops. We speculate that the distinctive structural difference in the bound complexes of apoCaM-Ng 13-49 and holoCaM-CaMKII delineates the importance of CaM's progressive mechanism of target binding on its Ca 2+ binding affinities. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Interaction of imatinib mesylate with human serum transferrin: The comparative spectroscopic studies

NASA Astrophysics Data System (ADS)

Śliwińska-Hill, Urszula

2017-02-01

Imatinib mesylate (Imt) is a tyrosine kinase inhibitor mainly used in the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia (Ph + CML). Human serum transferrin is the most abundant serum protein responsible for the transport of iron ions and many endogenous and exogenous ligands. In this study the mechanism of interactions between the imatinib mesylate and all states of transferrin (apo-Tf, Htf and holo-Tf) has been investigated by fluorescence, ultraviolet-visible (UV-vis), circular dichroism (CD) and zeta potential spectroscopic methods. Based on the experimental results it was proved that under physiological conditions the imatinib mesylate binds to the each form of transferrin with a binding constant c.a. 105 M- 1. The thermodynamic parameters indicate that hydrogen bonds and van der Waals were involved in the interaction of apo-Tf with the drug and hydrophobic and ionic strength participate in the reaction of Htf and holo-Tf with imatinib mesylate. Moreover, it was shown that common metal ions, Zn2 + and Ca2 + strongly influenced apo-Tf-Imt binding constant. The CD studies showed that there are no conformational changes in the secondary structure of the proteins. No significant changes in secondary structure of the proteins upon binding with the drug and instability of apo-Tf-Imt system are the desirable effects from pharmacological point of view.

ApoHRP-based assay to measure intracellular regulatory heme.

PubMed

Atamna, Hani; Brahmbhatt, Marmik; Atamna, Wafa; Shanower, Gregory A; Dhahbi, Joseph M

2015-02-01

The majority of the heme-binding proteins possess a "heme-pocket" that stably binds to heme. Usually known as housekeeping heme-proteins, they participate in a variety of metabolic reactions (e.g., catalase). Heme also binds with lower affinity to the "Heme-Regulatory Motifs" (HRM) in specific regulatory proteins. This type of heme binding is known as exchangeable or regulatory heme (RH). Heme binding to HRM proteins regulates their function (e.g., Bach1). Although there are well-established methods for assaying total cellular heme (e.g., heme-proteins plus RH), currently there is no method available for measuring RH independent of the total heme (TH). The current study describes and validates a new method to measure intracellular RH. This method is based on the reconstitution of apo-horseradish peroxidase (apoHRP) with heme to form holoHRP. The resulting holoHRP activity is then measured with a colorimetric substrate. The results show that apoHRP specifically binds RH but not with heme from housekeeping heme-proteins. The RH assay detects intracellular RH. Furthermore, using conditions that create positive (hemin) or negative (N-methyl protoporphyrin IX) controls for heme in normal human fibroblasts (IMR90), the RH assay shows that RH is dynamic and independent of TH. We also demonstrated that short-term exposure to subcytotoxic concentrations of lead (Pb), mercury (Hg), or amyloid-β (Aβ) significantly alters intracellular RH with little effect on TH. In conclusion the RH assay is an effective assay to investigate intracellular RH concentration and demonstrates that RH represents ∼6% of total heme in IMR90 cells.

H-Ferritin Is Preferentially Incorporated by Human Erythroid Cells through Transferrin Receptor 1 in a Threshold-Dependent Manner

PubMed Central

Sakamoto, Soichiro; Kawabata, Hiroshi; Masuda, Taro; Uchiyama, Tatsuki; Mizumoto, Chisaki; Ohmori, Katsuyuki; Koeffler, H. Phillip; Kadowaki, Norimitsu; Takaori-Kondo, Akifumi

2015-01-01

Ferritin is an iron-storage protein composed of different ratios of 24 light (L) and heavy (H) subunits. The serum level of ferritin is a clinical marker of the body’s iron level. Transferrin receptor (TFR)1 is the receptor not only for transferrin but also for H-ferritin, but how it binds two different ligands and the blood cell types that preferentially incorporate H-ferritin remain unknown. To address these questions, we investigated hematopoietic cell-specific ferritin uptake by flow cytometry. Alexa Fluor 488-labeled H-ferritin was preferentially incorporated by erythroid cells among various hematopoietic cell lines examined, and was almost exclusively incorporated by bone marrow erythroblasts among human primary hematopoietic cells of various lineages. H-ferritin uptake by erythroid cells was strongly inhibited by unlabeled H-ferritin but was only partially inhibited by a large excess of holo-transferrin. On the other hand, internalization of labeled holo-transferrin by these cells was not inhibited by H-ferritin. Chinese hamster ovary cells lacking functional endogenous TFR1 but expressing human TFR1 with a mutated RGD sequence, which is required for transferrin binding, efficiently incorporated H-ferritin, indicating that TFR1 has distinct binding sites for H-ferritin and holo-transferrin. H-ferritin uptake by these cells required a threshold level of cell surface TFR1 expression, whereas there was no threshold for holo-transferrin uptake. The requirement for a threshold level of TFR1 expression can explain why among primary human hematopoietic cells, only erythroblasts efficiently take up H-ferritin. PMID:26441243

Crystal structures of the NO sensor NsrR reveal how its iron-sulfur cluster modulates DNA binding

NASA Astrophysics Data System (ADS)

Volbeda, Anne; Dodd, Erin L.; Darnault, Claudine; Crack, Jason C.; Renoux, Oriane; Hutchings, Matthew I.; Le Brun, Nick E.; Fontecilla-Camps, Juan C.

2017-04-01

NsrR from Streptomyces coelicolor (Sc) regulates the expression of three genes through the progressive degradation of its [4Fe-4S] cluster on nitric oxide (NO) exposure. We report the 1.95 Å resolution crystal structure of dimeric holo-ScNsrR and show that the cluster is coordinated by the three invariant Cys residues from one monomer and, unexpectedly, Asp8 from the other. A cavity map suggests that NO displaces Asp8 as a cluster ligand and, while D8A and D8C variants remain NO sensitive, DNA binding is affected. A structural comparison of holo-ScNsrR with an apo-IscR-DNA complex shows that the [4Fe-4S] cluster stabilizes a turn between ScNsrR Cys93 and Cys99 properly oriented to interact with the DNA backbone. In addition, an apo ScNsrR structure suggests that Asn97 from this turn, along with Arg12, which forms a salt-bridge with Asp8, are instrumental in modulating the position of the DNA recognition helix region relative to its major groove.

Deciphering Cryptic Binding Sites on Proteins by Mixed-Solvent Molecular Dynamics.

PubMed

Kimura, S Roy; Hu, Hai Peng; Ruvinsky, Anatoly M; Sherman, Woody; Favia, Angelo D

2017-06-26

In recent years, molecular dynamics simulations of proteins in explicit mixed solvents have been applied to various problems in protein biophysics and drug discovery, including protein folding, protein surface characterization, fragment screening, allostery, and druggability assessment. In this study, we perform a systematic study on how mixtures of organic solvent probes in water can reveal cryptic ligand binding pockets that are not evident in crystal structures of apo proteins. We examine a diverse set of eight PDB proteins that show pocket opening induced by ligand binding and investigate whether solvent MD simulations on the apo structures can induce the binding site observed in the holo structures. The cosolvent simulations were found to induce conformational changes on the protein surface, which were characterized and compared with the holo structures. Analyses of the biological systems, choice of probes and concentrations, druggability of the resulting induced pockets, and application to drug discovery are discussed here.

Cross-linking mass spectrometry and mutagenesis confirm the functional importance of surface interactions between CYP3A4 and holo/apo cytochrome b(5).

PubMed

Zhao, Chunsheng; Gao, Qiuxia; Roberts, Arthur G; Shaffer, Scott A; Doneanu, Catalin E; Xue, Song; Goodlett, David R; Nelson, Sidney D; Atkins, William M

2012-11-27

Cytochrome b(5) (cyt b(5)) is one of the key components in the microsomal cytochrome P450 monooxygenase system. Consensus has not been reached about the underlying mechanism of cyt b(5) modulation of CYP catalysis. Both cyt b(5) and apo b(5) are reported to stimulate the activity of several P450 isoforms. In this study, the surface interactions of both holo and apo b(5) with CYP3A4 were investigated and compared for the first time. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions between the protein surfaces. Subsequently, the models of interaction of holo/apo b(5) with CYP3A4 were built using the identified interacting sites as constraints. Both cyt b(5) and apo b(5) were predicted to bind to the same groove on CYP3A4 with close contacts to the B-B' loop of CYP3A4, a substrate recognition site. Mutagenesis studies further confirmed that the interacting sites on CYP3A4 (Lys96, Lys127, and Lys421) are functionally important. Mutation of these residues reduced or abolished cyt b(5) binding affinity. The critical role of Arg446 on CYP3A4 in binding to cyt b(5) and/or cytochrome P450 reductase was also discovered. The results indicated that electrostatic interactions on the interface of the two proteins are functionally important. The results indicate that apo b(5) can dock with CYP3A4 in a manner analogous to that of holo b(5), so electron transfer from cyt b(5) is not required for its effects.

Conformational Transitions upon Ligand Binding: Holo-Structure Prediction from Apo Conformations

PubMed Central

Seeliger, Daniel; de Groot, Bert L.

2010-01-01

Biological function of proteins is frequently associated with the formation of complexes with small-molecule ligands. Experimental structure determination of such complexes at atomic resolution, however, can be time-consuming and costly. Computational methods for structure prediction of protein/ligand complexes, particularly docking, are as yet restricted by their limited consideration of receptor flexibility, rendering them not applicable for predicting protein/ligand complexes if large conformational changes of the receptor upon ligand binding are involved. Accurate receptor models in the ligand-bound state (holo structures), however, are a prerequisite for successful structure-based drug design. Hence, if only an unbound (apo) structure is available distinct from the ligand-bound conformation, structure-based drug design is severely limited. We present a method to predict the structure of protein/ligand complexes based solely on the apo structure, the ligand and the radius of gyration of the holo structure. The method is applied to ten cases in which proteins undergo structural rearrangements of up to 7.1 Å backbone RMSD upon ligand binding. In all cases, receptor models within 1.6 Å backbone RMSD to the target were predicted and close-to-native ligand binding poses were obtained for 8 of 10 cases in the top-ranked complex models. A protocol is presented that is expected to enable structure modeling of protein/ligand complexes and structure-based drug design for cases where crystal structures of ligand-bound conformations are not available. PMID:20066034

The Tom Core Complex

PubMed Central

Ahting, Uwe; Thun, Clemens; Hegerl, Reiner; Typke, Dieter; Nargang, Frank E.; Neupert, Walter; Nussberger, Stephan

1999-01-01

Translocation of nuclear-encoded preproteins across the outer membrane of mitochondria is mediated by the multicomponent transmembrane TOM complex. We have isolated the TOM core complex of Neurospora crassa by removing the receptors Tom70 and Tom20 from the isolated TOM holo complex by treatment with the detergent dodecyl maltoside. It consists of Tom40, Tom22, and the small Tom components, Tom6 and Tom7. This core complex was also purified directly from mitochondria after solubilization with dodecyl maltoside. The TOM core complex has the characteristics of the general insertion pore; it contains high-conductance channels and binds preprotein in a targeting sequence-dependent manner. It forms a double ring structure that, in contrast to the holo complex, lacks the third density seen in the latter particles. Three-dimensional reconstruction by electron tomography exhibits two open pores traversing the complex with a diameter of ∼2.1 nm and a height of ∼7 nm. Tom40 is the key structural element of the TOM core complex. PMID:10579717

Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity.

PubMed

Sayer, Christopher; Isupov, Michail N; Westlake, Aaron; Littlechild, Jennifer A

2013-04-01

The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases.

Molecular LEGO by domain-imprinting of cytochrome P450 BM3.

PubMed

Jetzschmann, K J; Yarman, A; Rustam, L; Kielb, P; Urlacher, V B; Fischer, A; Weidinger, I M; Wollenberger, U; Scheller, F W

2018-04-01

Electrosynthesis of the MIP nano-film after binding of the separated domains or holo-cytochrome BM3 via an engineered anchor should result in domain-specific cavities in the polymer layer. Both the two domains and the holo P450 BM3 have been bound prior polymer deposition via a N-terminal engineered his6-anchor to the electrode surface. Each step of MIP preparation was characterized by cyclic voltammetry of the redox-marker ferricyanide. Rebinding after template removal was evaluated by quantifying the suppression of the diffusive permeability of the signal for ferricyanide and by the NADH-dependent reduction of cytochrome c by the reductase domain (BMR). The working hypothesis is verified by the discrimination of the two domains by the respective MIPs: The holoenzyme P450 BM3 was ca. 5.5 times more effectively recognized by the film imprinted with the oxidase domain (BMO) as compared to the BMR-MIP or the non-imprinted polymer (NIP). Obviously, a cavity is formed during the imprinting process around the his 6 -tag-anchored BMR which cannot accommodate the broader BMO or the P450 BM3. The affinity of the MIP towards P450 BM3 is comparable with that to the monomer in solution. The his 6 -tagged P450 BM3 binds (30 percent) stronger which shows the additive effect of the interaction with the MIP and the binding to the electrode. Copyright © 2018. Published by Elsevier B.V.

Unfolding pathway of CotA-laccase and the role of copper on the prevention of refolding through aggregation of the unfolded state

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fernandes, Andre T.; Lopes, Carlos; Martins, Ligia O.

2012-06-08

Highlights: Black-Right-Pointing-Pointer CotA-laccase unfolds with an intermediate state. Black-Right-Pointing-Pointer Copper stabilizes the native and the intermediate state. Black-Right-Pointing-Pointer Copper binding to the unfolded state prevents refolding through protein aggregation. Black-Right-Pointing-Pointer Copper incorporation in CotA-laccase occurs as a later step during folding. -- Abstract: Copper is a redox-active metal and the main player in electron transfer reactions occurring in multicopper oxidases. The role of copper in the unfolding pathway and refolding of the multicopper oxidase CotA laccase in vitro was solved using double-jump stopped-flow experiments. Unfolding of apo- and holo-CotA was described as a three-state process with accumulation of an intermediatemore » in between the native and unfolded state. Copper stabilizes the native holo-CotA but also the intermediate state showing that copper is still bound to this state. Also, copper binds to unfolded holo-CotA in a non-native coordination promoting CotA aggregation and preventing refolding to the native structure. These results gather information on unfolding/folding pathways of multicopper oxidases and show that copper incorporation in vivo should be a tight controlled process as copper binding to the unfolded state under native conditions promotes protein aggregation.« less

Role of protein-glutathione contacts in defining glutaredoxin-3 [2Fe-2S] cluster chirality, ligand exchange and transfer chemistry.

PubMed

Sen, Sambuddha; Cowan, J A

2017-10-01

Monothiol glutaredoxins (Grx) serve as intermediate cluster carriers in iron-sulfur cluster trafficking. The [2Fe-2S]-bound holo forms of Grx proteins display cysteinyl coordination from exogenous glutathione (GSH), in addition to contact from protein-derived Cys. Herein, we report mechanistic studies that investigate the role of exogenous glutathione in defining cluster chirality, ligand exchange, and the cluster transfer chemistry of Saccharomyces cerevisiae Grx3. Systematic perturbations were introduced to the glutathione-binding site by substitution of conserved charged amino acids that form crucial electrostatic contacts with the glutathione molecule. Native Grx3 could also be reconstituted in the absence of glutathione, with either DTT, BME or free L-cysteine as the source of the exogenous Fe-S ligand contact, while retaining full functional reactivity. The delivery of the [2Fe-2S] cluster to Grx3 from cluster donor proteins such as Isa, Nfu, and a [2Fe-2S](GS) 4 complex, revealed that electrostatic contacts are of key importance for positioning the exogenous glutathione that in turn influences the chiral environment of the cluster. All Grx3 derivatives were reconstituted by standard chemical reconstitution protocols and found to transfer cluster to apo ferredoxin 1 (Fdx1) at rates comparable to native protein, even when using DTT, BME or free L-cysteine as a thiol source in place of GSH during reconstitution. Kinetic analysis of cluster transfer from holo derivatives to apo Fdx1 has led to a mechanistic model for cluster transfer chemistry of native holo Grx3, and identification of the likely rate-limiting step for the reaction.

The Active Form of the Saccharomyces cerevisiae Ribonucleotide Reductase Small Subunit Is a Heterodimer in Vitro and in Vivo†

PubMed Central

Perlstein, Deborah L.; Ge, Jie; Ortigosa, Allison D.; Robblee, John H.; Zhang, Zhen; Huang, Mingxia; Stubbe, JoAnne

2015-01-01

The class I ribonucleotide reductases (RNRs) are composed of two homodimeric subunits: R1 and R2. R2 houses a diferric-tyrosyl radical (Y•) cofactor. Saccharomyces cerevisiae has two R2s: Y2 (β2) and Y4 (β′2). Y4 is an unusual R2 because three residues required for iron binding have been mutated. While the heterodimer (ββ′) is thought to be the active form, several rnr4Δ strains are viable. To resolve this paradox, N-terminally epitope-tagged β and β′ were expressed in E. coli or integrated into the yeast genome. In vitro exchange studies reveal that when apo-(His6)-β2 (Hisβ2) is mixed with β′2, apo-Hisββ′ forms quantitatively within 2 min. In contrast, holo-ββ′ fails to exchange with apo-Hisβ2 to form holo-Hisββ and β′2. Isolation of genomically encoded tagged β or β′ from yeast extracts gave a 1:1 complex of β and β′, suggesting that ββ′ is the active form. The catalytic activity, protein concentrations, and Y• content of the rnr4Δ and wild type (wt) strains were compared to clarify the role of β′ in vivo. The Y• content of rnr4Δ is 15-fold less than that of wt, consistent with the observed low activity of rnr4Δ extracts (<0.01 nmol min−1 mg−1) versus wt (0.06 ± 0.01 nmol min−1 mg−1). FLAGβ2 isolated from the rnr4Δ strain has a specific activity of 2 nmol min−1 mg−1, similar to that of reconstituted apo-Hisβ2 (10 nmol min−1 mg−1), but significantly less than holo-Hisββ′ (~2000 nmol min−1 mg−1). These studies together demonstrate that β′ plays a crucial role in cluster assembly in vitro and in vivo and that the active form of the yeast R2 is ββ′. PMID:16285741

Performance characteristics of the ARCHITECT Active-B12 (Holotranscobalamin) assay.

PubMed

Merrigan, Stephen D; Owen, William E; Straseski, Joely A

2015-01-01

Vitamin B12 (cobalamin) is a necessary cofactor in methionine and succinyl-CoA metabolism. Studies estimate the deficiency prevalence as high as 30% in the elderly population. Ten to thirty percent of circulating cobalamin is bound to transcobalamin (holotranscobalamin, holoTC) which can readily enter cells and is therefore considered the bioactive form. The objective of our study was to evaluate the analytical performance of a high-throughput, automated holoTC assay (ARCHITECT i2000(SR) Active-B12 (Holotranscobalamin)) and compare it to other available methods. Manufacturer-specified limits of blank (LoB), detection (LoD), and quantitation (LoQ), imprecision, interference, and linearity were evaluated for the ARCHITECT HoloTC assay. Residual de-identified serum samples were used to compare the ARCHITECT HoloTC assay with the automated AxSYM Active-B12 (Holotranscobalamin) assay (Abbott Diagnostics) and the manual Active-B12 (Holotranscobalamin) Enzyme Immunoassay (EIA) (Axis-Shield Diagnostics, Dundee, Scotland, UK). Manufacturer's claims of LoB, LoD, LoQ, imprecision, interference, and linearity to the highest point tested (113.4 pmol/L) were verified for the ARCHITECT HoloTC assay. Method comparison of the ARCHITECT HoloTC to the AxSYM HoloTC produced the following Deming regression statistics: (ARCHITECT(HoloTc)) = 0.941 (AxSYM(HoloTC)) + 1.2 pmol/L, S(y/x) = 6.4, r = 0.947 (n = 98). Comparison to the Active-B12 EIA produced: (ARCHITECT(HoloTC)) = 1.105 (EIA(Active-B12)) - 6.8 pmol/L, S(y/x) = 11.0, r = 0.950 (n = 221). This assay performed acceptably for LoB, LoD, LoQ, imprecision, interference, linearity and method comparison to the predicate device (AxSYM). An additional comparison to a manual Active-B12 EIA method performed similarly, with minor exceptions. This study determined that the ARCHITECT HoloTC assay is suitable for routine clinical use, which provides a high-throughput alternative for automated testing of this emerging marker of cobalamin deficiency.

Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity

PubMed Central

Sayer, Christopher; Isupov, Michail N.; Westlake, Aaron; Littlechild, Jennifer A.

2013-01-01

The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-­aminotransferases. PMID:23519665

Mechanism and regulation of mycobactin fatty acyl-AMP ligase FadD33.

PubMed

Vergnolle, Olivia; Xu, Hua; Blanchard, John S

2013-09-27

Mycobacterial siderophores are critical components for bacterial virulence in the host. Pathogenic mycobacteria synthesize iron chelating siderophores named mycobactin and carboxymycobactin to extract intracellular macrophage iron. The two siderophores differ in structure only by a lipophilic aliphatic chain attached on the ε-amino group of the lysine mycobactin core, which is transferred by MbtK. Prior to acyl chain transfer, the lipophilic chain requires activation by a specific fatty acyl-AMP ligase FadD33 (also known as MbtM) and is then loaded onto phosphopantetheinylated acyl carrier protein (holo-MbtL) to form covalently acylated MbtL. We demonstrate that FadD33 prefers long chain saturated lipids and initial velocity studies showed that FadD33 proceeds via a Bi Uni Uni Bi ping-pong mechanism. Inhibition experiments suggest that, during the first half-reaction (adenylation), fatty acid binds first to the free enzyme, followed by ATP and the release of pyrophosphate to form the adenylate intermediate. During the second half-reaction (ligation), holo-MbtL binds to the enzyme followed by the release of products AMP and acylated MbtL. In addition, we characterized a post-translational regulation mechanism of FadD33 by the mycobacterial protein lysine acetyltransferase in a cAMP-dependent manner. FadD33 acetylation leads to enzyme inhibition, which can be reversed by the NAD(+)-dependent deacetylase, MSMEG_5175 (DAc1). To the best of our knowledge, this is the first time that bacterial siderophore synthesis has been shown to be regulated via post-translational protein acetylation.

The Solution Structure, Binding Properties, and Dynamics of the Bacterial Siderophore-binding Protein FepB*

PubMed Central

Chu, Byron C. H.; Otten, Renee; Krewulak, Karla D.; Mulder, Frans A. A.; Vogel, Hans J.

2014-01-01

The periplasmic binding protein (PBP) FepB plays a key role in transporting the catecholate siderophore ferric enterobactin from the outer to the inner membrane in Gram-negative bacteria. The solution structures of the 34-kDa apo- and holo-FepB from Escherichia coli, solved by NMR, represent the first solution structures determined for the type III class of PBPs. Unlike type I and II PBPs, which undergo large “Venus flytrap” conformational changes upon ligand binding, both forms of FepB maintain similar overall folds; however, binding of the ligand is accompanied by significant loop movements. Reverse methyl cross-saturation experiments corroborated chemical shift perturbation results and uniquely defined the binding pocket for gallium enterobactin (GaEnt). NMR relaxation experiments indicated that a flexible loop (residues 225–250) adopted a more rigid and extended conformation upon ligand binding, which positioned residues for optimal interactions with the ligand and the cytoplasmic membrane ABC transporter (FepCD), respectively. In conclusion, this work highlights the pivotal role that structural dynamics plays in ligand binding and transporter interactions in type III PBPs. PMID:25173704

Molecular requirements for RNA-induced silencing complex assembly in the Drosophila RNA interference pathway.

PubMed

Pham, John W; Sontheimer, Erik J

2005-11-25

Complexes in the Drosophila RNA-induced silencing complex (RISC) assembly pathway can be resolved using native gel electrophoresis, revealing an initiator called R1, an intermediate called R2, and an effector called R3 (now referred to as holo-RISC). Here we show that R1 forms when the Dicer-2/R2D2 heterodimer binds short interfering RNA (siRNA) duplexes. The heterodimer alone can initiate RISC assembly, indicating that other factors are dispensable for initiation. During assembly, R2 requires Argonaute 2 to convert into holo-RISC. This requirement is reminiscent of the RISC-loading complex, which also requires Argonaute 2 for assembly into RISC. We have compared R2 to the RISC-loading complex and show that the two complexes are similar in their sensitivities to ATP and to chemical modifications on siRNA duplexes, indicating that they are likely to be identical. We have examined the requirements for RISC formation and show that the siRNA 5'-termini are repeatedly monitored during RISC assembly, first by the Dcr-2/R2D2 heterodimer and again after R2 formation, before siRNA unwinding. The 2'-position of the 5'-terminal nucleotide also affects RISC assembly, because an siRNA strand bearing a 2'-deoxyribose at this position can inhibit the cognate strand from entering holo-RISC; in contrast, the 2'-deoxyribose-modified strand has enhanced activity in the RNA interference pathway.

Molecular dynamics simulation unveils the conformational flexibility of the interdomain linker in the bacterial transcriptional regulator GabR from Bacillus subtilis bound to pyridoxal 5’-phosphate

PubMed Central

Narzi, Daniele; Guidoni, Leonardo

2017-01-01

GabR from Bacillus subtilis is a transcriptional regulator belonging to the MocR subfamily of the GntR regulators. The structure of the MocR regulators is characterized by the presence of two domains: i) a N-terminal domain, about 60 residue long, possessing the winged-Helix-Turn-Helix (wHTH) architecture with DNA recognition and binding capability; ii) a C-terminal domain (about 350 residue) folded as the pyridoxal 5’-phosphate (PLP) dependent aspartate aminotransferase (AAT) with dimerization and effector binding functions. The two domains are linked to each other by a peptide bridge. Although structural and functional characterization of MocRs is proceeding at a fast pace, virtually nothing is know about the molecular changes induced by the effector binding and on how these modifications influence the properties of the regulator. An extensive molecular dynamics simulation on the crystallographic structure of the homodimeric B. subtilis GabR has been undertaken with the aim to envisage the role and the importance of conformational flexibility in the action of GabR. Molecular dynamics has been calculated for the apo (without PLP) and holo (with PLP bound) forms of the GabR. A comparison between the molecular dynamics trajectories calculated for the two GabR forms suggested that one of the wHTH domain detaches from the AAT-like domain in the GabR PLP-bound form. The most evident conformational change in the holo PLP-bound form is represented by the rotation and the subsequent detachment from the subunit surface of one of the wHTH domains. The movement is mediated by a rearrangement of the linker connecting the AAT domain possibly triggered by the presence of the negative charge of the PLP cofactor. This is the second most significant conformational modification. The C-terminal section of the linker docks into the “active site” pocket and establish stabilizing contacts consisting of hydrogen-bonds, salt-bridges and hydrophobic interactions. PMID:29253008

Future Of Visual Entertainment

NASA Astrophysics Data System (ADS)

Dryer, Ivan

1983-10-01

The development of new visual entertainment forms has and will continue to have a powerful impact on the direction of our society. Foremost among these new forms will be the Holo's--moving Holographic images of anything imaginable, projected in mid air (a room, a dome) and so lifelike they are virtually indistinguishable from "reality". The Holo's and space development will ultimately transform entertainment and in the process, humanity, too. Meanwhile, the seeds of these changes are now being planted in entertainment trends and innovations whose implications are just beginning to emerge.

Folding pathway of the pyridoxal 5′-phosphate C-S lyase MalY from Escherichia coli

PubMed Central

2005-01-01

MalY from Escherichia coli is a bifunctional dimeric PLP (pyridoxal 5′-phosphate) enzyme acting as a β-cystathionase and as a repressor of the maltose system. The spectroscopic and molecular properties of the holoenzyme, in the untreated and NaBH4-treated forms, and of the apoenzyme have been elucidated. A systematic study of the urea-induced unfolding of MalY has been monitored by gel filtration, cross-linking, ANS (8-anilino-1-naphthalenesulphonic acid) binding and by visible, near- and far-UV CD, fluorescence and NMR spectroscopies under equilibrium conditions. Unfolding proceeds in at least three stages. The first transition, occurring between 0 and 1 M urea, gives rise to a partially active dimeric species that binds PLP. The second equilibrium transition involving dimer dissociation, release of PLP and loss of lyase activity leads to the formation of a monomeric equilibrium intermediate. It is a partially unfolded molecule that retains most of the native-state secondary structure, binds significant amounts of ANS (a probe for exposed hydrophobic surfaces) and tends to self-associate. The self-associated aggregates predominate at urea concentrations of 2–4 M for holoMalY. The third step represents the complete unfolding of the enzyme. These results when compared with the urea-induced unfolding profiles of apoMalY and NaBH4-reduced holoenzyme suggest that the coenzyme group attached to the active-site lysine residue increases the stability of the dimeric enzyme. Both holo- and apo-MalY could be successfully refolded into the active enzyme with an 85% yield. Further refolding studies suggest that large misfolded soluble aggregates that cannot be refolded could be responsible for the incomplete re-activation. PMID:15823094

Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sayer, Christopher; Isupov, Michail N.; Westlake, Aaron

2013-04-01

The X-ray structures of two ω-aminotransferases from P. aeruginosa and C. violaceum in complex with an inhibitor offer the first detailed insight into the structural basis of the substrate specificity of these industrially important enzymes. The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes showmore » activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases.« less

A structural comparison of 'real' and 'model' calmodulin clarified allosteric interactions regulating domain motion.

PubMed

Shimoyama, Hiromitsu

2018-05-07

Calmodulin (CaM) is a multifunctional calcium-binding protein, which regulates various biochemical processes. CaM acts via structural changes and complex forming with its target enzymes. CaM has two globular domains (N-lobe and C-lobe) connected by a long linker region. Upon calcium binding, the N-lobe and C-lobe undergo local conformational changes, after that, entire CaM wraps the target enzyme through a large conformational change. However, the regulation mechanism, such as allosteric interactions regulating the conformational changes, is still unclear. In order to clarify the allosteric interactions, in this study, experimentally obtained 'real' structures are compared to 'model' structures lacking the allosteric interactions. As the allosteric interactions would be absent in calcium-free CaM (apo-CaM), allostery-eliminated calcium-bound CaM (holo-CaM) models were constructed by combining the apo-CaM's linker and the holo-CaM's N- and C-lobe. Before the comparison, the 'real' and 'model' structures were clustered and cluster-cluster relationship was determined by a principal component analysis. The structures were compared based on the relationship, then, a distance map and a contact probability analysis clarified that the inter-domain motion is regulated by several groups of inter-domain contacting residue pairs. The analyses suggested that these residues cause inter-domain translation and rotation, and as a consequence, the motion encourage structural diversity. The resultant diversity would contribute to the functional versatility of CaM.

The impact of highly hydrophobic material on the structure of transferrin and its ability to bind iron.

PubMed

Drug, E; Fadeev, L; Gozin, M

2011-05-30

Transferrin is a blood-plasma glycoprotein, which is responsible for ferric-ion delivery and which functions as the most important ferric pool in the body. The reversible complexation process of Fe(3+) ions is associated with conformational changes of the three-dimensional structure of the transferrin. This conformational dynamics is attributed to a partial unfolding of the N-lobe of the protein and could be described as a transition between the holo to the apo forms of the transferrin. The aim of the present work is to demonstrate the unprecedented ability of the transferrin to solubilize various polycyclic aromatic hydrocarbons in physiological solution and to explore the impact of these materials on the structure and functionality of the transferrin. The synthesis and characterization of novel materials, consisting of complexes between human transferrin and hydrophobic high-carbon-content compounds, is reported here for the first time. Furthermore, it is shown that the preparation of these complexes from holo-transferrin leads to an irreversible loss of the ferric ions from the protein. Analytical studies of these novel complexes may shed a light on the mechanism by which transferrin could lose its ability to bind and thus to transport and store iron. These findings clearly demonstrate a possible damaging impact of various hydrophobic pollutants, which can enter an organism by inhalation or ingestion, on the functionality of the transferrin. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Implicit ligand theory for relative binding free energies

NASA Astrophysics Data System (ADS)

Nguyen, Trung Hai; Minh, David D. L.

2018-03-01

Implicit ligand theory enables noncovalent binding free energies to be calculated based on an exponential average of the binding potential of mean force (BPMF)—the binding free energy between a flexible ligand and rigid receptor—over a precomputed ensemble of receptor configurations. In the original formalism, receptor configurations were drawn from or reweighted to the apo ensemble. Here we show that BPMFs averaged over a holo ensemble yield binding free energies relative to the reference ligand that specifies the ensemble. When using receptor snapshots from an alchemical simulation with a single ligand, the new statistical estimator outperforms the original.

Purification and characterisation of the fission yeast Ndc80 complex.

PubMed

Matsuo, Yuzy; Maurer, Sebastian P; Surrey, Thomas; Toda, Takashi

2017-07-01

The Ndc80 complex is a conserved outer kinetochore protein complex consisting of Ndc80 (Hec1), Nuf2, Spc24 and Spc25. This complex comprises a major, if not the sole, platform with which the plus ends of the spindle microtubules directly interact. In fission yeast, several studies indicate that multiple microtubule-associated proteins including the Dis1/chTOG microtubule polymerase and the Mal3/EB1 microtubule plus-end tracking protein directly or indirectly bind Ndc80, thereby ensuring stable kinetochore-microtubule attachment. However, the purification of the Ndc80 complex from this yeast has not been achieved, which hampers the in-depth investigation as to how the outer kinetochore attaches to the plus end of the spindle microtubule. Here we report the two-step purification of the fission yeast Ndc80 holo complex from bacteria. First, we purified separately two sub-complexes consisting of Ndc80-Nuf2 and Spc24-Spc25. Then, these two sub-complexes were mixed and applied to size-exclusion chromatography. The reconstituted Ndc80 holo complex is composed of four subunits with equal stoichiometry. The complex possesses microtubule-binding activity, and Total Internal Reflection Fluorescence (TIRF)-microscopy assays show that the complex binds the microtubule lattice. Interestingly, unlike the human complex, the fission yeast complex does not track depolymerising microtubule ends. Further analysis shows that under physiological ionic conditions, the Ndc80 holo complex does not detectably bind Dis1, but instead it interacts with Mal3/EB1, by which the Ndc80 complex tracks the growing microtubule plus end. This result substantiates the notion that the Ndc80 complex plays a crucial role in establishment of the dynamic kinetochore-microtubule interface by cooperating with chTOG and EB1. Copyright © 2017 The Francis Crick Institute. Published by Elsevier Inc. All rights reserved.

Kinetic and thermodynamic parameters for heat denaturation of human recombinant lactoferrin from rice.

PubMed

Castillo, Eduardo; Pérez, María Dolores; Franco, Indira; Calvo, Miguel; Sánchez, Lourdes

2012-06-01

Heat denaturation of recombinant human lactoferrin (rhLf) from rice with 3 different iron-saturation degrees, holo rhLf (iron-saturated), AsIs rhLf (60% iron saturation), and apo rhLf (iron-depleted), was studied. The 3 forms of rhLf were subjected to heat treatment, and the kinetic and thermodynamic parameters of the denaturation process were determined. Thermal denaturation of rhLf was assessed by measuring the loss of reactivity against specific antibodies. D(t) values (time to reduce 90% of immunoreactivity) decreased with increasing temperature of treatment for apo and holo rhLf, those values being higher for the iron-saturated form, which indicates that iron confers thermal stability to rhLf. However, AsIs rhLf showed a different behaviour with an increase in resistance to heat between 79 °C and 84 °C, so that the kinetic parameters could not be calculated. The heat denaturation process for apo and holo rhLf was best described assuming a reaction order of 1.5. The activation energy of the denaturation process was 648.20 kJ/mol for holo rhLf and 406.94 kJ/mol for apo rhLf, confirming that iron-depleted rhLf is more sensitive to heat treatment than iron-saturated rhLf.

Concurrent Increases and Decreases in Local Stability and Conformational Heterogeneity in Cu, Zn Superoxide Dismutase Variants Revealed by Temperature-Dependence of Amide Chemical Shifts.

PubMed

Doyle, Colleen M; Rumfeldt, Jessica A; Broom, Helen R; Sekhar, Ashok; Kay, Lewis E; Meiering, Elizabeth M

2016-03-08

The chemical shifts of backbone amide protons in proteins are sensitive reporters of local structural stability and conformational heterogeneity, which can be determined from their readily measured linear and nonlinear temperature-dependences, respectively. Here we report analyses of amide proton temperature-dependences for native dimeric Cu, Zn superoxide dismutase (holo pWT SOD1) and structurally diverse mutant SOD1s associated with amyotrophic lateral sclerosis (ALS). Holo pWT SOD1 loses structure with temperature first at its periphery and, while having extremely high global stability, nevertheless exhibits extensive conformational heterogeneity, with ∼1 in 5 residues showing evidence for population of low energy alternative states. The holo G93A and E100G ALS mutants have moderately decreased global stability, whereas V148I is slightly stabilized. Comparison of the holo mutants as well as the marginally stable immature monomeric unmetalated and disulfide-reduced (apo(2SH)) pWT with holo pWT shows that changes in the local structural stability of individual amides vary greatly, with average changes corresponding to differences in global protein stability measured by differential scanning calorimetry. Mutants also exhibit altered conformational heterogeneity compared to pWT. Strikingly, substantial increases as well as decreases in local stability and conformational heterogeneity occur, in particular upon maturation and for G93A. Thus, the temperature-dependence of amide shifts for SOD1 variants is a rich source of information on the location and extent of perturbation of structure upon covalent changes and ligand binding. The implications for potential mechanisms of toxic misfolding of SOD1 in disease and for general aspects of protein energetics, including entropy-enthalpy compensation, are discussed.

Expression of holo and apo forms of spinach acyl carrier protein-I in leaves of transgenic tobacco plants.

PubMed Central

Post-Beittenmiller, M A; Schmid, K M; Ohlrogge, J B

1989-01-01

Acyl carrier protein (ACP) is a chloroplast-localized cofactor of fatty acid synthesis, desaturation, and acyl transfer. We have transformed tobacco with a chimeric gene consisting of the tobacco ribulose-1,5-bisphosphate carboxylase promoter and transit peptide and the sequence encoding the mature spinach ACP-I. Spinach ACP-I was expressed in the transformed plants at levels twofold to threefold higher than the endogenous tobacco ACPs as determined by protein immunoblots and assays of ACP in leaf extracts. In addition to these elevated levels of the holo form, there were high levels of apoACP-I, a form lacking the 4'-phosphopantetheine prosthetic group and not previously detected in vivo. The mature forms of both apoACP-I and holoACP-I were located in the chloroplasts, indicating that the transit peptide was cleaved and that attachment of the prosthetic group was not required for uptake into the plastid. There were also significant levels of spinach acyl-ACP-I, demonstrating that spinach ACP-I participated in tobacco fatty acid metabolism. Lipid analyses of the transformed plants indicated that the increased ACP levels caused no significant alterations in leaf lipid biosynthesis. PMID:2535529

The effects of silver ions on copper metabolism in rats.

PubMed

Ilyechova, E Yu; Saveliev, A N; Skvortsov, A N; Babich, P S; Zatulovskaia, Yu A; Pliss, M G; Korzhevskii, D E; Tsymbalenko, N V; Puchkova, L V

2014-10-01

The influence of short and prolonged diet containing silver ions (Ag-diet) on copper metabolism was studied. Two groups of animals were used: one group of adult rats received a Ag-diet for one month (Ag-A1) and another group received a Ag-diet for 6 months from birth (Ag-N6). In Ag-A1 rats, the Ag-diet caused a dramatic decrease of copper status indexes that was manifested as ceruloplasmin-associated copper deficiency. In Ag-N6 rats, copper status indexes decreased only 2-fold as compared to control rats. In rats of both groups, silver entered the bloodstream and accumulated in the liver. Silver was incorporated into ceruloplasmin (Cp), but not SOD1. In the liver, a prolonged Ag-diet caused a decrease of the expression level of genes, associated with copper metabolism. Comparative spectrophotometric analysis of partially purified Cp fractions has shown that Cp from Ag-N6 rats was closer to holo-Cp by specific enzymatic activities and tertiary structure than Cp from Ag-A1 rats. However, Cp of Ag-N6 differs from control holo-Cp and Cp of Ag-A1 in its affinity to DEAE-Sepharose and in its binding properties to lectins. In the bloodstream of Ag-N6, two Cp forms are present as shown in pulse-experiments on rats with the liver isolated from circulation. One of the Cp isoforms is of hepatic origin, and the other is of extrahepatic origin; the latter is characterized by a faster rate of secretion than hepatic Cp. These data allowed us to suggest that the disturbance of holo-Cp formation in the liver was compensated by induction of extrahepatic Cp synthesis. The possible biological importance of these effects is discussed.

Interaction between holo transferrin and HSA-PPIX complex in the presence of lomefloxacin: An evaluation of PPIX aggregation in protein-protein interactions

NASA Astrophysics Data System (ADS)

Sattar, Zohreh; Iranfar, Hediye; Asoodeh, Ahmad; Saberi, Mohammad Reza; Mazhari, Mahboobeh; Chamani, Jamshidkhan

2012-11-01

Human serum albumin (HSA) and holo transferrin (TF) are two serum carrier proteins that are able to interact with each other, thereby altering their binding behavior toward their ligands. During the course of this study, the interaction between HSA-PPIX and TF, in the presence and absence of lomefloxacin (LMF), was for the first time investigated using different spectroscopic and molecular modeling techniques. Fluorescence spectroscopy experiments were performed in order to study conformational changes of proteins. The RLS technique was utilized to investigate the effect of LMF on J-aggregation of PPIX, which is the first report of its kind. Our findings present clear-cut evidence for the alteration of interactions between HSA and TF in the presence of PPIX and changes in drug-binding to HSA and HSA-PPIX complex upon interaction with TF. Moreover, molecular modeling studies suggested that the binding site for LMF became switched in the presence of PPIX, and that LMF bound to the site IIA of HSA. The obtained results should give new insight into research in this field and may cast some light on the dynamics of drugs in biological systems.

Crystal Structures of Copper-depleted and Copper-bound Fungal Pro-tyrosinase

PubMed Central

Fujieda, Nobutaka; Yabuta, Shintaro; Ikeda, Takuya; Oyama, Takuji; Muraki, Norifumi; Kurisu, Genji; Itoh, Shinobu

2013-01-01

Tyrosinase, a dinuclear copper monooxygenase/oxidase, plays a crucial role in the melanin pigment biosynthesis. The structure and functions of tyrosinase have so far been studied extensively, but the post-translational maturation process from the pro-form to the active form has been less explored. In this study, we provide the crystal structures of Aspergillus oryzae full-length pro-tyrosinase in the holo- and the apo-forms at 1.39 and 2.05 Å resolution, respectively, revealing that Phe513 on the C-terminal domain is accommodated in the substrate-binding site as a substrate analog to protect the dicopper active site from substrate access (proteolytic cleavage of the C-terminal domain or deformation of the C-terminal domain by acid treatment transforms the pro-tyrosinase to the active enzyme (Fujieda, N., Murata, M., Yabuta, S., Ikeda, T., Shimokawa, C., Nakamura, Y., Hata, Y., and Itoh, S. (2012) ChemBioChem. 13, 193–201 and Fujieda, N., Murata, M., Yabuta, S., Ikeda, T., Shimokawa, C., Nakamura, Y., Hata, Yl, and Itoh, S. (2013) J. Biol. Inorg. Chem. 18, 19–26). Detailed crystallographic analysis and structure-based mutational studies have shown that the copper incorporation into the active site is governed by three cysteines as follows: Cys92, which is covalently bound to His94 via an unusual thioether linkage in the holo-form, and Cys522 and Cys525 of the CXXC motif located on the C-terminal domain. Molecular mechanisms of the maturation processes of fungal tyrosinase involving the accommodation of the dinuclear copper unit, the post-translational His-Cys thioether cross-linkage formation, and the proteolytic C-terminal cleavage to produce the active tyrosinase have been discussed on the basis of the detailed structural information. PMID:23749993

Biochemical analyses are instrumental in identifying the impact of mutations on holo and/or apo-forms and on the region(s) of alanine:glyoxylate aminotransferase variants associated with Primary Hyperoxaluria Type I☆

PubMed Central

Oppici, Elisa; Montioli, Riccardo; Lorenzetto, Antonio; Bianconi, Silvia; Borri Voltattorni, Carla; Cellini, Barbara

2012-01-01

Primary Hyperoxaluria Type I (PH1) is a disorder of glyoxylate metabolism caused by mutations in the human AGXT gene encoding liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5′-phosphate (PLP) dependent enzyme. Previous investigations highlighted that, although PH1 is characterized by a significant variability in terms of enzymatic phenotype, the majority of the pathogenic variants are believed to share both structural and functional defects, as mainly revealed by data on AGT activity and expression level in crude cellular extracts. However, the knowledge of the defects of the AGT variants at a protein level is still poor. We therefore performed a side-by-side comparison between normal AGT and nine purified recombinant pathogenic variants in terms of catalytic activity, coenzyme binding mode and affinity, spectroscopic features, oligomerization, and thermal stability of both the holo- and apo-forms. Notably, we chose four variants in which the mutated residues are located in the large domain of AGT either within the active site and interacting with the coenzyme or in its proximity, and five variants in which the mutated residues are distant from the active site either in the large or in the small domain. Overall, this integrated analysis of enzymatic activity, spectroscopic and stability information is used to (i) reassess previous data obtained with crude cellular extracts, (ii) establish which form(s) (i.e. holoenzyme and/or apoenzyme) and region(s) (i.e. active site microenvironment, large and/or small domain) of the protein are affected by each mutation, and (iii) suggest the possible therapeutic approach for patients bearing the examined mutations. PMID:22018727

Biochemical analyses are instrumental in identifying the impact of mutations on holo and/or apo-forms and on the region(s) of alanine:glyoxylate aminotransferase variants associated with primary hyperoxaluria type I.

PubMed

Oppici, Elisa; Montioli, Riccardo; Lorenzetto, Antonio; Bianconi, Silvia; Borri Voltattorni, Carla; Cellini, Barbara

2012-01-01

Primary Hyperoxaluria Type I (PH1) is a disorder of glyoxylate metabolism caused by mutations in the human AGXT gene encoding liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP) dependent enzyme. Previous investigations highlighted that, although PH1 is characterized by a significant variability in terms of enzymatic phenotype, the majority of the pathogenic variants are believed to share both structural and functional defects, as mainly revealed by data on AGT activity and expression level in crude cellular extracts. However, the knowledge of the defects of the AGT variants at a protein level is still poor. We therefore performed a side-by-side comparison between normal AGT and nine purified recombinant pathogenic variants in terms of catalytic activity, coenzyme binding mode and affinity, spectroscopic features, oligomerization, and thermal stability of both the holo- and apo-forms. Notably, we chose four variants in which the mutated residues are located in the large domain of AGT either within the active site and interacting with the coenzyme or in its proximity, and five variants in which the mutated residues are distant from the active site either in the large or in the small domain. Overall, this integrated analysis of enzymatic activity, spectroscopic and stability information is used to (i) reassess previous data obtained with crude cellular extracts, (ii) establish which form(s) (i.e. holoenzyme and/or apoenzyme) and region(s) (i.e. active site microenvironment, large and/or small domain) of the protein are affected by each mutation, and (iii) suggest the possible therapeutic approach for patients bearing the examined mutations. Copyright © 2011 Elsevier Inc. All rights reserved.

The structural flexibility of the human copper chaperone Atox1: Insights from combined pulsed EPR studies and computations.

PubMed

Levy, Ariel R; Turgeman, Meital; Gevorkyan-Aiapetov, Lada; Ruthstein, Sharon

2017-08-01

Metallochaperones are responsible for shuttling metal ions to target proteins. Thus, a metallochaperone's structure must be sufficiently flexible both to hold onto its ion while traversing the cytoplasm and to transfer the ion to or from a partner protein. Here, we sought to shed light on the structure of Atox1, a metallochaperone involved in the human copper regulation system. Atox1 shuttles copper ions from the main copper transporter, Ctr1, to the ATP7b transporter in the Golgi apparatus. Conventional biophysical tools such as X-ray or NMR cannot always target the various conformational states of metallochaperones, owing to a requirement for crystallography or low sensitivity and resolution. Electron paramagnetic resonance (EPR) spectroscopy has recently emerged as a powerful tool for resolving biological reactions and mechanisms in solution. When coupled with computational methods, EPR with site-directed spin labeling and nanoscale distance measurements can provide structural information on a protein or protein complex in solution. We use these methods to show that Atox1 can accommodate at least four different conformations in the apo state (unbound to copper), and two different conformations in the holo state (bound to copper). We also demonstrate that the structure of Atox1 in the holo form is more compact than in the apo form. Our data provide insight regarding the structural mechanisms through which Atox1 can fulfill its dual role of copper binding and transfer. © 2017 The Protein Society.

Holo-Chidi video concentrator card

NASA Astrophysics Data System (ADS)

Nwodoh, Thomas A.; Prabhakar, Aditya; Benton, Stephen A.

2001-12-01

The Holo-Chidi Video Concentrator Card is a frame buffer for the Holo-Chidi holographic video processing system. Holo- Chidi is designed at the MIT Media Laboratory for real-time computation of computer generated holograms and the subsequent display of the holograms at video frame rates. The Holo-Chidi system is made of two sets of cards - the set of Processor cards and the set of Video Concentrator Cards (VCCs). The Processor cards are used for hologram computation, data archival/retrieval from a host system, and for higher-level control of the VCCs. The VCC formats computed holographic data from multiple hologram computing Processor cards, converting the digital data to analog form to feed the acousto-optic-modulators of the Media lab's Mark-II holographic display system. The Video Concentrator card is made of: a High-Speed I/O (HSIO) interface whence data is transferred from the hologram computing Processor cards, a set of FIFOs and video RAM used as buffer for data for the hololines being displayed, a one-chip integrated microprocessor and peripheral combination that handles communication with other VCCs and furnishes the card with a USB port, a co-processor which controls display data formatting, and D-to-A converters that convert digital fringes to analog form. The co-processor is implemented with an SRAM-based FPGA with over 500,000 gates and controls all the signals needed to format the data from the multiple Processor cards into the format required by Mark-II. A VCC has three HSIO ports through which up to 500 Megabytes of computed holographic data can flow from the Processor Cards to the VCC per second. A Holo-Chidi system with three VCCs has enough frame buffering capacity to hold up to thirty two 36Megabyte hologram frames at a time. Pre-computed holograms may also be loaded into the VCC from a host computer through the low- speed USB port. Both the microprocessor and the co- processor in the VCC can access the main system memory used to store control programs and data for the VCC. The Card also generates the control signals used by the scanning mirrors of Mark-II. In this paper we discuss the design of the VCC and its implementation in the Holo-Chidi system.

Disulfide bridge regulates ligand-binding site selectivity in liver bile acid-binding proteins.

PubMed

Cogliati, Clelia; Tomaselli, Simona; Assfalg, Michael; Pedò, Massimo; Ferranti, Pasquale; Zetta, Lucia; Molinari, Henriette; Ragona, Laura

2009-10-01

Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand-binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein-binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes.

Computing the binding affinity of Zn2+ in human carbonic anhydrase II on the basis of all-atom molecular dynamics simulations.

NASA Astrophysics Data System (ADS)

Wambo, Thierry; Rodriguez, Roberto

Human carbonic anhydrase II (hCAII) is a metalloenzyme with a Zinc cation at its binding site. The presence of the Zinc turns the protein into an efficient enzyme which catalyzes the reversible hydration of carbon dioxide into bicarbonate anion. Available X-ray structures of the apo-hCAII and holo-hCAII show no significant differences in the overall structure of these proteins. What difference, if any, is there between the structures of the hydrated apo-hCAII and holo? How can we use computer simulation to efficiently compute the binding affinity of Zinc to hCAII? We will present a scheme developed to compute the binding affinity of Zinc cation to hCAII on the basis of all-atom molecular dynamics simulation where Zinc is represented as a point charge and the CHARMM36 force field is used for running the dynamics of the system. Our computed binding affinity of the cation to hCAII is in good agreement with experiment, within the margin of error, while a look at the dynamics of the binding site suggests that in the absence of the Zinc, there is a re-organization of the nearby histidine residues which adopt a new distinct configuration. The authors are thankful for the NIH support through Grants GM084834 and GM060655. They also acknowledge the Texas Advanced Computing Center at the University of Texas at Austin for the supercomputing time. They thank Dr Liao Chen for his comments.

Structure-based mutational analysis of the 4'-phosphopantetheinyl transferases Sfp from Bacillus subtilis: carrier protein recognition and reaction mechanism.

PubMed

Mofid, Mohammad Reza; Finking, Robert; Essen, Lars Oliver; Marahiel, Mohamed A

2004-04-13

The activation of apo-peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases (NRPSs), apo-acyl carrier proteins (ACPs) of polyketide synthases (PKSs), and fatty acid synthases (FASs) to their active holo form is accomplished with dedicated 4'-phosphopantetheinyl transferases (PPTases). They catalyze the transfer of the essential prosthetic group 4'-phosphopantetheine (4'-Ppant) from coenzyme A (CoA) to a highly conserved serine residue in all PCPs and ACPs. PPTases, based on sequence and substrate specifity, have been classified into three types: bacterial holo-acyl carrier protein synthase (AcpS), fatty acid synthase of eukaryotes (FAS2) and Sfp, a PPTase of secondary metabolism. The recently solved crystal structures of AcpS and Sfp-type PPTases with CoA revealed a common alpha + beta-fold with a beta(1)alpha(3)beta(2) motif and similarities in CoA binding and polymerization mode. However, it was not possible to discern neither the PCP binding region of Sfp nor the priming reaction mechanism from the Sfp-CoA cocrystal. In this work, we provide a model for the reaction mechanism based on mutational analysis of Sfp that suggests a reaction mechanism in which the highly conserved E151 deprotonates the hydroxyl group of the invariant serine of PCP. That, in turn, acts as a nucleophile to attack the beta-phosphate of CoA. The Sfp mutants K112, E117, and K120 further revealed that the loop region between beta4 and alpha5 (residues T111-S124) in Sfp is the PCP binding region. Also, residues T44, K75, S89, H90, D107, E109, E151, and K155 that have been shown in the Sfp-CoA cocrystal structure to coordinate CoA are now all confirmed by mutational and biochemical analysis.

Evaluation of the technical performance of novel holotranscobalamin (holoTC) assays in a multicenter European demonstration project.

PubMed

Morkbak, Anne L; Heimdal, Randi M; Emmens, Kathleen; Molloy, Anne; Hvas, Anne-Mette; Schneede, Joern; Clarke, Robert; Scott, John M; Ueland, Per M; Nexo, Ebba

2005-01-01

A commercially available holotranscobalamin (holo-TC) radioimmunoassay (RIA) (Axis-Shield, Dundee, Scotland) was evaluated in four laboratories and compared with a holoTC ELISA run in one laboratory. The performance of the holoTC RIA assay was comparable in three of the four participating laboratories. The results from these three laboratories, involving at least 20 initial runs of "low", "medium" and "high" serum-based controls (mean holoTC concentrations 34, 60 and 110 pmol/L, respectively) yielded an intra-laboratory imprecision of 6-10%. No systematic inter-laboratory deviations were observed on runs involving 72 patient samples (holoTC concentration range 10-160 pmol/L). A fourth laboratory demonstrated higher assay imprecision for control samples and systematic deviation of results for the patient samples. Measurement of holoTC by ELISA showed an imprecision of 4-5%, and slightly higher mean values for the controls (mean holoTC concentrations 40, 70 and 114 pmol/L, respectively). Comparable results were obtained for the patient samples. The long-term intra-laboratory imprecision was 12% for the holoTC RIA and 6% for the ELISA. In conclusion, it would be prudent to check the calibration and precision prior to starting to use these holoTC assays in research or clinical practice. The results obtained using the holoTC RIA were similar to those obtained using the holoTC ELISA assay.

Free energy landscape remodeling of the cardiac pacemaker channel explains the molecular basis of familial sinus bradycardia

PubMed Central

Boulton, Stephen; Akimoto, Madoka; Akbarizadeh, Sam; Melacini, Giuseppe

2017-01-01

The hyperpolarization-activated and cyclic nucleotide-modulated ion channel (HCN) drives the pacemaker activity in the heart, and its malfunction can result in heart disorders. One such disorder, familial sinus bradycardia, is caused by the S672R mutation in HCN, whose electrophysiological phenotypes include a negative shift in the channel activation voltage and an accelerated HCN deactivation. The outcomes of these changes are abnormally low resting heart rates. However, the molecular mechanism underlying these electrophysiological changes is currently not fully understood. Crystallographic investigations indicate that the S672R mutation causes limited changes in the structure of the HCN intracellular gating tetramer, but its effects on protein dynamics are unknown. Here, we utilize comparative S672R versus WT NMR analyses to show that the S672R mutation results in extensive perturbations of the dynamics in both apo- and holo-forms of the HCN4 isoform, reflecting how S672R remodels the free energy landscape for the modulation of HCN4 by cAMP, i.e. the primary cyclic nucleotide modulator of HCN channels. We show that the S672R mutation results in a constitutive shift of the dynamic auto-inhibitory equilibrium toward inactive states of HCN4 and broadens the free-energy well of the apo-form, enhancing the millisecond to microsecond dynamics of the holo-form at sites critical for gating cAMP binding. These S672R-induced variations in dynamics provide a molecular basis for the electrophysiological phenotypes of this mutation and demonstrate that the pathogenic effects of the S672R mutation can be rationalized primarily in terms of modulations of protein dynamics. PMID:28174302

CDOCKER and lambda λ -dynamics for prospective prediction in D3R Grand Challenge 2

NASA Astrophysics Data System (ADS)

Ding, Xinqiang; Hayes, Ryan L.; Vilseck, Jonah Z.; Charles, Murchtricia K.; Brooks, Charles L.

2018-01-01

The opportunity to prospectively predict ligand bound poses and free energies of binding to the Farnesoid X Receptor in the D3R Grand Challenge 2 provided a useful exercise to evaluate CHARMM based docking (CDOCKER) and λ-dynamics methodologies for use in "real-world" applications in computer aided drug design. In addition to measuring their current performance, several recent methodological developments have been analyzed retrospectively to highlight best procedural practices in future applications. For pose prediction with CDOCKER, when the protein structure used for rigid receptor docking was close to the crystallographic holo structure, reliable poses were obtained. Benzimidazoles, with a known holo receptor structure, were successfully docked with an average RMSD of 0.97 Å. Other non-benzimidazole ligands displayed less accuracy largely because the receptor structures we chose for docking were too different from the experimental holo structures. However, retrospective analysis has shown that when these ligands were re-docked into their holo structures, the average RMSD dropped to 1.18 Å for all ligands. When sulfonamides and spiros were docked with the apo structure, which agrees more with their holo structure than the structures we chose, five out of six ligands were correctly docked. These docking results emphasize the need for flexible receptor docking approaches. For λ-dynamics techniques, including multisite λ-dynamics (MSλD), reasonable agreement with experiment was observed for the 33 ligands investigated; root mean square errors of 2.08 and 1.67 kcal/mol were obtained for free energy sets 1 and 2, respectively. Retrospectively, soft-core potentials, adaptive landscape flattening, and biasing potential replica exchange (BP-REX) algorithms were critical to model large substituent perturbations with sufficient precision and within restrictive timeframes, such as was required with participation in Grand Challenge 2. These developments, their associated benefits, and proposed procedures for their use in future applications are discussed.

CDOCKER and λ-dynamics for prospective prediction in D₃R Grand Challenge 2.

PubMed

Ding, Xinqiang; Hayes, Ryan L; Vilseck, Jonah Z; Charles, Murchtricia K; Brooks, Charles L

2018-01-01

The opportunity to prospectively predict ligand bound poses and free energies of binding to the Farnesoid X Receptor in the D3R Grand Challenge 2 provided a useful exercise to evaluate CHARMM based docking (CDOCKER) and [Formula: see text]-dynamics methodologies for use in "real-world" applications in computer aided drug design. In addition to measuring their current performance, several recent methodological developments have been analyzed retrospectively to highlight best procedural practices in future applications. For pose prediction with CDOCKER, when the protein structure used for rigid receptor docking was close to the crystallographic holo structure, reliable poses were obtained. Benzimidazoles, with a known holo receptor structure, were successfully docked with an average RMSD of 0.97 [Formula: see text]. Other non-benzimidazole ligands displayed less accuracy largely because the receptor structures we chose for docking were too different from the experimental holo structures. However, retrospective analysis has shown that when these ligands were re-docked into their holo structures, the average RMSD dropped to 1.18 [Formula: see text] for all ligands. When sulfonamides and spiros were docked with the apo structure, which agrees more with their holo structure than the structures we chose, five out of six ligands were correctly docked. These docking results emphasize the need for flexible receptor docking approaches. For [Formula: see text]-dynamics techniques, including multisite [Formula: see text]-dynamics (MS[Formula: see text]D), reasonable agreement with experiment was observed for the 33 ligands investigated; root mean square errors of 2.08 and 1.67 kcal/mol were obtained for free energy sets 1 and 2, respectively. Retrospectively, soft-core potentials, adaptive landscape flattening, and biasing potential replica exchange (BP-REX) algorithms were critical to model large substituent perturbations with sufficient precision and within restrictive timeframes, such as was required with participation in Grand Challenge 2. These developments, their associated benefits, and proposed procedures for their use in future applications are discussed.

Multiscale high-order/low-order (HOLO) algorithms and applications

NASA Astrophysics Data System (ADS)

Chacón, L.; Chen, G.; Knoll, D. A.; Newman, C.; Park, H.; Taitano, W.; Willert, J. A.; Womeldorff, G.

2017-02-01

We review the state of the art in the formulation, implementation, and performance of so-called high-order/low-order (HOLO) algorithms for challenging multiscale problems. HOLO algorithms attempt to couple one or several high-complexity physical models (the high-order model, HO) with low-complexity ones (the low-order model, LO). The primary goal of HOLO algorithms is to achieve nonlinear convergence between HO and LO components while minimizing memory footprint and managing the computational complexity in a practical manner. Key to the HOLO approach is the use of the LO representations to address temporal stiffness, effectively accelerating the convergence of the HO/LO coupled system. The HOLO approach is broadly underpinned by the concept of nonlinear elimination, which enables segregation of the HO and LO components in ways that can effectively use heterogeneous architectures. The accuracy and efficiency benefits of HOLO algorithms are demonstrated with specific applications to radiation transport, gas dynamics, plasmas (both Eulerian and Lagrangian formulations), and ocean modeling. Across this broad application spectrum, HOLO algorithms achieve significant accuracy improvements at a fraction of the cost compared to conventional approaches. It follows that HOLO algorithms hold significant potential for high-fidelity system scale multiscale simulations leveraging exascale computing.

Ligand binding induces a sharp decrease in hydrophobicity of folate binding protein assessed by 1-anilinonaphthalene-8-sulphonate which suppresses self-association of the hydrophobic apo-protein.

PubMed

Holm, Jan; Lawaetz, Anders J; Hansen, Steen I

2012-08-17

High affinity folate binding protein (FBP) regulates as a soluble protein and as a cellular receptor intracellular trafficking of folic acid, a vitamin of great importance to cell growth and division. We addressed two issues of potential importance to the biological function of FBP, a possible decrease of the surface hydrophobicity associated with the ligand-induced conformation change of FBP, and protein-inter-protein interactions involved in self-association of hydrophobic apo-FBP. The extrinsic fluorescent apolar dye 1-anilinonaphthalene-8-sulphonate (ANS) exhibited enhanced fluorescence intensity and a blueshift of emission maximum from 510-520 nm to 460-470 nm upon addition of apo-FBP indicating binding to a strongly hydrophobic environment. Neither enhancement of fluorescence nor blueshift of ANS emission maximum occurred when folate-ligated holo-FBP replaced apo-FBP. The drastic decrease in surface hydrophobicity of holo-FBP could have bearings on the biological function of FBP since changes in surface hydrophobicity have critical effects on the biological function of receptors and transport proteins. ANS interacts with exposed hydrophobic surfaces on proteins and may thereby block and prevent aggregation of proteins (chaperone-like effect). Hence, hydrophobic interactions seemed to participate in the concentration-dependent self-association of apo-FBP which was suppressed by high ANS concentrations in light scatter measurements. Copyright © 2012 Elsevier Inc. All rights reserved.

Transcellular transport of cobalamin in aortic endothelial cells.

PubMed

Hannibal, Luciana; Bolisetty, Keerthana; Axhemi, Armend; DiBello, Patricia M; Quadros, Edward V; Fedosov, Sergey; Jacobsen, Donald W

2018-05-09

Cobalamin [Cbl (or B 12 )] deficiency causes megaloblastic anemia and a variety of neuropathies. However, homeostatic mechanisms of cyanocobalamin (CNCbl) and other Cbls by vascular endothelial cells are poorly understood. Herein, we describe our investigation into whether cultured bovine aortic endothelial cells (BAECs) perform transcytosis of B 12 , namely, the complex formed between serum transcobalamin and B 12 , designated as holo-transcobalamin (holo-TC). We show that cultured BAECs endocytose [ 57 Co]-CNCbl-TC (source material) via the CD320 receptor. The bound Cbl is transported across the cell both via exocytosis in its free form, [ 57 Co]-CNCbl, and via transcytosis as [ 57 Co]-CNCbl-TC. Transcellular mobilization of Cbl occurred in a bidirectional manner. A portion of the endocytosed [ 57 Co]-CNCbl was enzymatically processed by methylmalonic aciduria combined with homocystinuria type C (cblC) with subsequent formation of hydroxocobalamin, methylcobalamin, and adenosylcobalamin, which were also transported across the cell in a bidirectional manner. This demonstrates that transport mechanisms for Cbl in vascular endothelial cells do not discriminate between various β-axial ligands of the vitamin. Competition studies with apoprotein- and holo-TC and holo-intrinsic factor showed that only holo-TC was effective at inhibiting transcellular transport of Cbl. Incubation of BAECs with a blocking antibody against the extracellular domain of the CD320 receptor inhibited uptake and transcytosis by ∼40%. This study reveals that endothelial cells recycle uncommitted intracellular Cbl for downstream usage by other cell types and suggests that the endothelium is self-sufficient for the specific acquisition and subsequent distribution of circulating B 12 via the CD320 receptor. We posit that the endothelial lining of the vasculature is an essential component for the maintenance of serum-tissue homeostasis of B 12 .-Hannibal, L., Bolisetty, K., Axhemi, A., DiBello, P. M., Quadros, E. V., Fedosov, S., Jacobsen, D. W. Transcellular transport of cobalamin in aortic endothelial cells.

Multiscale high-order/low-order (HOLO) algorithms and applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chacon, Luis; Chen, Guangye; Knoll, Dana Alan

Here, we review the state of the art in the formulation, implementation, and performance of so-called high-order/low-order (HOLO) algorithms for challenging multiscale problems. HOLO algorithms attempt to couple one or several high-complexity physical models (the high-order model, HO) with low-complexity ones (the low-order model, LO). The primary goal of HOLO algorithms is to achieve nonlinear convergence between HO and LO components while minimizing memory footprint and managing the computational complexity in a practical manner. Key to the HOLO approach is the use of the LO representations to address temporal stiffness, effectively accelerating the convergence of the HO/LO coupled system. Themore » HOLO approach is broadly underpinned by the concept of nonlinear elimination, which enables segregation of the HO and LO components in ways that can effectively use heterogeneous architectures. The accuracy and efficiency benefits of HOLO algorithms are demonstrated with specific applications to radiation transport, gas dynamics, plasmas (both Eulerian and Lagrangian formulations), and ocean modeling. Across this broad application spectrum, HOLO algorithms achieve significant accuracy improvements at a fraction of the cost compared to conventional approaches. It follows that HOLO algorithms hold significant potential for high-fidelity system scale multiscale simulations leveraging exascale computing.« less

Multiscale high-order/low-order (HOLO) algorithms and applications

DOE PAGES

Chacon, Luis; Chen, Guangye; Knoll, Dana Alan; ...

2016-11-11

Here, we review the state of the art in the formulation, implementation, and performance of so-called high-order/low-order (HOLO) algorithms for challenging multiscale problems. HOLO algorithms attempt to couple one or several high-complexity physical models (the high-order model, HO) with low-complexity ones (the low-order model, LO). The primary goal of HOLO algorithms is to achieve nonlinear convergence between HO and LO components while minimizing memory footprint and managing the computational complexity in a practical manner. Key to the HOLO approach is the use of the LO representations to address temporal stiffness, effectively accelerating the convergence of the HO/LO coupled system. Themore » HOLO approach is broadly underpinned by the concept of nonlinear elimination, which enables segregation of the HO and LO components in ways that can effectively use heterogeneous architectures. The accuracy and efficiency benefits of HOLO algorithms are demonstrated with specific applications to radiation transport, gas dynamics, plasmas (both Eulerian and Lagrangian formulations), and ocean modeling. Across this broad application spectrum, HOLO algorithms achieve significant accuracy improvements at a fraction of the cost compared to conventional approaches. It follows that HOLO algorithms hold significant potential for high-fidelity system scale multiscale simulations leveraging exascale computing.« less

Solving the crystal structure of human calcium-free S100Z: the siege and conquer of one of the last S100 family strongholds.

PubMed

Calderone, V; Fragai, M; Gallo, G; Luchinat, C

2017-06-01

The X-ray structure of human apo-S100Z has been solved and compared with that of the zebrafish calcium-bound S100Z, which is the closest in sequence. Human apo-S100A12, which shows only 43% sequence identity to human S100Z, has been used as template model to solve the crystallographic phase problem. Although a significant buried surface area between the two physiological dimers is present in the asymmetric unit of human apo-S100Z, the protein does not form the superhelical arrangement in the crystal as observed for the zebrafish calcium-bound S100Z and human calcium-bound S100A4. These findings further demonstrate that calcium plays a fundamental role in triggering quaternary structure formation in several S100s. Solving the X-ray structure of human apo-S100Z by standard molecular replacement procedures turned out to be a challenge and required trying different models and different software tools among which only one was successful. The model that allowed structure solution was that with one of the lowest sequence identity with the target protein among the S100 family in the apo state. Based on the previously solved zebrafish holo-S100Z, a putative human holo-S100Z structure has been then calculated through homology modeling; the differences between the experimental human apo and calculated holo structure have been compared to those existing for other members of the family.

Ensemble Generation and the Influence of Protein Flexibility on Geometric Tunnel Prediction in Cytochrome P450 Enzymes

PubMed Central

Kingsley, Laura J.; Lill, Markus A.

2014-01-01

Computational prediction of ligand entry and egress paths in proteins has become an emerging topic in computational biology and has proven useful in fields such as protein engineering and drug design. Geometric tunnel prediction programs, such as Caver3.0 and MolAxis, are computationally efficient methods to identify potential ligand entry and egress routes in proteins. Although many geometric tunnel programs are designed to accommodate a single input structure, the increasingly recognized importance of protein flexibility in tunnel formation and behavior has led to the more widespread use of protein ensembles in tunnel prediction. However, there has not yet been an attempt to directly investigate the influence of ensemble size and composition on geometric tunnel prediction. In this study, we compared tunnels found in a single crystal structure to ensembles of various sizes generated using different methods on both the apo and holo forms of cytochrome P450 enzymes CYP119, CYP2C9, and CYP3A4. Several protein structure clustering methods were tested in an attempt to generate smaller ensembles that were capable of reproducing the data from larger ensembles. Ultimately, we found that by including members from both the apo and holo data sets, we could produce ensembles containing less than 15 members that were comparable to apo or holo ensembles containing over 100 members. Furthermore, we found that, in the absence of either apo or holo crystal structure data, pseudo-apo or –holo ensembles (e.g. adding ligand to apo protein throughout MD simulations) could be used to resemble the structural ensembles of the corresponding apo and holo ensembles, respectively. Our findings not only further highlight the importance of including protein flexibility in geometric tunnel prediction, but also suggest that smaller ensembles can be as capable as larger ensembles at capturing many of the protein motions important for tunnel prediction at a lower computational cost. PMID:24956479

On the molecular interaction between lactoferrin and the dye Red HE-3B. A novel approach for docking a charged and highly flexible molecule to protein surfaces

NASA Astrophysics Data System (ADS)

Grasselli, Mariano; Cascone, Osvaldo; Anspach, F. Birger; Delfino, Jose M.

2002-12-01

Lactoferrin (Lf) is a non-heme, iron binding protein present in many physiological fluids of vertebrates where its main role is the microbicidal activity. It has been isolated by different methods, including dye-affinity chromatography. Red HE-3B is one of the most common triazinic dyes applied in protein purification, but scant knowledge is available on structural details and on the energetics of its interaction with proteins. In this work we present a computational approach useful for identifying possible binding sites for Red HE-3B in apo and holo forms of Lfs from human and bovine source. A new geometrical description of Red HE-3B is introduced which greatly simplifies the conformational analysis. This approach proved to be of particular advantage for addressing conformational ensembles of highly flexible molecules. Predictions from this analysis were correlated with experimentally observed dye-binding sites, as mapped by protection from proteolysis in Red HE-3B/Lf complexes. This method could bear relevance for the screening of possible dye-binding sites in proteins whose structure is known and as a potential tool for the design of engineered protein variants which could be purified by dye-affinity chromatography.

On the molecular interaction between lactoferrin and the dye Red HE-3b. A novel approach for docking a charged and highly flexible molecule to protein surfaces.

PubMed

Grasselli, Mariano; Cascone, Osvaldo; Birger Anspach, F; Delfino, Jose M

2002-12-01

Lactoferrin (Lf) is a non-heme, iron binding protein present in many physiological fluids of vertebrates where its main role is the microbicidal activity. It has been isolated by different methods, including dye-affinity chromatography. Red HE-3B is one of the most common triazinic dyes applied in protein purification, but scant knowledge is available on structural details and on the energetics of its interaction with proteins. In this work we present a computational approach useful for identifying possible binding sites for Red HE-3B in apo and holo forms of Lfs from human and bovine source. A new geometrical description of Red HE-3B is introduced which greatly simplifies the conformational analysis. This approach proved to be of particular advantage for addressing conformational ensembles of highly flexible molecules. Predictions from this analysis were correlated with experimentally observed dye-binding sites, as mapped by protection from proteolysis in Red HE-3B/Lf complexes. This method could bear relevance for the screening of possible dye-binding sites in proteins whose structure is known and as a potential tool for the design of engineered protein variants which could be purified by dye-affinity chromatography.

Metal retention in human transferrin: consequences of solvent composition in analytical sample preparation methods.

PubMed

Quarles, C Derrick; Randunu, K Manoj; Brumaghim, Julia L; Marcus, R Kenneth

2011-10-01

The analysis of metal-binding proteins requires careful sample manipulation to ensure that the metal-protein complex remains in its native state and the metal retention is preserved during sample preparation or analysis. Chemical analysis for the metal content in proteins typically involves some type of liquid chromatography/electrophoresis separation step coupled with an atomic (i.e., inductively coupled plasma-optical emission spectroscopy or -mass spectrometry) or molecular (i.e., electrospray ionization-mass spectrometry) analysis step that requires altered-solvent introduction techniques. UV-VIS absorbance is employed here to monitor the iron content in human holo-transferrin (Tf) under various solvent conditions, changing polarity, pH, ionic strength, and the ionic and hydrophobic environment of the protein. Iron loading percentages (i.e. 100% loading equates to 2 Fe(3+):1 Tf) were quantitatively determined to evaluate the effect of solvent composition on the retention of Fe(3+) in Tf. Maximum retention of Fe(3+) was found in buffered (20 mM Tris) solutions (96 ± 1%). Exposure to organic solvents and deionized H(2)O caused release of ~23-36% of the Fe(3+) from the binding pocket(s) at physiological pH (7.4). Salt concentrations similar to separation conditions used for ion exchange had little to no effect on Fe(3+) retention in holo-Tf. Unsurprisingly, changes in ionic strength caused by additions of guanidine HCl (0-10 M) to holo-Tf resulted in unfolding of the protein and loss of Fe(3+) from Tf; however, denaturing and metal loss was found not to be an instantaneous process for additions of 1-5 M guanidinium to Tf. In contrast, complete denaturing and loss of Fe(3+) was instantaneous with ≥6 M additions of guanidinium, and denaturing and loss of iron from Tf occurred in parallel proportions. Changes to the hydrophobicity of Tf (via addition of 0-14 M urea) had less effect on denaturing and release of Fe(3+) from the Tf binding pocket compared to changes in ionic strength. This journal is © The Royal Society of Chemistry 2011

Acyl carrier proteins from sunflower (Helianthus annuus L.) seeds and their influence on FatA and FatB acyl-ACP thioesterase activities.

PubMed

Aznar-Moreno, Jose A; Venegas-Calerón, Mónica; Martínez-Force, Enrique; Garcés, Rafael; Salas, Joaquín J

2016-08-01

The kinetics of acyl-ACP thioesterases from sunflower importantly changed when endogenous ACPs were used. Sunflower FatB was much more specific towards saturated acyl-ACPs when assayed with them. Acyl carrier proteins (ACPs) are small (~9 kDa), soluble, acidic proteins involved in fatty acid synthesis in plants and bacteria. ACPs bind to fatty acids through a thioester bond, generating the acyl-ACP lipoproteins that are substrates for fatty acid synthase (FAS) complexes, and that are required for fatty acid chain elongation, acting as important intermediates in de novo fatty acid synthesis in plants. Plants, usually express several ACP isoforms with distinct functionalities. We report here the cloning of three ACPs from developing sunflower seeds: HaACP1, HaACP2, and HaACP3. These proteins were plastidial ACPs expressed strongly in seeds, and as such they are probably involved in the synthesis of sunflower oil. The recombinant sunflower ACPs were expressed in bacteria but they were lethal to the prokaryote host. Thus, they were finally produced using the GST gene fusion system, which allowed the apo-enzyme to be produced and later activated to the holo form. Radiolabelled acyl-ACPs from the newly cloned holo-ACP forms were also synthesized and used to characterize the activity of recombinant sunflower FatA and FatB thioesterases, important enzymes in plant fatty acids synthesis. The activity of these enzymes changed significantly when the endogenous ACPs were used. Thus, FatA importantly increased its activity levels, whereas FatB displayed a different specificity profile, with much high activity levels towards saturated acyl-CoA derivatives. All these data pointed to an important influence of the ACP moieties on the activity of enzymes involved in lipid synthesis.

Caspase-9 holoenzyme is a specific and optimal pro-caspase-3 processing machine

PubMed Central

Yin, Qian; Park, Hyun Ho; Chung, Jee Y.; Lin, Su-Chang; Lo, Yu-Chih; da Graca, Li S.; Jiang, Xuejun; Wu, Hao

2010-01-01

Summary Caspase-9 activation is critical for intrinsic cell death. The activity of caspase-9 is increased dramatically upon association with the apoptosome and the apoptosome bound caspase-9 is the caspase-9 holoenzyme (C9Holo). In this study, we use quantitative enzymatic assays to fully characterize C9Holo and a leucine-zipper linked dimeric caspase-9 (LZ-C9). We surprisingly show that LZ-C9 is more active than C9Holo for the optimal caspase-9 peptide substrate LEHD-AFC, but is much less active than C9Holo for the physiological substrate pro-caspase-3. The measured Km values of C9Holo and LZ-C9 for LEHD-AFC are similar, demonstrating that dimerization is sufficient for catalytic activation of caspase-9. The lower activity of C9Holo against LEHD-AFC may be attributed to incomplete C9Holo assembly. However, the measured Km of C9Holo for pro-caspase-3 is much lower than that of LZ-C9. Therefore, in addition to dimerization, the apoptosome activates caspase-9 by enhancing its affinity for pro-caspase-3, which is important for pro-caspase-3 activation at the physiological concentration. PMID:16630893

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chacón, L., E-mail: chacon@lanl.gov; Chen, G.; Knoll, D.A.

We review the state of the art in the formulation, implementation, and performance of so-called high-order/low-order (HOLO) algorithms for challenging multiscale problems. HOLO algorithms attempt to couple one or several high-complexity physical models (the high-order model, HO) with low-complexity ones (the low-order model, LO). The primary goal of HOLO algorithms is to achieve nonlinear convergence between HO and LO components while minimizing memory footprint and managing the computational complexity in a practical manner. Key to the HOLO approach is the use of the LO representations to address temporal stiffness, effectively accelerating the convergence of the HO/LO coupled system. The HOLOmore » approach is broadly underpinned by the concept of nonlinear elimination, which enables segregation of the HO and LO components in ways that can effectively use heterogeneous architectures. The accuracy and efficiency benefits of HOLO algorithms are demonstrated with specific applications to radiation transport, gas dynamics, plasmas (both Eulerian and Lagrangian formulations), and ocean modeling. Across this broad application spectrum, HOLO algorithms achieve significant accuracy improvements at a fraction of the cost compared to conventional approaches. It follows that HOLO algorithms hold significant potential for high-fidelity system scale multiscale simulations leveraging exascale computing.« less

Statistical Profiling of One Promiscuous Protein Binding Site: Illustrated by Urokinase Catalytic Domain.

PubMed

Cerisier, Natacha; Regad, Leslie; Triki, Dhoha; Petitjean, Michel; Flatters, Delphine; Camproux, Anne-Claude

2017-10-01

While recent literature focuses on drug promiscuity, the characterization of promiscuous binding sites (ability to bind several ligands) remains to be explored. Here, we present a proteochemometric modeling approach to analyze diverse ligands and corresponding multiple binding sub-pockets associated with one promiscuous binding site to characterize protein-ligand recognition. We analyze both geometrical and physicochemical profile correspondences. This approach was applied to examine the well-studied druggable urokinase catalytic domain inhibitor binding site, which results in a large number of complex structures bound to various ligands. This approach emphasizes the importance of jointly characterizing pocket and ligand spaces to explore the impact of ligand diversity on sub-pocket properties and to establish their main profile correspondences. This work supports an interest in mining available 3D holo structures associated with a promiscuous binding site to explore its main protein-ligand recognition tendency. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Investigation of glutathione-derived electrostatic and hydrogen-bonding interactions and their role in defining Grx5 [2Fe-2S] cluster optical spectra and transfer chemistry.

PubMed

Sen, Sambuddha; Bonfio, Claudia; Mansy, Sheref S; Cowan, J A

2018-03-01

Human glutaredoxin 5 (Grx5) is one of the core components of the Isc (iron-sulfur cluster) assembly and trafficking machinery, and serves as an intermediary cluster carrier, putatively delivering cluster from the Isu scaffold protein to target proteins. The tripeptide glutathione is intimately involved in this role, providing cysteinyl coordination to the iron center of the Grx5-bound [2Fe-2S] cluster. Grx5 has a well-defined glutathione-binding pocket with protein amino acid residues providing many ionic and hydrogen binding contacts to the bound glutathione. In this report, we investigated the importance of these interactions in cluster chirality and exchange reactivity by systematically perturbing the crucial contacts by use of natural and non-natural amino acid substitutions to disrupt the binding contacts from both the protein and glutathione. Native Grx5 could be reconstituted with all of the glutathione analogs used, as well as other thiol ligands, such as DTT or L-cysteine, by in vitro chemical reconstitution, and the holo proteins were found to transfer [2Fe-2S] cluster to apo ferredoxin 1 at comparable rates. However, the circular dichroism spectra of these derivatives displayed prominent differences that reflect perturbations in local cluster chirality. These studies provided a detailed molecular understanding of glutathione-protein interactions in holo Grx5 that define both cluster spectroscopy and exchange chemistry.

Biological and functional relevance of CASP predictions.

PubMed

Liu, Tianyun; Ish-Shalom, Shirbi; Torng, Wen; Lafita, Aleix; Bock, Christian; Mort, Matthew; Cooper, David N; Bliven, Spencer; Capitani, Guido; Mooney, Sean D; Altman, Russ B

2018-03-01

Our goal is to answer the question: compared with experimental structures, how useful are predicted models for functional annotation? We assessed the functional utility of predicted models by comparing the performances of a suite of methods for functional characterization on the predictions and the experimental structures. We identified 28 sites in 25 protein targets to perform functional assessment. These 28 sites included nine sites with known ligand binding (holo-sites), nine sites that are expected or suggested by experimental authors for small molecule binding (apo-sites), and Ten sites containing important motifs, loops, or key residues with important disease-associated mutations. We evaluated the utility of the predictions by comparing their microenvironments to the experimental structures. Overall structural quality correlates with functional utility. However, the best-ranked predictions (global) may not have the best functional quality (local). Our assessment provides an ability to discriminate between predictions with high structural quality. When assessing ligand-binding sites, most prediction methods have higher performance on apo-sites than holo-sites. Some servers show consistently high performance for certain types of functional sites. Finally, many functional sites are associated with protein-protein interaction. We also analyzed biologically relevant features from the protein assemblies of two targets where the active site spanned the protein-protein interface. For the assembly targets, we find that the features in the models are mainly determined by the choice of template. © 2017 The Authors Proteins: Structure, Function and Bioinformatics Published by Wiley Periodicals, Inc.

Structures of rat cytosolic PEPCK: insight into the mechanism of phosphorylation and decarboxylation of oxaloacetic acid.

PubMed

Sullivan, Sarah M; Holyoak, Todd

2007-09-04

The structures of the rat cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK) reported in the PEPCK-Mn2+, -Mn2+-oxaloacetic acid (OAA), -Mn2+-OAA-Mn2+-guanosine-5'-diphosphate (GDP), and -Mn2+-Mn2+-guanosine-5'-tri-phosphate (GTP) complexes provide insight into the mechanism of phosphoryl transfer and decarboxylation mediated by this enzyme. OAA is observed to bind in a number of different orientations coordinating directly to the active site metal. The Mn2+-OAA and Mn2+-OAA-Mn2+GDP structures illustrate inner-sphere coordination of OAA to the manganese ion through the displacement of two of the three water molecules coordinated to the metal in the holo-enzyme by the C3 and C4 carbonyl oxygens. In the PEPCK-Mn2+-OAA complex, an alternate bound conformation of OAA is present. In this conformation, in addition to the previous interactions, the C1 carboxylate is directly coordinated to the active site Mn2+, displacing all of the waters coordinated to the metal in the holo-enzyme. In the PEPCK-Mn2+-GTP structure, the same water molecule displaced by the C1 carboxylate of OAA is displaced by one of the gamma-phosphate oxygens of the triphosphate nucleotide. The structures are consistent with a mechanism of direct in-line phosphoryl transfer, supported by the observed stereochemistry of the reaction. In the catalytically competent binding mode, the C1 carboxylate of OAA is sandwiched between R87 and R405 in an environment that would serve to facilitate decarboxylation. In the reverse reaction, these two arginines would form the CO2 binding site. Comparison of the Mn2+-OAA-Mn2+GDP and Mn2+-Mn2+GTP structures illustrates a marked difference in the bound conformations of the nucleotide substrates in which the GTP nucleotide is bound in a high-energy state resulting from the eclipsing of all three of the phosphoryl groups along the triphosphate chain. This contrasts a previously determined structure of PEPCK in complex with a triphosphate nucleotide analogue in which the analogue mirrors the conformation of GDP as opposed to GTP. Last, the structures illustrate a correlation between conformational changes in the P-loop, the nucleotide binding site, and the active site lid that are important for catalysis.

Oxidation of the N-terminal domain of the wheat metallothionein Ec -1 leads to the formation of three distinct disulfide bridges.

PubMed

Tarasava, Katsiaryna; Chesnov, Serge; Freisinger, Eva

2016-05-01

Metallothioneins (MTs) are low molecular weight proteins, characterized by a high cysteine content and the ability to coordinate large amounts of d(10) metal ions, for example, Zn(II), Cd(II), and Cu(I), in form of metal-thiolate clusters. Depending on intracellular conditions such as redox potential or metal ion concentrations, MTs can occur in various states ranging from the fully metal-loaded holo- to the metal-free apo-form. The Cys thiolate groups in the apo-form can be either reduced or be involved in disulfide bridges. Although oxidation-mediated Zn(II) release might be a possible mechanism for the regulation of Zn(II) availability by MTs, no concise information regarding the associated pathways and the structure of oxidized apo-MT forms is available. Using the well-studied Zn2 γ-Ec -1 domain of the wheat Zn6 Ec -1 MT we attempt here to answer several question regarding the structure and biophysical properties of oxidized MT forms, such as: (1) does disulfide bond formation increase the stability against proteolysis, (2) is the overall peptide backbone fold similar for the holo- and the oxidized apo-MT form, and (3) are disulfide bridges specifically or randomly formed? Our investigations show that oxidation leads to three distinct disulfide bridges independently of the applied oxidation conditions and of the initial species used for oxidation, that is, the apo- or the holo-form. In addition, the oxidized apo-form is as stable against proteolysis as Zn2 γ-Ec -1, rendering the currently assumed degradation of oxidized MTs unlikely and suggesting a role of the oxidation process for the extension of protein lifetime in absence of sufficient amounts of metal ions. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 295-308, 2016. © 2016 Wiley Periodicals, Inc.

Iron Release from Soybean Seed Ferritin Induced by Cinnamic Acid Derivatives.

PubMed

Sha, Xuejiao; Chen, Hai; Zhang, Jingsheng; Zhao, Guanghua

2018-05-04

Plant ferritin represents a novel class of iron supplement, which widely co-exists with phenolic acids in a plant diet. However, there are few reports on the effect of these phenolic acids on function of ferritin. In this study, we demonstrated that cinnamic acid derivatives, as widely occurring phenolic acids, can induce iron release from holo soybean seed ferritin (SSF) in a structure-dependent manner. The ability of the iron release from SSF by five cinnamic acids follows the sequence of Cinnamic acid > Chlorogenic acid > Ferulic acid > p -Coumaric acid > Trans -Cinnamic acid. Fluorescence titration in conjunction with dialysis results showed that all of these five compounds have a similar, weak ability to bind with protein, suggesting that their protein-binding ability is not related to their iron release activity. In contrast, both Fe 2+ -chelating activity and reducibility of these cinnamic acid derivatives are in good agreement with their ability to induce iron release from ferritin. These studies indicate that cinnamic acid and its derivatives could have a negative effect on iron stability of holo soybean seed ferritin in diet, and the Fe 2+ -chelating activity and reducibility of cinnamic acid and its derivatives have strong relations to the iron release of soybean seed ferritin.

Ligand Binding Induces Conformational Changes in Human Cellular Retinol-binding Protein 1 (CRBP1) Revealed by Atomic Resolution Crystal Structures.

PubMed

Silvaroli, Josie A; Arne, Jason M; Chelstowska, Sylwia; Kiser, Philip D; Banerjee, Surajit; Golczak, Marcin

2016-04-15

Important in regulating the uptake, storage, and metabolism of retinoids, cellular retinol-binding protein 1 (CRBP1) is essential for trafficking vitamin A through the cytoplasm. However, the molecular details of ligand uptake and targeted release by CRBP1 remain unclear. Here we report the first structure of CRBP1 in a ligand-free form as well as ultra-high resolution structures of this protein bound to either all-trans-retinol or retinylamine, the latter a therapeutic retinoid that prevents light-induced retinal degeneration. Superpositioning of human apo- and holo-CRBP1 revealed major differences within segments surrounding the entrance to the retinoid-binding site. These included α-helix II and hairpin turns between β-strands βC-βD and βE-βF as well as several side chains, such as Phe-57, Tyr-60, and Ile-77, that change their orientations to accommodate the ligand. Additionally, we mapped hydrogen bond networks inside the retinoid-binding cavity and demonstrated their significance for the ligand affinity. Analyses of the crystallographic B-factors indicated several regions with higher backbone mobility in the apoprotein that became more rigid upon retinoid binding. This conformational flexibility of human apo-CRBP1 facilitates interaction with the ligands, whereas the more rigid holoprotein structure protects the labile retinoid moiety during vitamin A transport. These findings suggest a mechanism of induced fit upon ligand binding by mammalian cellular retinol-binding proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Visualization of a radical B 12 enzyme with its G-protein chaperone

DOE PAGES

Jost, Marco; Cracan, Valentin; Hubbard, Paul A.; ...

2015-02-09

G-protein metallochaperones ensure fidelity during cofactor assembly for a variety of metalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogenase, and thus have both medical and biofuel development applications. In this paper, we present crystal structures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, which reveal the molecular architecture of a G-protein metallochaperone in complex with its target protein. These structures show that conserved G-protein elements become ordered upon target protein association, creating the molecular pathways that both sense and report on the cofactor loading state. Structures determined of both apo- and holo-forms ofmore » IcmF depict both open and closed enzyme states, in which the cofactor-binding domain is alternatively positioned for cofactor loading and for catalysis. Finally and notably, the G protein moves as a unit with the cofactor-binding domain, providing a visualization of how a chaperone assists in the sequestering of a precious cofactor inside an enzyme active site.« less

The Crystal Structures of Apo and cAMP-Bound GlxR from Corynebacterium glutamicum Reveal Structural and Dynamic Changes upon cAMP Binding in CRP/FNR Family Transcription Factors

PubMed Central

Townsend, Philip D.; Jungwirth, Britta; Pojer, Florence; Bußmann, Michael; Money, Victoria A.; Cole, Stewart T.; Pühler, Alfred; Tauch, Andreas; Bott, Michael; Cann, Martin J.; Pohl, Ehmke

2014-01-01

The cyclic AMP-dependent transcriptional regulator GlxR from Corynebacterium glutamicum is a member of the super-family of CRP/FNR (cyclic AMP receptor protein/fumarate and nitrate reduction regulator) transcriptional regulators that play central roles in bacterial metabolic regulatory networks. In C. glutamicum, which is widely used for the industrial production of amino acids and serves as a non-pathogenic model organism for members of the Corynebacteriales including Mycobacterium tuberculosis, the GlxR homodimer controls the transcription of a large number of genes involved in carbon metabolism. GlxR therefore represents a key target for understanding the regulation and coordination of C. glutamicum metabolism. Here we investigate cylic AMP and DNA binding of GlxR from C. glutamicum and describe the crystal structures of apo GlxR determined at a resolution of 2.5 Å, and two crystal forms of holo GlxR at resolutions of 2.38 and 1.82 Å, respectively. The detailed structural analysis and comparison of GlxR with CRP reveals that the protein undergoes a distinctive conformational change upon cyclic AMP binding leading to a dimer structure more compatible to DNA-binding. As the two binding sites in the GlxR homodimer are structurally identical dynamic changes upon binding of the first ligand are responsible for the allosteric behavior. The results presented here show how dynamic and structural changes in GlxR lead to optimization of orientation and distance of its two DNA-binding helices for optimal DNA recognition. PMID:25469635

MCAT is not required for in vitro polyketide synthesis in a minimal actinorhodin polyketide synthase from Streptomyces coelicolor.

PubMed

Matharu, A L; Cox, R J; Crosby, J; Byrom, K J; Simpson, T J

1998-12-01

It has been proposed that Streptomyces malonyl CoA: holo acyl carrier protein transacylases (MCATs) provide a link between fatty acid and polyketide biosynthesis. Two recent studies have provided evidence that the presence of MCAT is essential for polyketide synthesis to proceed in reconstituted minimal polyketide synthases (PKSs). In contrast to this, we previously showed that the holo acyl carrier proteins (ACPs) from type II PKSs are capable of catalytic self-malonylation in the presence of malonyl CoA, which suggests that MCAT might not be necessary for polyketide biosynthesis. We reconstituted a homologous actinorhodin (act) type II minimal PKS in vitro. When act holo-ACP is present in limiting concentrations, MCAT is required by the synthase complex in order for polyketide biosynthesis to proceed. When holo-ACP is present in excess, however, efficient polyketide synthesis proceeds without MCAT. The rate of polyketide production increases with holo-ACP concentration, but at low ACP concentration or equimolar AC:KS:CLF (KS, ketosynthase; CLF, chain length determining factor) concentrations this rate is significantly lower than expected, indicating that free holo-ACP is sequestered by the KS/CLF complex. The rate of polyketide biosynthesis is dictated by the ratio of holo-ACP to KS and CLF, as well as by the total protein concentration. There is no absolute requirement for MCAT in polyketide biosynthesis in vitro, although the role of MCAT during polyketide synthesis in vivo remains an open question. MCAT might be responsible for the rate enhancement of malonyl transfer at very low free holo-ACP concentrations or it could be required to catalyse the transfer of malonyl groups from malonyl CoA to sequestered holo-ACP.

Structural characterization of Staphylococcus aureus biotin protein ligase and interaction partners: an antibiotic target.

PubMed

Pendini, Nicole R; Yap, Min Y; Traore, D A K; Polyak, Steven W; Cowieson, Nathan P; Abell, Andrew; Booker, Grant W; Wallace, John C; Wilce, Jacqueline A; Wilce, Matthew C J

2013-06-01

The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug-resistant pathogens. Staphylococcus aureus BPL (SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here, we report the structural analysis of both holo- and apo-forms of SaBPL using X-ray crystallography. We also present small-angle X-ray scattering data of SaBPL in complex with its biotin-carboxyl carrier protein substrate as well as the SaBPL:DNA complex that underlies repression. This has revealed the molecular basis of ligand (biotinyl-5'-AMP) binding and conformational changes associated with catalysis and repressor function. These data provide new information to better understand the bifunctional activities of SaBPL and to inform future strategies for antibiotic discovery. © 2013 The Protein Society.

Structural characterization of Staphylococcus aureus biotin protein ligase and interaction partners: An antibiotic target

PubMed Central

Pendini, Nicole R; Yap, Min Y; Polyak, Steven W; Cowieson, Nathan P; Abell, Andrew; Booker, Grant W; Wallace, John C; Wilce, Jacqueline A; Wilce, Matthew C J

2013-01-01

The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug-resistant pathogens. Staphylococcus aureus BPL (SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here, we report the structural analysis of both holo- and apo-forms of SaBPL using X-ray crystallography. We also present small-angle X-ray scattering data of SaBPL in complex with its biotin-carboxyl carrier protein substrate as well as the SaBPL:DNA complex that underlies repression. This has revealed the molecular basis of ligand (biotinyl-5′-AMP) binding and conformational changes associated with catalysis and repressor function. These data provide new information to better understand the bifunctional activities of SaBPL and to inform future strategies for antibiotic discovery. PMID:23559560

Vitamin B12 absorption judged by measurement of holotranscobalamin, active vitamin B12: evaluation of a commercially available EIA kit.

PubMed

Greibe, Eva; Nexo, Ebba

2011-11-01

Active vitamin B12 absorption is followed by an increase in holotranscobalamin (holoTC) upon loading with a high physiological dose of the vitamin (the CobaSorb test). This study evaluates the use of a newly launched EIA kit for measurement of holoTC (active B12) in relation to the CobaSorb test. Intra-assay imprecision and linearity of the EIA kit was examined, employing serum pools of increasing holoTC concentrations. For the CobaSorb test, holoTC was measured before and after loading with 3-times 9 μg of vitamin B12 employing both the in-house ELISA and the EIA kit (n=25). The EIA kit showed an intra-assay CV between 2.2% and 5.8% for holoTC values ranging from 21 to 80 pmol/L. Employing diluted serum samples resulted in spurious high values of holoTC. The EIA kit performed well in relation to the CobaSorb test and classified the patients studied as capable of absorbing vitamin B12 (n=10) or not (n=15), as did the in-house ELISA. The Active B12 (holoTC) EIA kit proved suitable for use with the CobaSorb test, but not for analysis of diluted serum samples.

Augmented Reality Technology Using Microsoft HoloLens in Anatomic Pathology.

PubMed

Hanna, Matthew G; Ahmed, Ishtiaque; Nine, Jeffrey; Prajapati, Shyam; Pantanowitz, Liron

2018-05-01

Context Augmented reality (AR) devices such as the Microsoft HoloLens have not been well used in the medical field. Objective To test the HoloLens for clinical and nonclinical applications in pathology. Design A Microsoft HoloLens was tested for virtual annotation during autopsy, viewing 3D gross and microscopic pathology specimens, navigating whole slide images, telepathology, as well as real-time pathology-radiology correlation. Results Pathology residents performing an autopsy wearing the HoloLens were remotely instructed with real-time diagrams, annotations, and voice instruction. 3D-scanned gross pathology specimens could be viewed as holograms and easily manipulated. Telepathology was supported during gross examination and at the time of intraoperative consultation, allowing users to remotely access a pathologist for guidance and to virtually annotate areas of interest on specimens in real-time. The HoloLens permitted radiographs to be coregistered on gross specimens and thereby enhanced locating important pathologic findings. The HoloLens also allowed easy viewing and navigation of whole slide images, using an AR workstation, including multiple coregistered tissue sections facilitating volumetric pathology evaluation. Conclusions The HoloLens is a novel AR tool with multiple clinical and nonclinical applications in pathology. The device was comfortable to wear, easy to use, provided sufficient computing power, and supported high-resolution imaging. It was useful for autopsy, gross and microscopic examination, and ideally suited for digital pathology. Unique applications include remote supervision and annotation, 3D image viewing and manipulation, telepathology in a mixed-reality environment, and real-time pathology-radiology correlation.

High-Order/Low-Order methods for ocean modeling

DOE PAGES

Newman, Christopher; Womeldorff, Geoff; Chacón, Luis; ...

2015-06-01

In this study, we examine a High Order/Low Order (HOLO) approach for a z-level ocean model and show that the traditional semi-implicit and split-explicit methods, as well as a recent preconditioning strategy, can easily be cast in the framework of HOLO methods. The HOLO formulation admits an implicit-explicit method that is algorithmically scalable and second-order accurate, allowing timesteps much larger than the barotropic time scale. We show how HOLO approaches, in particular the implicit-explicit method, can provide a solid route for ocean simulation to heterogeneous computing and exascale environments.

Active B12: a rapid, automated assay for holotranscobalamin on the Abbott AxSYM analyzer.

PubMed

Brady, Jeff; Wilson, Lesley; McGregor, Lynda; Valente, Edward; Orning, Lars

2008-03-01

Conventional tests for vitamin B(12) deficiency measure total serum vitamin B12, whereas only that portion of vitamin B12 carried by transcobalamin (holotranscobalamin) is metabolically active. Measurement of holotranscobalamin (holoTC) may be more diagnostically accurate for detecting B(12) deficiency that requires therapy. We developed an automated assay for holoTC that can be used on the Abbott AxSYM immunoassay analyzer. AxSYM Active B12 is a 2-step sandwich microparticle enzyme immunoassay. In step 1, a holoTC-specific antibody immobilized onto latex microparticles captures holoTC in samples of serum or plasma. In step 2, the captured holoTC is detected with a conjugate of alkaline phosphatase and antiTC antibody. Neither apoTC nor haptocorrin exhibited detectable cross-reactivity. The detection limit was < or = 0.1 pmol/L. Within-run and total imprecision (CV ranges) were 3.4%-5.1% and 6.3%-8.5%, respectively. Assay CVs were < 20% from at least 3 pmol/L to 107 pmol/L. With diluted serum samples, measured concentrations were 104%-114% of the expected values in the working range of the assay. No interference from bilirubin, hemoglobin, triglycerides, erythrocytes, rheumatoid factor, or total protein was detected at expected (abnormal) concentrations. A comparison of the AxSYM Active B12 assay with a commercial RIA for holoTC yielded the regression equation: AxSYM = 0.98RIA + 4.7 pmol/L (S(y x), 11.4 pmol/L; n = 204). Assay throughput was 45 tests/h. A 95% reference interval of 19-134 pmol/L holoTC was established with samples from 292 healthy individuals. The AxSYM Active B12 assay allows rapid, precise, sensitive, specific, and automated measurement of human holoTC in serum and plasma.

A missense mutation in TCN2 is associated with decreased risk for congenital heart defects and may increase cellular uptake of vitamin B12 via Megalin.

PubMed

Li, Peiqiang; Huang, Lijuan; Zheng, Yufang; Pan, Xuedong; Peng, Rui; Jiang, Yueming; Finnell, Richard H; Li, Haijie; Qiao, Bin; Wang, Hong-Yan

2017-08-15

Deregulation of folate and vitamin B12 (VB12) metabolism contributes to the risk of congenital heart defects (CHDs). Transcobalamin (TCN2) is essential for transporting VB12 from blood to cells as TCN2-bound VB12 (holo-TC) is the only form for somatic cellular uptake. In this study, we performed an association study between common polymorphisms in 46 one carbon metabolism genes and CHD in 412 CHDs and 213 controls. Only two significant association signals in coding regions were identified: FTCD c.1470C>T & TCN2 c.230A>T. The only missense mutation, TCN2 c.230A>T, was further validated in 412 CHDs and 1177 controls. TCN2 c.230T is significantly associated with reduced CHD risk in North Chinese (odds ratio = 0.67, P = 4.62e-05), compared with the 230A allele. Interestingly, the mean level of plasma holo-TC in women with the TA genotype was 1.77-fold higher than that in women with the AA genotype. Further analysis suggested that c.230A>T enhanced the cellular uptake of holo-TC via the LRP2 receptor. Our results determined that a functional polymorphism in TCN2 contributes to the prevalence of CHDs. TCN2 c.230A>T is significantly associated with a reduced CHD risk, likely due to TCN2 c.230T improving the interaction between holo-TC and its LRP2 receptor.

A missense mutation in TCN2 is associated with decreased risk for congenital heart defects and may increase cellular uptake of vitamin B12 via Megalin

PubMed Central

Zheng, Yufang; Pan, Xuedong; Peng, Rui; Jiang, Yueming; Finnell, Richard H.; Li, Haijie; Qiao, Bin; Wang, Hong-Yan

2017-01-01

Deregulation of folate and vitamin B12 (VB12) metabolism contributes to the risk of congenital heart defects (CHDs). Transcobalamin (TCN2) is essential for transporting VB12 from blood to cells as TCN2-bound VB12 (holo-TC) is the only form for somatic cellular uptake. In this study, we performed an association study between common polymorphisms in 46 one carbon metabolism genes and CHD in 412 CHDs and 213 controls. Only two significant association signals in coding regions were identified: FTCD c.1470C>T & TCN2 c.230A>T. The only missense mutation, TCN2 c.230A>T, was further validated in 412 CHDs and 1177 controls. TCN2 c.230T is significantly associated with reduced CHD risk in North Chinese (odds ratio = 0.67, P = 4.62e-05), compared with the 230A allele. Interestingly, the mean level of plasma holo-TC in women with the TA genotype was 1.77-fold higher than that in women with the AA genotype. Further analysis suggested that c.230A>T enhanced the cellular uptake of holo-TC via the LRP2 receptor. Our results determined that a functional polymorphism in TCN2 contributes to the prevalence of CHDs. TCN2 c.230A>T is significantly associated with a reduced CHD risk, likely due to TCN2 c.230T improving the interaction between holo-TC and its LRP2 receptor. PMID:28903415

Inhibitory Effects of Lactoferrin on Growth and Biofilm Formation of Porphyromonas gingivalis and Prevotella intermedia▿

PubMed Central

Wakabayashi, Hiroyuki; Yamauchi, Koji; Kobayashi, Tetsuo; Yaeshima, Tomoko; Iwatsuki, Keiji; Yoshie, Hiromasa

2009-01-01

Lactoferrin (LF) is an iron-binding antimicrobial protein present in saliva and gingival crevicular fluids, and it is possibly associated with host defense against oral pathogens, including periodontopathic bacteria. In the present study, we evaluated the in vitro effects of LF-related agents on the growth and biofilm formation of two periodontopathic bacteria, Porphyromonas gingivalis and Prevotella intermedia, which reside as biofilms in the subgingival plaque. The planktonic growth of P. gingivalis and P. intermedia was suppressed for up to 5 h by incubation with ≥130 μg/ml of human LF (hLF), iron-free and iron-saturated bovine LF (apo-bLF and holo-bLF, respectively), and ≥6 μg/ml of bLF-derived antimicrobial peptide lactoferricin B (LFcin B); but those effects were weak after 8 h. The biofilm formation of P. gingivalis and P. intermedia over 24 h was effectively inhibited by lower concentrations (≥8 μg/ml) of various iron-bound forms (the apo, native, and holo forms) of bLF and hLF but not LFcin B. A preformed biofilm of P. gingivalis and P. intermedia was also reduced by incubation with various iron-bound bLFs, hLF, and LFcin B for 5 h. In an examination of the effectiveness of native bLF when it was used in combination with four antibiotics, it was found that treatment with ciprofloxacin, clarithromycin, and minocycline in combination with native bLF for 24 h reduced the amount of a preformed biofilm of P. gingivalis compared with the level of reduction achieved with each agent alone. These results demonstrate the antibiofilm activity of LF with lower iron dependency against P. gingivalis and P. intermedia and the potential usefulness of LF for the prevention and treatment of periodontal diseases and as adjunct therapy for periodontal diseases. PMID:19451301

Structure of the thermophilic l-Arabinose isomerase from Geobacillus kaustophilus reveals metal-mediated intersubunit interactions for activity and thermostability.

PubMed

Choi, Jin Myung; Lee, Yong-Jik; Cao, Thinh-Phat; Shin, Sun-Mi; Park, Min-Kyu; Lee, Han-Seung; di Luccio, Eric; Kim, Seong-Bo; Lee, Sang-Jae; Lee, Sang Jun; Lee, Sung Haeng; Lee, Dong-Woo

2016-04-15

Thermophilic l-arabinose isomerase (AI), which catalyzes the interconversion of l-arabinose and l-ribulose, can be used to produce d-tagatose, a sugar substitute, from d-galactose. Unlike mesophilic AIs, thermophilic AIs are highly dependent on divalent metal ions for their catalytic activity and thermostability at elevated temperatures. However, the molecular basis underlying the substrate preferences and metal requirements of multimeric AIs remains unclear. Here we report the first crystal structure of the apo and holo forms of thermophilic Geobacillus kaustophilus AI (GKAI) in hexamer form. The structures, including those of GKAI in complex with l-arabitol, and biochemical analyses revealed not only how the substrate-binding site of GKAI is formed through displacement of residues at the intersubunit interface when it is bound to Mn(2+), but also revealed the water-mediated H-bonding networks that contribute to the structural integrity of GKAI during catalysis. These observations suggest metal-mediated isomerization reactions brought about by intersubunit interactions at elevated temperatures are responsible for the distinct active site features that promote the substrate specificity and thermostability of thermophilic AIs. Copyright © 2016 Elsevier Inc. All rights reserved.

Redox Control of the Human Iron-Sulfur Repair Protein MitoNEET Activity via Its Iron-Sulfur Cluster*

PubMed Central

Golinelli-Cohen, Marie-Pierre; Lescop, Ewen; Mons, Cécile; Gonçalves, Sergio; Clémancey, Martin; Santolini, Jérôme; Guittet, Eric; Blondin, Geneviève; Latour, Jean-Marc; Bouton, Cécile

2016-01-01

Human mitoNEET (mNT) is the first identified Fe-S protein of the mammalian outer mitochondrial membrane. Recently, mNT has been implicated in cytosolic Fe-S repair of a key regulator of cellular iron homeostasis. Here, we aimed to decipher the mechanism by which mNT triggers its Fe-S repair capacity. By using tightly controlled reactions combined with complementary spectroscopic approaches, we have determined the differential roles played by both the redox state of the mNT cluster and dioxygen in cluster transfer and protein stability. We unambiguously demonstrated that only the oxidized state of the mNT cluster triggers cluster transfer to a generic acceptor protein and that dioxygen is neither required for the cluster transfer reaction nor does it affect the transfer rate. In the absence of apo-acceptors, a large fraction of the oxidized holo-mNT form is converted back to reduced holo-mNT under low oxygen tension. Reduced holo-mNT, which holds a [2Fe-2S]+ with a global protein fold similar to that of the oxidized form is, by contrast, resistant in losing its cluster or in transferring it. Our findings thus demonstrate that mNT uses an iron-based redox switch mechanism to regulate the transfer of its cluster. The oxidized state is the “active state,” which reacts promptly to initiate Fe-S transfer independently of dioxygen, whereas the reduced state is a “dormant form.” Finally, we propose that the redox-sensing function of mNT is a key component of the cellular adaptive response to help stress-sensitive Fe-S proteins recover from oxidative injury. PMID:26887944

Apoglobin Stability Is the Major Factor Governing both Cell-free and in Vivo Expression of Holomyoglobin*♦

PubMed Central

Samuel, Premila P.; Smith, Lucian P.; Phillips, George N.; Olson, John S.

2015-01-01

Expression levels in animal muscle tissues and in Escherichia coli vary widely for naturally occurring mammalian myoglobins (Mb). To explore this variation, we developed an in vitro transcription and wheat germ extract-based translation assay to examine quantitatively the factors that govern expression of holoMb. We constructed a library of naturally occurring Mbs from two terrestrial and four deep-diving aquatic mammals and three distal histidine mutants designed to enhance apoglobin stability but decrease hemin affinity. A strong linear correlation is observed between cell-free expression levels of holo-metMb variants and their corresponding apoglobin stabilities, which were measured independently by guanidine HCl-induced unfolding titrations using purified proteins. In contrast, there is little dependence of expression on hemin affinity. Our results confirm quantitatively that deep diving mammals have highly stable Mbs that express to higher levels in animal myocytes, E. coli, and the wheat germ cell-free system than Mbs from terrestrial mammals. Our theoretical analyses show that the rate of aggregation of unfolded apoMb is very large, and as a result, the key factor for high level expression of holoMb, and presumably other heme proteins, is an ultra high fraction of folded, native apoglobin that is capable of rapidly binding hemin. This fraction is determined by the overall equilibrium folding constant and not hemin affinity. These results also demonstrate that the cell-free transcription/translation system can be used as a high throughput platform to screen for apoglobin stability without the need to generate large amounts of protein for in vitro unfolding measurements. PMID:26205820

Mycobacterium tuberculosis WhiB1 is an essential DNA-binding protein with a nitric oxide sensitive iron-sulphur cluster

PubMed Central

Smith, Laura J.; Stapleton, Melanie R.; Fullstone, Gavin J. M.; Crack, Jason C.; Thomson, Andrew J.; Le Brun, Nick E.; Hunt, Debbie M.; Harvey, Evelyn; Adinolfi, Salvatore; Buxton, Roger S.; Green, Jeffrey

2010-01-01

Mycobacterium tuberculosis is a major pathogen that has the ability to establish, and emerge from, a persistent state. Wbl family proteins are associated with developmental processes in actinomycetes, and M. tuberculosis has seven such proteins. Here it is shown that the M. tuberculosis H37Rv whiB1 gene is essential. The WhiB1 protein possesses a [4Fe-4S]2+ cluster that is stable in air but reacts rapidly with eight equivalents of nitric oxide to yield two dinuclear dinitrosyl-iron thiol complexes. The [4Fe-4S] form of WhiB1 did not bind whiB1 promoter DNA, but the reduced and oxidized apo-WhiB1, and nitric oxide-treated holo-WhiB1 did bind to DNA. Mycobacterium smegmatis RNA polymerase induced transcription of whiB1 in vitro; however in the presence of apo-WhiB1 transcription was severely inhibited, irrespective of the presence or absence of the CRP protein Rv3676, which is known to activate whiB1 expression. Footprinting suggested that autorepression of whiB1 is achieved by apo-WhiB1 binding at a region that overlaps the core promoter elements. A model incorporating regulation of whiB1 expression in response to nitric oxide and cAMP is discussed with implications for sensing two important signals in establishing M. tuberculosis infections. PMID:20929442

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yee, Ben Chung; Wollaber, Allan Benton; Haut, Terry Scot

The high-order low-order (HOLO) method is a recently developed moment-based acceleration scheme for solving time-dependent thermal radiative transfer problems, and has been shown to exhibit orders of magnitude speedups over traditional time-stepping schemes. However, a linear stability analysis by Haut et al. (2015 Haut, T. S., Lowrie, R. B., Park, H., Rauenzahn, R. M., Wollaber, A. B. (2015). A linear stability analysis of the multigroup High-Order Low-Order (HOLO) method. In Proceedings of the Joint International Conference on Mathematics and Computation (M&C), Supercomputing in Nuclear Applications (SNA) and the Monte Carlo (MC) Method; Nashville, TN, April 19–23, 2015. American Nuclear Society.)more » revealed that the current formulation of the multigroup HOLO method was unstable in certain parameter regions. Since then, we have replaced the intensity-weighted opacity in the first angular moment equation of the low-order (LO) system with the Rosseland opacity. Furthermore, this results in a modified HOLO method (HOLO-R) that is significantly more stable.« less

A stable 1D multigroup high-order low-order method

DOE PAGES

Yee, Ben Chung; Wollaber, Allan Benton; Haut, Terry Scot; ...

2016-07-13

The high-order low-order (HOLO) method is a recently developed moment-based acceleration scheme for solving time-dependent thermal radiative transfer problems, and has been shown to exhibit orders of magnitude speedups over traditional time-stepping schemes. However, a linear stability analysis by Haut et al. (2015 Haut, T. S., Lowrie, R. B., Park, H., Rauenzahn, R. M., Wollaber, A. B. (2015). A linear stability analysis of the multigroup High-Order Low-Order (HOLO) method. In Proceedings of the Joint International Conference on Mathematics and Computation (M&C), Supercomputing in Nuclear Applications (SNA) and the Monte Carlo (MC) Method; Nashville, TN, April 19–23, 2015. American Nuclear Society.)more » revealed that the current formulation of the multigroup HOLO method was unstable in certain parameter regions. Since then, we have replaced the intensity-weighted opacity in the first angular moment equation of the low-order (LO) system with the Rosseland opacity. Furthermore, this results in a modified HOLO method (HOLO-R) that is significantly more stable.« less

Regulation of expression of the ada gene controlling the adaptive response. Interactions with the ada promoter of the Ada protein and RNA polymerase.

PubMed

Sakumi, K; Sekiguchi, M

1989-01-20

The Ada protein of Escherichia coli catalyzes transfer of methyl groups from methylated DNA to its own molecule, and the methylated form of Ada protein promotes transcription of its own gene, ada. Using an in vitro reconstituted system, we found that both the sigma factor and the methylated Ada protein are required for transcription of the ada gene. To elucidate molecular mechanisms involved in the regulation of the ada transcription, we investigated interactions of the non-methylated and methylated forms of Ada protein and the RNA polymerase holo enzyme (the core enzyme and sigma factor) with a DNA fragment carrying the ada promoter region. Footprinting analyses revealed that the methylated Ada protein binds to a region from positions -63 to -31, which includes the ada regulatory sequence AAAGCGCA. No firm binding was observed with the non-methylated Ada protein, although some DNase I-hypersensitive sites were produced in the promoter by both types of Ada protein. RNA polymerase did bind to the promoter once the methylated Ada protein had bound to the upstream sequence. To correlate these phenomena with the process in vivo, we used the DNAs derived from promoter-defective mutants. No binding of Ada protein nor of RNA polymerase occurred with a mutant DNA having a C to G substitution at position -47 within the ada regulatory sequence. In the case of a -35 box mutant with a T to A change at position -34, the methylated Ada protein did bind to the ada regulatory sequence, yet there was no RNA polymerase binding. Thus, the binding of the methylated Ada protein to the upstream region apparently facilitates binding of the RNA polymerase to the proper region of the promoter. The Ada protein possesses two known methyl acceptor sites, Cys69 and Cys321. The role of methylation of each cysteine residue was investigated using mutant forms of the Ada protein. The Ada protein with the cysteine residue at position 69 replaced by alanine was incapable of binding to the ada promoter even when the cysteine residue at position 321 of the protein was methylated. When the Ada protein with alanine at position 321 was methylated, it acquired the potential to bind to the ada promoter. These results are compatible with the notion that methylation of the cysteine residue at position 69 causes a conformational change of the Ada protein, thereby facilitating binding of the protein to the upstream regulatory sequence.

Combined indicator of vitamin B12 status: modification for missing biomarkers and folate status, and recommendations for revised cut-points

USDA-ARS?s Scientific Manuscript database

Background: We propose a novel approach to diagnose B12 status by combining four blood markers: total B12 (B12), holo-transcobalamin (holoTC), methylmalonic acid (MMA) and total homocysteine (tHcy). Combined B12 status is expressed as cB12=log10[(holoTC•B12)/(MMA•Hcy)]–(reference, age function). Her...

Structures of holo wild-type human cellular retinol-binding protein II (hCRBPII) bound to retinol and retinal

PubMed Central

Nossoni, Zahra; Assar, Zahra; Yapici, Ipek; Nosrati, Meisam; Wang, Wenjing; Berbasova, Tetyana; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James

2014-01-01

Cellular retinol-binding proteins (CRBPs) I and II, which are members of the intracellular lipid-binding protein (iLBP) family, are retinoid chaperones that are responsible for the intracellular transport and delivery of both retinol and retinal. Although structures of retinol-bound CRBPI and CRBPII are known, no structure of a retinal-bound CRBP has been reported. In addition, the retinol-bound human CRBPII (hCRBPII) structure shows partial occupancy of a non­canonical conformation of retinol in the binding pocket. Here, the structure of retinal-bound hCRBPII and the structure of retinol-bound hCRBPII with retinol fully occupying the binding pocket are reported. It is further shown that the retinoid derivative seen in both the zebrafish CRBP and the hCRBPII structures is likely to be the product of flux-dependent and wavelength-dependent X-ray damage during data collection. The structures of retinoid-bound CRBPs are compared and contrasted, and rationales for the differences in binding affinities for retinal and retinol are provided. PMID:25478840

Structures of holo wild-type human cellular retinol-binding protein II (hCRBPII) bound to retinol and retinal.

PubMed

Nossoni, Zahra; Assar, Zahra; Yapici, Ipek; Nosrati, Meisam; Wang, Wenjing; Berbasova, Tetyana; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James

2014-12-01

Cellular retinol-binding proteins (CRBPs) I and II, which are members of the intracellular lipid-binding protein (iLBP) family, are retinoid chaperones that are responsible for the intracellular transport and delivery of both retinol and retinal. Although structures of retinol-bound CRBPI and CRBPII are known, no structure of a retinal-bound CRBP has been reported. In addition, the retinol-bound human CRBPII (hCRBPII) structure shows partial occupancy of a noncanonical conformation of retinol in the binding pocket. Here, the structure of retinal-bound hCRBPII and the structure of retinol-bound hCRBPII with retinol fully occupying the binding pocket are reported. It is further shown that the retinoid derivative seen in both the zebrafish CRBP and the hCRBPII structures is likely to be the product of flux-dependent and wavelength-dependent X-ray damage during data collection. The structures of retinoid-bound CRBPs are compared and contrasted, and rationales for the differences in binding affinities for retinal and retinol are provided.

An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, William J; Senkovich, Olga; Chattopadhyay, Debasish

2009-06-08

The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips tomore » the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step. We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2{angstrom} resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in the substrate-free conformation. Orientation of the substrate with respect to the active site histidine and serine (in the mutant enzyme) also varies in different subunits. The structures of the C. parvum GAPDH ternary complex and other GAPDH complexes demonstrate the plasticity of the substrate binding site. We propose that the active site of GAPDH can accommodate the substrate in multiple conformations at multiple locations during the initial encounter. However, the C-3 phosphate group clearly prefers the 'new Pi' site for initial binding in the active site.« less

NMR studies reveal the role of biomembranes in modulating ligand binding and release by intracellular bile acid binding proteins.

PubMed

Pedò, Massimo; Löhr, Frank; D'Onofrio, Mariapina; Assfalg, Michael; Dötsch, Volker; Molinari, Henriette

2009-12-18

Bile acid molecules are transferred vectorially between basolateral and apical membranes of hepatocytes and enterocytes in the context of the enterohepatic circulation, a process regulating whole body lipid homeostasis. This work addresses the role of the cytosolic lipid binding proteins in the intracellular transfer of bile acids between different membrane compartments. We present nuclear magnetic resonance (NMR) data describing the ternary system composed of the bile acid binding protein, bile acids, and membrane mimetic systems, such as anionic liposomes. This work provides evidence that the investigated liver bile acid binding protein undergoes association with the anionic membrane and binding-induced partial unfolding. The addition of the physiological ligand to the protein-liposome mixture is capable of modulating this interaction, shifting the equilibrium towards the free folded holo protein. An ensemble of NMR titration experiments, based on nitrogen-15 protein and ligand observation, confirm that the membrane and the ligand establish competing binding equilibria, modulating the cytoplasmic permeability of bile acids. These results support a mechanism of ligand binding and release controlled by the onset of a bile salt concentration gradient within the polarized cell. The location of a specific protein region interacting with liposomes is highlighted.

Mechanism and Substrate Recognition of Human Holo ACP Synthase

PubMed Central

Bunkoczi, Gabor; Pasta, Saloni; Joshi, Anil; Wu, Xiaoqiu; Kavanagh, Kathryn L.; Smith, Stuart; Oppermann, Udo

2007-01-01

Summary Mammals utilize a single phosphopantetheinyl transferase for the posttranslational modification of at least three different apoproteins: the carrier protein components of cytosolic and mitochondrial fatty acid synthases and the aminoadipate semialdehyde reductase involved in lysine degradation. We determined the crystal structure of the human phosphopantetheinyl transferase, a eukaryotic phosphopantetheinyl transferase characterized, complexed with CoA and Mg2+, and in ternary complex with CoA and ACP. The involvement of key residues in ligand binding and catalysis was confirmed by mutagenesis and kinetic analysis. Human phosphopantetheinyl transferase exhibits an α/β fold and 2-fold pseudosymmetry similar to the Sfp phosphopantetheinyl transferase from Bacillus subtilis. Although the bound ACP exhibits a typical four-helix structure, its binding is unusual in that it is facilitated predominantly by hydrophobic interactions. A detailed mechanism is proposed describing the substrate binding and catalytic process. PMID:18022563

Structural Basis for Flip-Flop Action of Thiamin Pyrophosphate-dependent Enzymes Revealed by Human Pyruvate Dehydrogenase

NASA Technical Reports Server (NTRS)

Ciszak, Ewa M.; Korotchkina, Lioubov G.; Dominiak, Paulina M.; Sidhu, Sukdeep; Patel, Mulchand S.

2003-01-01

The derivative of vitamin B1, thiamin pyrophosphate, is a cofactor of enzymes performing catalysis in pathways of energy production. In alpha (sub 2) beta (sub 2)-heterotetrameric human pyruvate dehydrogenase, this cofactor is used to cleave the C(sup alpha) -C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites has not yet been understood. To understand the mechanism of action of this enzyme, we determined the crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95-Angstrom resolution. We propose a model for the flip-flop action of this enzyme through a concerted approximately 2-Angstrom shuttle-like motion of its heterodimers. Similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase with functionally related enzymes suggests that this newly defined shuttle-like motion of domains is common to the family of thiamin pyrophosphate-dependent enzymes.

Prediction of fatty acid-binding residues on protein surfaces with three-dimensional probability distributions of interacting atoms.

PubMed

Mahalingam, Rajasekaran; Peng, Hung-Pin; Yang, An-Suei

2014-08-01

Protein-fatty acid interaction is vital for many cellular processes and understanding this interaction is important for functional annotation as well as drug discovery. In this work, we present a method for predicting the fatty acid (FA)-binding residues by using three-dimensional probability density distributions of interacting atoms of FAs on protein surfaces which are derived from the known protein-FA complex structures. A machine learning algorithm was established to learn the characteristic patterns of the probability density maps specific to the FA-binding sites. The predictor was trained with five-fold cross validation on a non-redundant training set and then evaluated with an independent test set as well as on holo-apo pair's dataset. The results showed good accuracy in predicting the FA-binding residues. Further, the predictor developed in this study is implemented as an online server which is freely accessible at the following website, http://ismblab.genomics.sinica.edu.tw/. Copyright © 2014 Elsevier B.V. All rights reserved.

Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator

NASA Astrophysics Data System (ADS)

Deng, Zengqin; Wang, Qing; Liu, Zhao; Zhang, Manfeng; Machado, Ana Carolina Dantas; Chiu, Tsu-Pei; Feng, Chong; Zhang, Qi; Yu, Lin; Qi, Lei; Zheng, Jiangge; Wang, Xu; Huo, Xinmei; Qi, Xiaoxuan; Li, Xiaorong; Wu, Wei; Rohs, Remo; Li, Ying; Chen, Zhongzhou

2015-07-01

Ferric uptake regulator (Fur) plays a key role in the iron homeostasis of prokaryotes, such as bacterial pathogens, but the molecular mechanisms and structural basis of Fur-DNA binding remain incompletely understood. Here, we report high-resolution structures of Magnetospirillum gryphiswaldense MSR-1 Fur in four different states: apo-Fur, holo-Fur, the Fur-feoAB1 operator complex and the Fur-Pseudomonas aeruginosa Fur box complex. Apo-Fur is a transition metal ion-independent dimer whose binding induces profound conformational changes and confers DNA-binding ability. Structural characterization, mutagenesis, biochemistry and in vivo data reveal that Fur recognizes DNA by using a combination of base readout through direct contacts in the major groove and shape readout through recognition of the minor-groove electrostatic potential by lysine. The resulting conformational plasticity enables Fur binding to diverse substrates. Our results provide insights into metal ion activation and substrate recognition by Fur that suggest pathways to engineer magnetotactic bacteria and antipathogenic drugs.

Low holo-transcobalamin levels are prevalent in vegetarians and is associated with coronary artery disease in Indian population.

PubMed

Basak, Trayambak; Garg, Gaurav; Bhardwaj, Nitin; Tanwar, Vinay Singh; Seth, Sandeep; Karthikeyan, Ganesan; Sengupta, Shantanu

2016-07-01

Coronary artery disease (CAD) has been increasing alarmingly in India. We had earlier shown that vitamin B12 deficiency is associated with CAD in Indian population. However, only about a quarter of the total vitamin B12 is internalised in the cells by the proteins transcobalamin II. Vitamin B12-bound transcobalamin II (holotranscobalamin, holoTC) is thus referred to as biologically active B12. In this study, we ascertained the levels of holoTC in 501 CAD cases and 1253 healthy controls and for the first time show that holoTC levels are significantly lower (p = 2.57E-4) in CAD (26.81 pmol/l) cases as compared to controls (29.97 pmol/l).

Determination of the cellular retinoic-acid-binding protein in dysplastic epithelia of the cervix uteri, differentiated into apo and holo forms.

PubMed

Volz-Köster, S; Volz, J; Kiefer, A; Biesalski, H K

2000-01-01

The appearance of the cervical mucosa is regulated by different factors including retinoic acid. Hormone-dependent alteration of the cervix uteri mucosa is accompanied by a decrease or increase of cytoplasmatic retinoic-acid-binding protein (CRABP). To elucidate whether this hormone-dependent alteration of CRABP is preserved in the case of neoplasms of the cervix uteri, we measured the level of total and apo-CRABP in normal and neoplastically transformed cervical cells. In a prospective pilot study, standardised biopsies of normal epithelium and cervical intra-epithelial neoplasm grade 3 (CIN III) were taken from 24 patients. A newly developed method was used to determine the intra-epithelial level of apo- and total CRABP. The concentration of total CRABP in normal squamous epithelium compared with that in intra-epithelial neoplasm grade 3 is very significantly lower in the CIN III areas (normal: 3.66 +/- 1.46 pmol/ mg wet weight +/- SD; CIN III 1.43 +/- 0.59 pmol/mg P < 0.01). In addition CRABP in the apo form is lower in normal than in neoplastic epithelium (Wilcoxon test for paired non-parametric values: P < 0.05; mean for all patients: normal: 1.65 + 0.82 pmol/mg; CIN III: 1.14 +/- 0.23 pmol/mg). From our results we conclude that, in neoplastically transformed cells, the hormone-dependent CRABP cycle is interrupted. Whether this has consequences for the further development of the neoplastic cells has to be elucidated.

Holographic Rovers: Augmented Reality and the Microsoft HoloLens

NASA Technical Reports Server (NTRS)

Toler, Laura

2017-01-01

Augmented Reality is an emerging field in technology, and encompasses Head Mounted Displays, smartphone apps, and even projected images. HMDs include the Meta 2, Magic Leap, Avegant Light Field, and the Microsoft HoloLens, which is evaluated specifically. The Microsoft HoloLens is designed to be used as an AR personal computer, and is being optimized with that goal in mind. Microsoft allied with the Unity3D game engine to create an SDK for interested application developers that can be used in the Unity environment.

Conformational analysis of HAMLET, the folding variant of human alpha-lactalbumin associated with apoptosis.

PubMed

Casbarra, Annarita; Birolo, Leila; Infusini, Giuseppe; Dal Piaz, Fabrizio; Svensson, Malin; Pucci, Piero; Svanborg, Catharina; Marino, Gennaro

2004-05-01

A combination of hydrogen/deuterium (H/D) exchange and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the conformation in solution of HAMLET, the folding variant of human alpha-lactalbumin, complexed to oleic acid, that induces apoptosis in tumor and immature cells. Although near- and far-UV CD and fluorescence spectroscopy were not able to discriminate between HAMLET and apo-alpha-lactalbumin, H/D exchange experiments clearly showed that they correspond to two distinct conformational states, with HAMLET incorporating a greater number of deuterium atoms than the apo and holo forms. Complementary proteolysis experiments revealed that HAMLET and apo are both accessible to proteases in the beta-domain but showed substantial differences in accessibility to proteases at specific sites. The overall results indicated that the conformational changes associated with the release of Ca2+ are not sufficient to induce the HAMLET conformation. Metal depletion might represent the first event to produce a partial unfolding in the beta-domain of alpha-lactalbumin, but some more unfolding is needed to generate the active conformation HAMLET, very likely allowing the protein to bind the C18:1 fatty acid moiety. On the basis of these data, a putative binding site of the oleic acid, which stabilizes the HAMLET conformation, is proposed.

The HOLO Series: Critical Ground-Based Demonstrations of Holographic Scanning Lidars

NASA Technical Reports Server (NTRS)

Wilkerson, Thomas D.; Sanders, Jason A.; Andrus, Ionio Q.; Schwemmer, Geary K.; Miller, David O.; Guerra, David; Schnick, Jeffrey; Moody, Stephen E.

2000-01-01

Results of two lidar measurement campaigns are presented, HOLO-1 (Utah, March 1999) and HOLO-2 (New Hampshire, June 1999). These tests demonstrate the ability of lidars utilizing holographic optical elements (HOEs) to determine tropospheric wind velocity and direction at cloud altitude. Several instruments were employed. HOLO-1 used the 1,064 mm transmission-HOE lidar (HARLIE, Goddard Space Flight Center), a zenith-staring 532 nm lidar (AROL-2, Utah State University), and a wide-field video camera (SkyCam) for imagery of clouds overhead. HOLO-2 included these instruments plus the 532 nm reflection-HOE lidar (PHASERS, St. Anselm College). HARLIE and PHASERS scan the sky at constant cone angles of 45 deg. and 42 deg. from normal, respectively. The progress of clouds and entire cloud fields across the sky is tracked by the repetitive conical scans of the HOE lidars. AROL-2 provides the attitude information enabling the SkyCam cloud images to be analyzed for independent data on cloud motion. Data from the HOE lidars are reduced by means of correlations, visualization by animation techniques, and kinematic diagrams of cloud feature motion. Excellent agreement is observed between the HOE lidar results and those obtained with video imagery and lidar ranging.

The structure of apo and holo forms of xylose reductase, a dimeric aldo-keto reductase from Candida tenuis.

PubMed

Kavanagh, Kathryn L; Klimacek, Mario; Nidetzky, Bernd; Wilson, David K

2002-07-16

Xylose reductase is a homodimeric oxidoreductase dependent on NADPH or NADH and belongs to the largely monomeric aldo-keto reductase superfamily of proteins. It catalyzes the first step in the assimilation of xylose, an aldose found to be a major constituent monosaccharide of renewable plant hemicellulosic material, into yeast metabolic pathways. It does this by reducing open chain xylose to xylitol, which is reoxidized to xylulose by xylitol dehydrogenase and metabolically integrated via the pentose phosphate pathway. No structure has yet been determined for a xylose reductase, a dimeric aldo-keto reductase or a family 2 aldo-keto reductase. The structures of the Candida tenuis xylose reductase apo- and holoenzyme, which crystallize in spacegroup C2 with different unit cells, have been determined to 2.2 A resolution and an R-factor of 17.9 and 20.8%, respectively. Residues responsible for mediating the novel dimeric interface include Asp-178, Arg-181, Lys-202, Phe-206, Trp-313, and Pro-319. Alignments with other superfamily members indicate that these interactions are conserved in other dimeric xylose reductases but not throughout the remainder of the oligomeric aldo-keto reductases, predicting alternate modes of oligomerization for other families. An arrangement of side chains in a catalytic triad shows that Tyr-52 has a conserved function as a general acid. The loop that folds over the NAD(P)H cosubstrate is disordered in the apo form but becomes ordered upon cosubstrate binding. A slow conformational isomerization of this loop probably accounts for the observed rate-limiting step involving release of cosubstrate. Xylose binding (K(m) = 87 mM) is mediated by interactions with a binding pocket that is more polar than a typical aldo-keto reductase. Modeling of xylose into the active site of the holoenzyme using ordered waters as a guide for sugar hydroxyls suggests a convincing mode of substrate binding.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Halavaty, Andrei S.; Northwestern University, Chicago, IL 60611; Kim, Youngchang

The structural characterization of acyl-carrier-protein synthase (AcpS) from three different pathogenic microorganisms is reported. One interesting finding of the present work is a crystal artifact related to the activity of the enzyme, which fortuitously represents an opportunity for a strategy to design a potential inhibitor of a pathogenic AcpS. Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holomore » form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpS{sub SA}), Vibrio cholerae (AcpS{sub VC}) and Bacillus anthracis (AcpS{sub BA}) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpS{sub BA} is emphasized because of the two 3′, 5′-adenosine diphosphate (3′, 5′-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3′, 5′-ADP is bound as the 3′, 5′-ADP part of CoA in the known structures of the CoA–AcpS and 3′, 5′-ADP–AcpS binary complexes. The position of the second 3′, 5′-ADP has never been described before. It is in close proximity to the first 3′, 5′-ADP and the ACP-binding site. The coordination of two ADPs in AcpS{sub BA} may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP.« less

Conversion of pre-RISC to holo-RISC by Ago2 during assembly of RNAi complexes

PubMed Central

Kim, Kevin; Lee, Young Sik; Carthew, Richard W.

2007-01-01

In the Drosophila RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) direct Argonaute2 (Ago2), an endonuclease, within the RNA-induced silencing complex (RISC) to cleave complementary mRNA targets. In vitro studies have shown that, for each siRNA duplex, RISC retains only one strand, the guide, and releases the other, the passenger, to form a holo-RISC complex. Here, we have isolated a new Ago2 mutant allele and provide, for the first time, in vivo evidence that endogenous Ago2 slicer activity is important to mount an RNAi response in Drosophila. We demonstrate in vivo that efficient removal of the passenger strand from RISC requires the cleavage activity of Ago2. We have also identified a new intermediate complex in the RISC assembly pathway, pre-RISC, in which Ago2 is stably bound to double-stranded siRNA. PMID:17123955

Color silver halide hologram production and mastering

NASA Astrophysics Data System (ADS)

Bjelkhagen, Hans I.; Huang, Qiang

1997-04-01

Color reflection holograms recorded with the Denisyuk geometry have been demonstrated by the recently formed HOLOS Corporation in New Hampshire. The Slavich red-green-blue (RGB) sensitized ultra-high resolution silver halide emulsion was used for the hologram recording. The employed laser wavelengths were 647 nm, 532 nm, and 476 nm, generated by an argon ion, a frequency doubled Nd:YAG, and a krypton ion laser, respectively. A beam combination mechanism with dichroic filters enabled a simultaneous RGB exposure, which made the color balance and overall exposure energy easy to control as well as simplifying the recording procedure. HOLOS has been producing limited edition color holograms in various sizes from 4' X 5' to 12' X 16'. A 30 foot long optical table and high power lasers will enable HOLOS to record color holograms up to the size of one meter square in the near future. Various approaches have been investigated in generating color hologram masters which have sufficiently high diffraction efficiency to contact copy the color images onto photopolymer materials. A specially designed test object including the 1931 CIE chromaticity diagram, a rainbow ribbon cable, pure yellow dots, and a cloisonne elephant was used for color recording experiments. In addition, the Macbeth Color Checker chart was used. Both colorimetric evaluation and scattering noise measurements were performed using the PR-650 Photo Research SpectraScan SpectraCalorimeter.

Visualization of molecular structures using HoloLens-based augmented reality

PubMed Central

Hoffman, MA; Provance, JB

2017-01-01

Biological molecules and biologically active small molecules are complex three dimensional structures. Current flat screen monitors are limited in their ability to convey the full three dimensional characteristics of these molecules. Augmented reality devices, including the Microsoft HoloLens, offer an immersive platform to change how we interact with molecular visualizations. We describe a process to incorporate the three dimensional structures of small molecules and complex proteins into the Microsoft HoloLens using aspirin and the human leukocyte antigen (HLA) as examples. Small molecular structures can be introduced into the HoloStudio application, which provides native support for rotating, resizing and performing other interactions with these molecules. Larger molecules can be imported through the Unity gaming development platform and then Microsoft Visual Developer. The processes described here can be modified to import a wide variety of molecular structures into augmented reality systems and improve our comprehension of complex structural features. PMID:28815109

Optical architecture of HoloLens mixed reality headset

NASA Astrophysics Data System (ADS)

Kress, Bernard C.; Cummings, William J.

2017-06-01

HoloLens by Microsoft Corp. is the world's first untethered Mixed Reality (MR) Head Mounted Display (HMD) system, released to developers in March 2016 as a Development Kit. We review in this paper the various display requirements and subsequent optical hardware choices we made for HoloLens. Its main achievements go along performance and comfort for the user: it is the first fully untethered MR headset, with the highest angular resolution and the industry's largest eyebox. It has the first inside-out global sensor fusion system including precise head tracking and 3D mapping all controlled by a fully custom on-board GPU. Based on such achievements, HoloLens came out as the most advanced MR system today. Additional features may be implemented in next generations MR headsets, leading to the ultimate experience for the user, and securing the upcoming fabulous AR/MR market predicted by most analysts.

PoLi: A Virtual Screening Pipeline Based On Template Pocket And Ligand Similarity

PubMed Central

Roy, Ambrish; Srinivasan, Bharath; Skolnick, Jeffrey

2015-01-01

Often in pharmaceutical research, the goal is to identify small molecules that can interact with and appropriately modify the biological behavior of a new protein target. Unfortunately, most proteins lack both known structures and small molecule binders, prerequisites of many virtual screening, VS, approaches. For such proteins, ligand homology modeling, LHM, that copies ligands from homologous and perhaps evolutionarily distant template proteins, has been shown to be a powerful VS approach to identify possible binding ligands. However, if we want to target a specific pocket for which there is no homologous holo template protein structure, then LHM will not work. To address this issue, in a new pocket based approach, PoLi, we generalize LHM by exploiting the fact that the number of distinct small molecule ligand binding pockets in proteins is small. PoLi identifies similar ligand binding pockets in a holo-template protein library, selectively copies relevant parts of template ligands and uses them for VS. In practice, PoLi is a hybrid structure and ligand based VS algorithm that integrates 2D fingerprint-based and 3D shape-based similarity metrics for improved virtual screening performance. On standard DUD and DUD-E benchmark databases, using modeled receptor structures, PoLi achieves an average enrichment factor of 13.4 and 9.6 respectively, in the top 1% of the screened library. In contrast, traditional docking based VS using AutoDock Vina and homology-based VS using FINDSITEfilt have an average enrichment of 1.6 (3.0) and 9.0 (7.9) on the DUD (DUD-E) sets respectively. Experimental validation of PoLi predictions on dihydrofolate reductase, DHFR, using differential scanning fluorimetry, DSF, identifies multiple ligands with diverse molecular scaffolds, thus demonstrating the advantage of PoLi over current state-of-the-art VS methods. PMID:26225536

HoloVir: A Workflow for Investigating the Diversity and Function of Viruses in Invertebrate Holobionts

PubMed Central

Laffy, Patrick W.; Wood-Charlson, Elisha M.; Turaev, Dmitrij; Weynberg, Karen D.; Botté, Emmanuelle S.; van Oppen, Madeleine J. H.; Webster, Nicole S.; Rattei, Thomas

2016-01-01

Abundant bioinformatics resources are available for the study of complex microbial metagenomes, however their utility in viral metagenomics is limited. HoloVir is a robust and flexible data analysis pipeline that provides an optimized and validated workflow for taxonomic and functional characterization of viral metagenomes derived from invertebrate holobionts. Simulated viral metagenomes comprising varying levels of viral diversity and abundance were used to determine the optimal assembly and gene prediction strategy, and multiple sequence assembly methods and gene prediction tools were tested in order to optimize our analysis workflow. HoloVir performs pairwise comparisons of single read and predicted gene datasets against the viral RefSeq database to assign taxonomy and additional comparison to phage-specific and cellular markers is undertaken to support the taxonomic assignments and identify potential cellular contamination. Broad functional classification of the predicted genes is provided by assignment of COG microbial functional category classifications using EggNOG and higher resolution functional analysis is achieved by searching for enrichment of specific Swiss-Prot keywords within the viral metagenome. Application of HoloVir to viral metagenomes from the coral Pocillopora damicornis and the sponge Rhopaloeides odorabile demonstrated that HoloVir provides a valuable tool to characterize holobiont viral communities across species, environments, or experiments. PMID:27375564

Structural basis of AMPK regulation by adenine nucleotides and glycogen

DOE PAGES

Li, Xiaodan; Wang, Lili; Zhou, X. Edward; ...

2014-11-21

AMP-activated protein kinase (AMPK) is a central cellular energy sensor and regulator of energy homeostasis, and a promising drug target for the treatment of diabetes, obesity, and cancer. Here we present low-resolution crystal structures of the human α1β2γ1 holo-AMPK complex bound to its allosteric modulators AMP and the glycogen-mimic cyclodextrin, both in the phosphorylated (4.05 Å) and non-phosphorylated (4.60 Å) state. In addition, we have solved a 2.95 Å structure of the human kinase domain (KD) bound to the adjacent autoinhibitory domain (AID) and have performed extensive biochemical and mutational studies. Altogether, these studies illustrate an underlying mechanism of allostericmore » AMPK modulation by AMP and glycogen, whose binding changes the equilibria between alternate AID (AMP) and carbohydrate-binding module (glycogen) interactions.« less

Solution Structure and Backbone Dynamics of Human Liver Fatty Acid Binding Protein: Fatty Acid Binding Revisited

PubMed Central

Cai, Jun; Lücke, Christian; Chen, Zhongjing; Qiao, Ye; Klimtchuk, Elena; Hamilton, James A.

2012-01-01

Liver fatty acid binding protein (L-FABP), a cytosolic protein most abundant in liver, is associated with intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein family, L-FABP is of particular interest as it can i), bind two fatty acid molecules simultaneously and ii), accommodate a variety of bulkier physiological ligands such as bilirubin and fatty acyl CoA. To better understand the promiscuous binding and transport properties of L-FABP, we investigated structure and dynamics of human L-FABP with and without bound ligands by means of heteronuclear NMR. The overall conformation of human L-FABP shows the typical β-clam motif. Binding of two oleic acid (OA) molecules does not alter the protein conformation substantially, but perturbs the chemical shift of certain backbone and side-chain protons that are involved in OA binding according to the structure of the human L-FABP/OA complex. Comparison of the human apo and holo L-FABP structures revealed no evidence for an “open-cap” conformation or a “swivel-back” mechanism of the K90 side chain upon ligand binding, as proposed for rat L-FABP. Instead, we postulate that the lipid binding process in L-FABP is associated with backbone dynamics. PMID:22713574

Human lactoferrin stimulates skin keratinocyte function and wound re-epithelialization.

PubMed

Tang, L; Wu, J J; Ma, Q; Cui, T; Andreopoulos, F M; Gil, J; Valdes, J; Davis, S C; Li, J

2010-07-01

Human lactoferrin (hLF), a member of the transferrin family, is known for its antimicrobial and anti-inflammatory effects. Recent studies on various nonskin cell lines indicate that hLF may have a stimulatory effect on cell proliferation. To study the potential role of hLF in wound re-epithelialization. The effects of hLF on cell growth, migration, attachment and survival were assessed, with a rice-derived recombinant hLF (holo-rhLF), using proliferation analysis, scratch migration assay, calcein-AM/propidium iodide staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) method, respectively. The mechanisms of hLF on cell proliferation and migration were explored using specific pathway inhibitors. The involvement of lactoferrin receptor low-density lipoprotein receptor-related protein 1 (LRP1) was examined with RNA interference technique. An in vivo swine second-degree burn wound model was also used to assess wound re-epithelialization. Studies revealed that holo-rhLF significantly stimulated keratinocyte proliferation which could be blocked by mitogen-activated protein kinase (MAPK) kinase 1 inhibitor. Holo-rhLF also showed strong promoting effects on keratinocyte migration, which could be blocked by either inhibition of the MAPK, Src and Rho/ROCK pathways, or downregulation of the LRP1 receptor. With cells under starving or 12-O-tetradecanoylphorbol-13-acetate exposure, the addition of holo-rhLF was found greatly to increase cell viability and inhibit cell apoptosis. Additionally, holo-rhLF significantly increased the rate of wound re-epithelialization in swine second-degree burn wounds. Our studies demonstrate the direct effects of holo-rhLF on wound re-epithelialization including the enhancement of keratinocyte proliferation and migration as well as the protection of cells from apoptosis. The data strongly indicate its potential therapeutic applications in wound healing.

Visualisation Enhancement of HoloCatT Matrix

NASA Astrophysics Data System (ADS)

Rosli, Nor Azlin; Mohamed, Azlinah; Khan, Rahmattullah

Graphology and personality psychology are two different analyses approach perform by two different groups of people, but addresses the personality of the person that were analyzed. It is of interest to visualize a system that would aid personality identification given information visualization of these two domains. Therefore, a research in identifying the relationship between those two domains has been carried out by producing the HoloCatT Matrix, a combination of graphology features and a selected personality traits approach. The objectives of this research are to identify new features of the existing HoloCatT Matrix and validate the new version of matrix with two (2) related group of experts. A set of questionnaire has been distributed to a group of Personologist to identify the relationship and an interview has been done with a Graphologist in validating the matrix. Based on the analysis, 87.5% of the relation confirmed by both group of experts and subsequently the third (3rd) version of HoloCatT Matrix is obtained.

Holo-analysis.

PubMed

Rosen, G D

2006-06-01

Meta-analysis is a vague descriptor used to encompass very diverse methods of data collection analysis, ranging from simple averages to more complex statistical methods. Holo-analysis is a fully comprehensive statistical analysis of all available data and all available variables in a specified topic, with results expressed in a holistic factual empirical model. The objectives and applications of holo-analysis include software production for prediction of responses with confidence limits, translation of research conditions to praxis (field) circumstances, exposure of key missing variables, discovery of theoretically unpredictable variables and interactions, and planning future research. Holo-analyses are cited as examples of the effects on broiler feed intake and live weight gain of exogenous phytases, which account for 70% of variation in responses in terms of 20 highly significant chronological, dietary, environmental, genetic, managemental, and nutrient variables. Even better future accountancy of variation will be facilitated if and when authors of papers routinely provide key data for currently neglected variables, such as temperatures, complete feed formulations, and mortalities.

Digital holographic microscopy

NASA Astrophysics Data System (ADS)

Barkley, Solomon; Dimiduk, Thomas; Manoharan, Vinothan

Digital holographic microscopy is a 3D optical imaging technique with high temporal ( ms) and spatial ( 10 nm) precision. However, its adoption as a characterization technique has been limited due to the inherent difficulty of recovering 3D data from the holograms. Successful analysis has traditionally required substantial knowledge about the sample being imaged (for example, the approximate positions of particles in the field of view), as well as expertise in scattering theory. To overcome the obstacles to widespread adoption of holographic microscopy, we developed HoloPy - an open source python package for analysis of holograms and scattering data. HoloPy uses Bayesian statistical methods to determine the geometry and properties of discrete scatterers from raw holograms. We demonstrate the use of HoloPy to measure the dynamics of colloidal particles at interfaces, to ascertain the structures of self-assembled colloidal particles, and to track freely swimming bacteria. The HoloPy codebase is thoroughly tested and well-documented to facilitate use by the broader experimental community. This research is supported by NSF Grant DMR-1306410 and NSERC.

Reactivation of mutant p53: Constraints on mechanism highlighted by principal component analysis of the DNA binding domain.

PubMed

Ouaray, Zahra; ElSawy, Karim M; Lane, David P; Essex, Jonathan W; Verma, Chandra

2016-10-01

Most p53 mutations associated with cancer are located in its DNA binding domain (DBD). Many structures (X-ray and NMR) of this domain are available in the protein data bank (PDB) and a vast conformational heterogeneity characterizes the various free and complexed states. The major difference between the apo and the holo-complexed states appears to lie in the L1 loop. In particular, the conformations of this loop appear to depend intimately on the sequence of DNA to which it binds. This conclusion builds upon recent observations that implicate the tetramerization and the C-terminal domains (respectively TD and Cter) in DNA binding specificity. Detailed PCA analysis of the most recent collection of DBD structures from the PDB have been carried out. In contrast to recommendations that small molecules/drugs stabilize the flexible L1 loop to rescue mutant p53, our study highlights a need to retain the flexibility of the p53 DNA binding surface (DBS). It is the adaptability of this region that enables p53 to engage in the diverse interactions responsible for its functionality. Proteins 2016; 84:1443-1461. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Holos: A collaborative environment for similarity-based holistic approaches.

PubMed

Lê, Tâm Minh; Brard, Margot; Lê, Sébastien

2017-10-01

Through this article, we aim to introduce Holos-a new collaborative environment that allows researchers to carry out experiments based on similarity assessments between stimuli, such as in projective-mapping and sorting tasks. An important feature of Holos is its capacity to assess real-time individual processes during the task. Within the Holos environment, researchers can design experiments on its platform, which can handle four kinds of stimuli: concepts, images, sounds, and videos. In addition, researchers can share their study resources within the scientific community, including stimuli, experimental protocols, and/or the data collected. With a dedicated Android application combined with a tactile human-machine interface, subjects can perform experiments using a tablet to obtain similarity measures between stimuli. On the tablet, the stimuli are displayed as icons that can be dragged with one finger to position them, depending on the ways they are perceived. By recording the x,y coordinates of the stimuli while subjects move the icons, the obtained data can reveal the cognitive processes of the subjects during the experiment. Such data, named digit-tracking data, can be analyzed with the SensoMineR package. In this article, we describe how researchers can design an experiment, how subjects can perform the experiment, and how digit-tracking data can be statistically analyzed within the Holos environment. At the end of the article, a short exemplary experiment is presented.

Structures of three polycystic kidney disease-like domains from Clostridium histolyticum collagenases ColG and ColH

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bauer, Ryan; Janowska, Katarzyna; Taylor, Kelly

Clostridium histolyticumcollagenases ColG and ColH are segmental enzymes that are thought to be activated by Ca 2+-triggered domain reorientation to cause extensive tissue destruction. The collagenases consist of a collagenase module (s1), a variable number of polycystic kidney disease-like (PKD-like) domains (s2a and s2b in ColH and s2 in ColG) and a variable number of collagen-binding domains (s3 in ColH and s3a and s3b in ColG). The X-ray crystal structures of Ca 2+-bound holo s2b (1.4 Å resolution,R= 15.0%,R free= 19.1%) and holo s2a (1.9 Å resolution,R= 16.3%,R free= 20.7%), as well as of Ca 2+-free apo s2a (1.8 Åmore » resolution,R= 20.7%,R free= 27.2%) and two new forms of N-terminally truncated apo s2 (1.4 Å resolution,R= 16.9%,R free= 21.2%; 1.6 Å resolution,R= 16.2%,R free= 19.2%), are reported. The structurally similar PKD-like domains resemble the V-set Ig fold. In addition to a conserved β-bulge, the PKD-like domains feature a second bulge that also changes the allegiance of the subsequent β-strand. This β-bulge and the genesis of a Ca 2+pocket in the archaeal PKD-like domain suggest a close kinship between bacterial and archaeal PKD-like domains. Different surface properties and indications of different dynamics suggest unique roles for the PKD-like domains in ColG and in ColH. Surface aromatic residues found on ColH s2a-s2b, but not on ColG s2, may provide the weak interaction in the biphasic collagen-binding mode previously found in s2b-s3.B-factor analyses suggest that in the presence of Ca 2+the midsection of s2 becomes more flexible but the midsections of s2a and s2b stay rigid. The different surface properties and dynamics of the domains suggest that the PKD-like domains of M9B bacterial collagenase can be grouped into either a ColG subset or a ColH subset. The conserved properties of PKD-like domains in ColG and in ColH include Ca 2+binding. Conserved residues not only interact with Ca 2+, but also position the Ca 2+-interacting water molecule. Ca 2+aligns the N-terminal linker approximately parallel to the major axis of the domain. Ca 2+binding also increases stability against heat and guanidine hydrochloride, and may improve the longevity in the extracellular matrix. The results of this study will further assist in developing collagen-targeting vehicles for various signal molecules.« less

Structures of three polycystic kidney disease-like domains from Clostridium histolyticum collagenases ColG and ColH

DOE PAGES

Bauer, Ryan; Janowska, Katarzyna; Taylor, Kelly; ...

2015-03-01

Clostridium histolyticumcollagenases ColG and ColH are segmental enzymes that are thought to be activated by Ca 2+-triggered domain reorientation to cause extensive tissue destruction. The collagenases consist of a collagenase module (s1), a variable number of polycystic kidney disease-like (PKD-like) domains (s2a and s2b in ColH and s2 in ColG) and a variable number of collagen-binding domains (s3 in ColH and s3a and s3b in ColG). The X-ray crystal structures of Ca 2+-bound holo s2b (1.4 Å resolution,R= 15.0%,R free= 19.1%) and holo s2a (1.9 Å resolution,R= 16.3%,R free= 20.7%), as well as of Ca 2+-free apo s2a (1.8 Åmore » resolution,R= 20.7%,R free= 27.2%) and two new forms of N-terminally truncated apo s2 (1.4 Å resolution,R= 16.9%,R free= 21.2%; 1.6 Å resolution,R= 16.2%,R free= 19.2%), are reported. The structurally similar PKD-like domains resemble the V-set Ig fold. In addition to a conserved β-bulge, the PKD-like domains feature a second bulge that also changes the allegiance of the subsequent β-strand. This β-bulge and the genesis of a Ca 2+pocket in the archaeal PKD-like domain suggest a close kinship between bacterial and archaeal PKD-like domains. Different surface properties and indications of different dynamics suggest unique roles for the PKD-like domains in ColG and in ColH. Surface aromatic residues found on ColH s2a-s2b, but not on ColG s2, may provide the weak interaction in the biphasic collagen-binding mode previously found in s2b-s3.B-factor analyses suggest that in the presence of Ca 2+the midsection of s2 becomes more flexible but the midsections of s2a and s2b stay rigid. The different surface properties and dynamics of the domains suggest that the PKD-like domains of M9B bacterial collagenase can be grouped into either a ColG subset or a ColH subset. The conserved properties of PKD-like domains in ColG and in ColH include Ca 2+binding. Conserved residues not only interact with Ca 2+, but also position the Ca 2+-interacting water molecule. Ca 2+aligns the N-terminal linker approximately parallel to the major axis of the domain. Ca 2+binding also increases stability against heat and guanidine hydrochloride, and may improve the longevity in the extracellular matrix. The results of this study will further assist in developing collagen-targeting vehicles for various signal molecules.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wollaber, Allan Benton; Park, HyeongKae; Lowrie, Robert Byron

Moment-based acceleration via the development of “high-order, low-order” (HO-LO) algorithms has provided substantial accuracy and efficiency enhancements for solutions of the nonlinear, thermal radiative transfer equations by CCS-2 and T-3 staff members. Accuracy enhancements over traditional, linearized methods are obtained by solving a nonlinear, timeimplicit HO-LO system via a Jacobian-free Newton Krylov procedure. This also prevents the appearance of non-physical maximum principle violations (“temperature spikes”) associated with linearization. Efficiency enhancements are obtained in part by removing “effective scattering” from the linearized system. In this highlight, we summarize recent work in which we formally extended the HO-LO radiation algorithm to includemore » operator-split radiation-hydrodynamics.« less

The Genres of "Shouted Speech" in Cheke Holo.

ERIC Educational Resources Information Center

Boswell, Freddy

Speech genres in Cheke Holo (CH) have not been studied extensively. Speech genres related to shouted speech in CH deserves more study because it is commonly used. Culturally speaking, shouted speech is a natural expression of the importance and centrality of CH community and surrounding authority structures, and has a very strong hortatory…

Interactive Molecular Graphics for Augmented Reality Using HoloLens.

PubMed

Müller, Christoph; Krone, Michael; Huber, Markus; Biener, Verena; Herr, Dominik; Koch, Steffen; Reina, Guido; Weiskopf, Daniel; Ertl, Thomas

2018-06-13

Immersive technologies like stereo rendering, virtual reality, or augmented reality (AR) are often used in the field of molecular visualisation. Modern, comparably lightweight and affordable AR headsets like Microsoft's HoloLens open up new possibilities for immersive analytics in molecular visualisation. A crucial factor for a comprehensive analysis of molecular data in AR is the rendering speed. HoloLens, however, has limited hardware capabilities due to requirements like battery life, fanless cooling and weight. Consequently, insights from best practises for powerful desktop hardware may not be transferable. Therefore, we evaluate the capabilities of the HoloLens hardware for modern, GPU-enabled, high-quality rendering methods for the space-filling model commonly used in molecular visualisation. We also assess the scalability for large molecular data sets. Based on the results, we discuss ideas and possibilities for immersive molecular analytics. Besides more obvious benefits like the stereoscopic rendering offered by the device, this specifically includes natural user interfaces that use physical navigation instead of the traditional virtual one. Furthermore, we consider different scenarios for such an immersive system, ranging from educational use to collaborative scenarios.

HoloGondel: in situ cloud observations on a cable car in the Swiss Alps using a holographic imager

NASA Astrophysics Data System (ADS)

Beck, Alexander; Henneberger, Jan; Schöpfer, Sarah; Fugal, Jacob; Lohmann, Ulrike

2017-02-01

In situ observations of cloud properties in complex alpine terrain where research aircraft cannot sample are commonly conducted at mountain-top research stations and limited to single-point measurements. The HoloGondel platform overcomes this limitation by using a cable car to obtain vertical profiles of the microphysical and meteorological cloud parameters. The main component of the HoloGondel platform is the HOLographic Imager for Microscopic Objects (HOLIMO 3G), which uses digital in-line holography to image cloud particles. Based on two-dimensional images the microphysical cloud parameters for the size range from small cloud particles to large precipitation particles are obtained for the liquid and ice phase. The low traveling velocity of a cable car on the order of 10 m s-1 allows measurements with high spatial resolution; however, at the same time it leads to an unstable air speed towards the HoloGondel platform. Holographic cloud imagers, which have a sample volume that is independent of the air speed, are therefore well suited for measurements on a cable car. Example measurements of the vertical profiles observed in a liquid cloud and a mixed-phase cloud at the Eggishorn in the Swiss Alps in the winters 2015 and 2016 are presented. The HoloGondel platform reliably observes cloud droplets larger than 6.5 µm, partitions between cloud droplets and ice crystals for a size larger than 25 µm and obtains a statistically significantly size distribution for every 5 m in vertical ascent.

Falsely Elevated Serum Vitamin B12 Levels Were Associated with the Severity and Prognosis of Chronic Viral Liver Disease

PubMed Central

Sugihara, Takaaki; Koda, Masahiko; Okamoto, Toshiaki; Miyoshi, Kenichi; Matono, Tomomitsu; Oyama, Kenji; Hosho, Keiko; Okano, Jun-ichi; Isomoto, Hajime; Murawaki, Yoshikazu

2017-01-01

Background Vitamin B12 is stored primarily in the liver, and highly elevated serum vitamin B12 levels occur in acute hepatitis and severe alcoholic liver disease. We evaluated the relationship between vitamin B12 levels and liver disease severity and long term prognosis in patients with chronic viral hepatitis and cirrhosis. Methods We enrolled 90 patients (57 men, 33 women) with chronic viral hepatitis and cirrhosis who admitted to our hospital as a prospective cohort study. Overall, 37 patients had chronic hepatitis and 53 had cirrhosis (Child-Pugh A 33, B 13, and C 7); 57 patients had primary liver cancer. Serum vitamin B12 concentration and holotranscobalamin (holoTC) II (active form of vitamin B12) were determined and followed prospectively for at least 5 years. Results Mean total serum vitamin B12 concentration was significantly higher in Child-Pugh C (1308 ± 599 pg/mL) compared to those with chronic hepatitis (655 ± 551 pg/mL), Child-Pugh A (784 ± 559 pg/mL), and Child-Pugh B (660 ± 464 pg/mL) (P = 0.036) Presence of primary liver cancer also influenced serum vitamin B12 levels [657 (167–2956) vs. 432 (189–2956); P = 0.015]. Patients were divided into quartiles by vitamin B12 level. Patients without primary liver cancer in quartile 4 (≥ 880 pg/mL) demonstrated significantly poorer prognosis than those in quartiles 1–3 (< 880 pg/mL) (P = 0.023). The percentage of holohaptocorrin (holoHC) [(total vitamin B12 – holoTC II) × 100] was significantly higher in Child-Pugh B and C 86 (80–87)% than chronic hepatitis and Child-Pugh A 77 (31–89)% (P = 0.006) Multivariate analysis indicated serum vitamin B12 levels (HR = 1.001, P = 0.029) as a prognostic factor. Conclusion Falsely elevated serum vitamin B12 levels mainly composed of increased holoHC were associated with severity (Child-Pugh C and primary liver cancer) and prognosis in chronic viral liver disease. PMID:28331419

Behavior of Solvent-Exposed Hydrophobic Groove in the Anti-Apoptotic Bcl-XL Protein: Clues for Its Ability to Bind Diverse BH3 Ligands from MD Simulations

PubMed Central

Sankararamakrishnan, Ramasubbu

2013-01-01

Bcl-XL is a member of Bcl-2 family of proteins involved in the regulation of intrinsic pathway of apoptosis. Its overexpression in many human cancers makes it an important target for anti-cancer drugs. Bcl-XL interacts with the BH3 domain of several pro-apoptotic Bcl-2 partners. This helical bundle protein has a pronounced hydrophobic groove which acts as a binding region for the BH3 domains. Eight independent molecular dynamics simulations of the apo/holo forms of Bcl-XL were carried out to investigate the behavior of solvent-exposed hydrophobic groove. The simulations used either a twin-range cut-off or particle mesh Ewald (PME) scheme to treat long-range interactions. Destabilization of the BH3 domain-containing helix H2 was observed in all four twin-range cut-off simulations. Most of the other major helices remained stable. The unwinding of H2 can be related to the ability of Bcl-XL to bind diverse BH3 ligands. The loss of helical character can also be linked to the formation of homo- or hetero-dimers in Bcl-2 proteins. Several experimental studies have suggested that exposure of BH3 domain is a crucial event before they form dimers. Thus unwinding of H2 seems to be functionally very important. The four PME simulations, however, revealed a stable helix H2. It is possible that the H2 unfolding might occur in PME simulations at longer time scales. Hydrophobic residues in the hydrophobic groove are involved in stable interactions among themselves. The solvent accessible surface areas of bulky hydrophobic residues in the groove are significantly buried by the loop LB connecting the helix H2 and subsequent helix. These observations help to understand how the hydrophobic patch in Bcl-XL remains stable in the solvent-exposed state. We suggest that both the destabilization of helix H2 and the conformational heterogeneity of loop LB are important factors for binding of diverse ligands in the hydrophobic groove of Bcl-XL. PMID:23468841

Isomers in the excited state of electron-transferring flavoprotein from Megasphaera elsdenii: spectral resolution from the time-resolved fluorescence spectra.

PubMed

Sato, Kyosuke; Nishina, Yasuzo; Shiga, Kiyoshi; Tanaka, Fumio

2008-02-27

Electron-transferring flavoprotein (Holo-ETF) from Megasphaera elsdenii contains two FAD's, one of which easily dissociates to form Iso-ETF (contains one FAD). Time-resolved fluorescence of FAD in Iso-ETF, and Holo-ETF were measured at 5 degrees C and 25 degrees C. Wavelength-dependent fluorescence decays of the both ETF at 5 degrees C and 25 degrees C were analyzed to resolve them into two independent spectra. It was found that Iso-ETF displayed two spectra with lifetime of 0.605 ns (emission peak, 508 nm) and with lifetime of 1.70 ns (emission peak, 540 nm) at 5 degrees C, and with lifetime of 0.693 ns (emission peak, 508 nm) and with lifetime of 2.75 ns (emission peak, 540 nm) at 25 degrees C. Holo-ETF displayed two spectra with lifetime of 0.739 ns (emission peak, 508 nm) and with lifetime of 2.06 ns (emission peak, 545 nm) at 5 degrees C, and with lifetime of 0.711 ns (emission peak, 527 nm) and with lifetime of 3.08 ns (emission peak, 540 nm) at 25 degrees C. Thus fluorescence lifetimes of every spectrum increased upon elevating temperature. Emission peaks Iso-ETF did not change much upon elevating temperature. Activation enthalpy changes, activation entropy changes and activation Gibbs energy changes of quenching rates all displayed negative. Two emission species in the both ETF may be hydrogen-bonding isomers, because isoalloxazine ring of FAD contains four hydrogen acceptors and one donor.

AutoDockFR: Advances in Protein-Ligand Docking with Explicitly Specified Binding Site Flexibility

PubMed Central

Ravindranath, Pradeep Anand; Forli, Stefano; Goodsell, David S.; Olson, Arthur J.; Sanner, Michel F.

2015-01-01

Automated docking of drug-like molecules into receptors is an essential tool in structure-based drug design. While modeling receptor flexibility is important for correctly predicting ligand binding, it still remains challenging. This work focuses on an approach in which receptor flexibility is modeled by explicitly specifying a set of receptor side-chains a-priori. The challenges of this approach include the: 1) exponential growth of the search space, demanding more efficient search methods; and 2) increased number of false positives, calling for scoring functions tailored for flexible receptor docking. We present AutoDockFR–AutoDock for Flexible Receptors (ADFR), a new docking engine based on the AutoDock4 scoring function, which addresses the aforementioned challenges with a new Genetic Algorithm (GA) and customized scoring function. We validate ADFR using the Astex Diverse Set, demonstrating an increase in efficiency and reliability of its GA over the one implemented in AutoDock4. We demonstrate greatly increased success rates when cross-docking ligands into apo receptors that require side-chain conformational changes for ligand binding. These cross-docking experiments are based on two datasets: 1) SEQ17 –a receptor diversity set containing 17 pairs of apo-holo structures; and 2) CDK2 –a ligand diversity set composed of one CDK2 apo structure and 52 known bound inhibitors. We show that, when cross-docking ligands into the apo conformation of the receptors with up to 14 flexible side-chains, ADFR reports more correctly cross-docked ligands than AutoDock Vina on both datasets with solutions found for 70.6% vs. 35.3% systems on SEQ17, and 76.9% vs. 61.5% on CDK2. ADFR also outperforms AutoDock Vina in number of top ranking solutions on both datasets. Furthermore, we show that correctly docked CDK2 complexes re-create on average 79.8% of all pairwise atomic interactions between the ligand and moving receptor atoms in the holo complexes. Finally, we show that down-weighting the receptor internal energy improves the ranking of correctly docked poses and that runtime for AutoDockFR scales linearly when side-chain flexibility is added. PMID:26629955

Ubiquitous distribution of fluorescent protein in muscles of four species and two subspecies of eel (genus Anguilla).

PubMed

Funahashi, Aki; Itakura, Takao; Hassanin Abeer, A I; Komatsu, Masaharu; Hayashi, Seiichi; Kaminishi, Yoshio

2017-03-01

In this study, the localization of fluorescent protein (FP) was characterized in the muscles of four species and two subspecies of eels Anguilla anguilla, A. australis, A. bicolor bicolor (b.), A. bicolor pacifica (p.) and A. mossambica in addition to the previously reported A. japonica. The open reading frame of each eel FP was 417 bp encoding 139 amino acid residues. The deduced amino acid sequences among the four species and two subspecies exhibited 91.4-100% identity, and belonged to the fatty-acid-binding protein (FABP) family. The gene structure of eel FPs in A. japonica, A. anguilla, A. australis, A. bicolor b., A. bicolor p. and A. mossambica have four exons and three introns, and were common to that of FABP family. The apo eel FPs expressed by Escherichia coli with recombinant eel FP genes were analysed for the fluorescent properties in the presence of bilirubin. The excitation and emission spectra of holo eel FPs had the maximum wavelengths of 490-496 and 527-530 nm, respectively. The holo eel FPs indicated that the fluorescent intensities were stronger in A. japonica and A. bicolor than in A. mossambica, A. australis and A. anguilla. The comparison of amino acid sequences revealed two common substitutions in A. mossambica, A. australis and A. anguilla with weak fluorescent intensity.

Structural stability of E. coli transketolase to temperature and pH denaturation.

PubMed

Jahromi, Raha R F; Morris, Phattaraporn; Martinez-Torres, Ruben J; Dalby, Paul A

2011-09-10

We have previously shown that the denaturation of TK with urea follows a non-aggregating though irreversible denaturation pathway in which the cofactor binding appears to become altered but without dissociating, then followed at higher urea by partial denaturation of the homodimer prior to any further unfolding or dissociation of the two monomers. Urea is not typically present during biocatalysis, whereas access to TK enzymes that retain activity at increased temperature and extreme pH would be useful for operation under conditions that increase substrate and product stability or solubility. To provide further insight into the underlying causes of its deactivation in process conditions, we have characterised the effects of temperature and pH on the structure, stability, aggregation and activity of Escherichia coli transketolase. The activity of TK was initially found to progressively improve after pre-incubation at increasing temperatures. Loss of activity at higher temperature and low pH resulted primarily from protein denaturation and subsequent irreversible aggregation. By contrast, high pH resulted in the formation of a native-like state that was only partially inactive. The apo-TK enzyme structure content also increased at pH 9 to converge on that of the holo-TK. While cofactor dissociation was previously proposed for high pH deactivation, the observed structural changes in apo-TK but not holo-TK indicate a more complex mechanism. Copyright © 2011 Elsevier B.V. All rights reserved.

Understanding the EF-hand closing pathway using non-biased interatomic potentials.

PubMed

Dupuis, L; Mousseau, Normand

2012-01-21

The EF-hand superfamily of proteins is characterized by the presence of calcium binding helix-loop-helix structures. Many of these proteins undergo considerable motion responsible for a wide range of properties upon binding but the exact mechanism at the root of this motion is not fully understood. Here, we use an unbiased accelerated multiscale simulation scheme, coupled with two force fields - CHARMM-EEF1 and the extended OPEP - to explore in details the closing pathway, from the unbound holo state to the closed apo state, of two EF-hand proteins, the Calmodulin and Troponin C N-terminal nodules. Based on a number of closing simulations for these two sequences, we show that the EF-hand β-scaffold, identified as crucial by Grabarek for the EF-hand opening driven by calcium binding, is also important in closing the EF-hand. We also show the crucial importance of the phenylalanine situated at the end of first EF-hand helix, and identify an intermediate state modulating its behavior, providing a detailed picture of the closing mechanism for these two representatives of EF-hand proteins. © 2012 American Institute of Physics

Dissociation of Heme–Globin Complexes by Blackbody Infrared Radiative Dissociation: Molecular Specificity in the Gas Phase?

PubMed Central

Gross, Deborah S.; Zhao, Yuexing; Williams, Evan R.

2005-01-01

The temperature dependence of the unimolecular kinetics for dissociation of the heme group from holo-myoglobin (Mb) and holo-hemoglobin α-chain (Hb-α) was investigated with blackbody infrared radiative dissociation (BIRD). The rate constant for dissociation of the 9 + charge state of Mb formed by electrospray ionization from a “pseudo-native” solution is 60% lower than that of Hb-α at each of the temperatures investigated. In solutions of pH 5.5–8.0, the thermal dissociation rate for Mb is also lower than that of HB-α (Hargrove, M. S. et al. J. Biol. Chem. 1994, 269, 4207–4214). Thus, Mb is thermally more stable with respect to heme loss than Hb-α both in the gas phase and in solution. The Arrhenius activation parameters for both dissociation processes are indistinguishable within the current experimental error (activation energy 0.9 eV and pre-exponential factor of 108–10 s−1). The 9+ to 12+ charge states of Mb have similar Arrhenius parameters when these ions are formed from pseudo-native solutions. In contrast, the activation energies and pre-exponential factors decrease from 0.8 to 0.3 eV and 107 to 102 s−1, respectively, for the 9 + to 12 + charge states formed from acidified solutions in which at least 50% of the secondary structure is lost. These results demonstrate that gas-phase Mb ions retain clear memory of the composition of the solution from which they are formed and that these differences can be probed by BIRD. PMID:16479269

Dissociation of heme-globin complexes by blackbody infrared radiative dissociation: molecular specificity in the gas phase?

PubMed

Gross, D S; Zhao, Y; Williams, E R

1997-05-01

The temperature dependence of the unimolecular kinetics for dissociation of the heme group from holo-myoglobin (Mb) and holo-hemoglobin alpha-chain (Hb-alpha) was investigated with blackbody infrared radiative dissociation (BIRD). The rate constant for dissociation of the 9 + charge state of Mb formed by electrospray ionization from a "pseudo-native" solution is 60% lower than that of Hb-alpha at each of the temperatures investigated. In solutions of pH 5.5-8.0, the thermal dissociation rate for Mb is also lower than that of HB-alpha (Hargrove, M. S. et al. J. Biol. Chem.1994, 269, 4207-4214). Thus, Mb is thermally more stable with respect to heme loss than Hb-alpha both in the gas phase and in solution. The Arrhenius activation parameters for both dissociation processes are indistinguishable within the current experimental error (activation energy 0.9 eV and pre-exponential factor of 10(8-10) s(-1)). The 9+ to 12+ charge states of Mb have similar Arrhenius parameters when these ions are formed from pseudo-native solutions. In contrast, the activation energies and pre-exponential factors decrease from 0.8 to 0.3 eV and 10(7) to 10(2) s(-1), respectively, for the 9 + to 12 + charge states formed from acidified solutions in which at least 50% of the secondary structure is lost. These results demonstrate that gas-phase Mb ions retain clear memory of the composition of the solution from which they are formed and that these differences can be probed by BIRD.

The N Termini of a-Subunit Isoforms Are Involved in Signaling between Vacuolar H+-ATPase (V-ATPase) and Cytohesin-2*

PubMed Central

Hosokawa, Hiroyuki; Dip, Phat Vinh; Merkulova, Maria; Bakulina, Anastasia; Zhuang, Zhenjie; Khatri, Ashok; Jian, Xiaoying; Keating, Shawn M.; Bueler, Stephanie A.; Rubinstein, John L.; Randazzo, Paul A.; Ausiello, Dennis A.; Grüber, Gerhard; Marshansky, Vladimir

2013-01-01

Previously, we reported an acidification-dependent interaction of the endosomal vacuolar H+-ATPase (V-ATPase) with cytohesin-2, a GDP/GTP exchange factor (GEF), suggesting that it functions as a pH-sensing receptor. Here, we have studied the molecular mechanism of signaling between the V-ATPase, cytohesin-2, and Arf GTP-binding proteins. We found that part of the N-terminal cytosolic tail of the V-ATPase a2-subunit (a2N), corresponding to its first 17 amino acids (a2N(1–17)), potently modulates the enzymatic GDP/GTP exchange activity of cytohesin-2. Moreover, this peptide strongly inhibits GEF activity via direct interaction with the Sec7 domain of cytohesin-2. The structure of a2N(1–17) and its amino acids Phe5, Met10, and Gln14 involved in interaction with Sec7 domain were determined by NMR spectroscopy analysis. In silico docking experiments revealed that part of the V-ATPase formed by its a2N(1–17) epitope competes with the switch 2 region of Arf1 and Arf6 for binding to the Sec7 domain of cytohesin-2. The amino acid sequence alignment and GEF activity studies also uncovered the conserved character of signaling between all four (a1–a4) a-subunit isoforms of mammalian V-ATPase and cytohesin-2. Moreover, the conserved character of this phenomenon was also confirmed in experiments showing binding of mammalian cytohesin-2 to the intact yeast V-ATPase holo-complex. Thus, here we have uncovered an evolutionarily conserved function of the V-ATPase as a novel cytohesin-signaling receptor. PMID:23288846

Characterization of the Deoxynucleotide Triphosphate Triphosphohydrolase (dNTPase) Activity of the EF1143 Protein from Enterococcus faecalis and Crystal Structure of the Activator-Substrate Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vorontsov, Ivan I.; Minasov, George; Kiryukhina, Olga

2012-06-19

The EF1143 protein from Enterococcus faecalis is a distant homolog of deoxynucleotide triphosphate triphosphohydrolases (dNTPases) from Escherichia coli and Thermus thermophilus. These dNTPases are important components in the regulation of the dNTP pool in bacteria. Biochemical assays of the EF1143 dNTPase activity demonstrated nonspecific hydrolysis of all canonical dNTPs in the presence of Mn{sup 2+}. In contrast, with Mg{sup 2+} hydrolysis required the presence of dGTP as an effector, activating the degradation of dATP and dCTP with dGTP also being consumed in the reaction with dATP. The crystal structure of EF1143 and dynamic light scattering measurements in solution revealed amore » tetrameric oligomer as the most probable biologically active unit. The tetramer contains four dGTP specific allosteric regulatory sites and four active sites. Examination of the active site with the dATP substrate suggests an in-line nucleophilic attack on the {alpha}-phosphate center as a possible mechanism of the hydrolysis and two highly conserved residues, His-129 and Glu-122, as an acid-base catalytic dyad. Structural differences between EF1143 apo and holo forms revealed mobility of the {alpha}3 helix that can regulate the size of the active site binding pocket and could be stabilized in the open conformation upon formation of the tetramer and dGTP effector binding.« less

Interactions of Escherichia coli σ70 within the transcription elongation complex

PubMed Central

Daube, Shirley S.; von Hippel, Peter H.

1999-01-01

A functional transcription elongation complex can be formed without passing through a promoter by adding a complementary RNA primer and core Escherichia coli RNA polymerase in trans to an RNA-primed synthetic bubble-duplex DNA framework. This framework consists of a double-stranded DNA sequence with an internal noncomplementary DNA “bubble” containing a hybridized RNA primer. On addition of core polymerase and the requisite NTPs, the RNA primer is extended in a process that manifests most of the properties of in vitro transcription elongation. This synthetic elongation complex can also be assembled by using holo rather than core RNA polymerase, and in this study we examine the interactions and fate of the σ70 specificity subunit of the holopolymerase in the assembly process. We show that the addition of holopolymerase to the bubble-duplex construct triggers the dissociation of the sigma factor from some complexes, whereas in others the RNA oligomer is released into solution instead. These results are consistent with an allosteric competition between σ70 and the nascent RNA strand within the elongation complex and suggest that both cannot be bound to the core polymerase simultaneously. However, the dissociation of σ70 from the complex can also be stimulated by binding of the holopolymerase to the DNA bubble duplex in the absence of a hybridized RNA primer, suggesting that the binding of the core polymerase to the bubble-duplex construct also triggers a conformational change that additionally weakens the sigma–core interaction. PMID:10411885

Computational approaches for deciphering the equilibrium and kinetic properties of iron transport proteins.

PubMed

Abdizadeh, H; Atilgan, A R; Atilgan, C; Dedeoglu, B

2017-11-15

With the advances in three-dimensional structure determination techniques, high quality structures of the iron transport proteins transferrin and the bacterial ferric binding protein (FbpA) have been deposited in the past decade. These are proteins of relatively large size, and developments in hardware and software have only recently made it possible to study their dynamics using standard computational resources. We review computational techniques towards understanding the equilibrium and kinetic properties of iron transport proteins under different environmental conditions. At the level of detail that requires quantum chemical treatments, the octahedral geometry around iron has been scrutinized and it has been established that the iron coordinating tyrosines are in an unusual deprotonated state. At the atomistic level, both the N-lobe and the full bilobal structure of transferrin have been studied under varying conditions of pH, ionic strength and binding of other metal ions by molecular dynamics (MD) simulations. These studies have allowed questions to be answered, among others, on the function of second shell residues in iron release, the role of synergistic anions in preparing the active site for iron binding, and the differences between the kinetics of the N- and the C-lobe. MD simulations on FbpA have led to the detailed observation of the binding kinetics of phosphate to the apo form, and to the conformational preferences of the holo form under conditions mimicking the environmental niches provided by the periplasmic space. To study the dynamics of these proteins with their receptors, one must resort to coarse-grained methodologies, since these systems are prohibitively large for atomistic simulations. A study of the complex of human transferrin (hTf) with its pathogenic receptor by such methods has revealed a potential mechanistic explanation for the defense mechanism that arises in evolutionary warfare. Meanwhile, the motions in the transferrin receptor bound hTf have been shown to disfavor apo hTf dissociation, explaining why the two proteins remain in complex during the recycling process from the endosome to the cell surface. Open problems and possible technological applications related to metal ion binding-release in iron transport proteins that may be handled by hybrid use of quantum mechanical, MD and coarse-grained approaches are discussed.

Combined indicator of vitamin B 12 status: modification for missing biomarkers and folate status and recommendations for revised cut-points

USDA-ARS?s Scientific Manuscript database

Background: A novel approach to determine vitamin B 12 status is to combine four blood markers: total B 12 (B 12 ), holotranscobalamin (holoTC), methylmalonic acid (MMA) and total homocysteine (tHcy). This combined indicator of B 12 status is expressed as cB 12 = log 10 [(holoTC · B 12 )/ (MMA · Hcy...

HoloLens

NASA Image and Video Library

2016-02-20

ISS046e043637 (02/20/2016) --- NASA astronaut Scott Kelly tweeted out this image to his followers Feb 20, 2016 with the tag: "This #Saturday morning checked out the @Microsoft #HoloLens aboard @Space_Station! Wow! #YearInSpace ". The device is part of NASA’s project Sidekick which is exploring the use of augmented reality to reduce crew training requirements and increase the efficiency at which astronauts can work in space.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cody, Vivian, E-mail: cody@hwi.buffalo.edu; University of Buffalo, Buffalo, NY 14260; Pace, Jim

The structures of mouse DHFR holo enzyme and a ternary complex with NADPH and a potent inhibitor are described. It has been shown that 2, 4-diamino-6-arylmethylpteridines and 2, 4-diamino-5-arylmethylpyrimidines containing an O-carboxylalkyloxy group in the aryl moiety are potent and selective inhibitors of the dihydrofolate reductase (DHFR) from opportunistic pathogens such as Pneumocystis carinii, the causative agent of Pneumocystis pneumonia in HIV/AIDS patients. In order to understand the structure–activity profile observed for a series of substituted dibenz[b, f]azepine antifolates, the crystal structures of mouse DHFR (mDHFR; a mammalian homologue) holo and ternary complexes with NADPH and the inhibitor 2, 4-diamino-6-(2′-hydroxydibenz[b,more » f]azepin-5-yl)methylpteridine were determined to 1.9 and 1.4 Å resolution, respectively. Structural data for the ternary complex with the potent O-(3-carboxypropyl) inhibitor PT684 revealed no electron density for the O-carboxylalkyloxy side chain. The side chain was either cleaved or completely disordered. The electron density fitted the less potent hydroxyl compound PT684a. Additionally, cocrystallization of mDHFR with NADPH and the less potent 2′-(4-carboxybenzyl) inhibitor PT682 showed no electron density for the inhibitor and resulted in the first report of a holoenzyme complex despite several attempts at crystallization of a ternary complex. Modeling data of PT682 in the active site of mDHFR and P. carinii DHFR (pcDHFR) indicate that binding would require ligand-induced conformational changes to the enzyme for the inhibitor to fit into the active site or that the inhibitor side chain would have to adopt an alternative binding mode to that observed for other carboxyalkyloxy inhibitors. These data also show that the mDHFR complexes have a decreased active-site volume as reflected in the relative shift of helix C (residues 59–64) by 0.6 Å compared with pcDHFR ternary complexes. These data are consistent with the greater inhibitory potency against pcDHFR.« less

Physics holo.lab learning experience: using smartglasses for augmented reality labwork to foster the concepts of heat conduction

NASA Astrophysics Data System (ADS)

Strzys, M. P.; Kapp, S.; Thees, M.; Klein, P.; Lukowicz, P.; Knierim, P.; Schmidt, A.; Kuhn, J.

2018-05-01

Fundamental concepts of thermodynamics rely on abstract physical quantities such as energy, heat and entropy, which play an important role in the process of interpreting thermal phenomena and statistical mechanics. However, these quantities are not covered by human visual perception, and since heat sensation is purely qualitative and easy to deceive, an intuitive understanding often is lacking. Today immersive technologies like head-mounted displays of the newest generation, especially HoloLens, allow for high-quality augmented reality learning experiences, which can overcome this gap in human perception by presenting different representations of otherwise invisible quantities directly in the field of view of the user on the experimental apparatus, which simultaneously avoids a split-attention effect. In a mixed reality (MR) scenario as presented in this paper—which we call a holo.lab—human perception can be extended to the thermal regime by presenting false-color representations of the temperature of objects as a virtual augmentation directly on the real object itself in real-time. Direct feedback to experimental actions of the users in the form of different representations allows for immediate comparison to theoretical principles and predictions and therefore is supposed to intensify the theory–experiment interactions and to increase students’ conceptual understanding. We tested this technology for an experiment on thermal conduction of metals in the framework of undergraduate laboratories. A pilot study with treatment and control groups (N = 59) showed a small positive effect of MR on students’ performance measured with a standardized concept test for thermodynamics, pointing to an improvement of the understanding of the underlying physical concepts. These findings indicate that complex experiments could benefit even more from augmentation. This motivates us to enrich further experiments with MR.

Regulation of the ATPase activity of ABCE1 from Pyrococcus abyssi by Fe-S cluster status and Mg²⁺: implication for ribosomal function.

PubMed

Sims, Lynn M; Igarashi, Robert Y

2012-08-15

Ribosomal function is dependent on multiple proteins. The ABCE1 ATPase, a unique ABC superfamily member that bears two Fe₄S₄ clusters, is crucial for ribosomal biogenesis and recycling. Here, the ATPase activity of the Pyrococcus abyssi ABCE1 (PabABCE1) was studied using both apo- (without reconstituted Fe-S clusters) and holo- (with full complement of Fe-S clusters reconstituted post-purification) forms, and is shown to be jointly regulated by the status of Fe-S clusters and Mg²⁺. Typically ATPases require Mg²⁺, as is true for PabABCE1, but Mg²⁺ also acts as a negative allosteric effector that modulates ATP affinity of PabABCE1. Physiological [Mg²⁺] inhibits the PabABCE1 ATPase (K(i) of ∼1 μM) for both apo- and holo-PabABCE1. Comparative kinetic analysis of Mg²⁺ inhibition shows differences in degree of allosteric regulation between the apo- and holo-PabABCE1 where the apparent ATP K(m) of apo-PabABCE1 increases >30-fold from ∼30 μM to over 1 mM with M²⁺. This effect would significantly convert the ATPase activity of PabABCE1 from being independent of cellular energy charge (φ) to being dependent on φ with cellular [Mg²⁺]. These findings uncover intricate overlapping effects by both [Mg²⁺] and the status of Fe-S clusters that regulate ABCE1's ATPase activity with implications to ribosomal function. Copyright © 2012 Elsevier Inc. All rights reserved.

Homotropic Cooperativity from the Activation Pathway of the Allosteric Ligand-Responsive Regulatory Protein TRAP†

PubMed Central

Kleckner, Ian R.; McElroy, Craig A.; Kuzmic, Petr; Gollnick, Paul; Foster, Mark P.

2014-01-01

The trp RNA-binding Attenuation Protein (TRAP) assembles into an 11-fold symmetric ring that regulates transcription and translation of trp-mRNA in bacilli via heterotropic allosteric activation by the amino acid tryptophan (Trp). Whereas nuclear magnetic resonance studies have revealed that Trp-induced activation coincides with both μs-ms rigidification and local structural changes in TRAP, the pathway of binding of the 11 Trp ligands to the TRAP ring remains unclear. Moreover, because each of eleven bound Trp molecules is completely surrounded by protein, its release requires flexibility of Trp-bound (holo) TRAP. Here, we used stopped-flow fluorescence to study the kinetics of Trp binding by Bacillus stearothermophilus TRAP over a range of temperatures and we observed well-separated kinetic steps. These data were analyzed using non-linear least-squares fitting of several two- and three-step models. We found that a model with two binding steps best describes the data, although the structural equivalence of the binding sites in TRAP implies a fundamental change in the time-dependent structure of the TRAP rings upon Trp binding. Application of the two binding step model reveals that Trp binding is much slower than the diffusion limit, suggesting a gating mechanism that depends on the dynamics of apo TRAP. These data also reveal that Trp dissociation from the second binding mode is much slower than after the first Trp binding mode, revealing insight into the mechanism for positive homotropic allostery, or cooperativity. Temperature dependent analyses reveal that both binding modes imbue increases in bondedness and order toward a more compressed active state. These results provide insight into mechanisms of cooperative TRAP activation, and underscore the importance of protein dynamics for ligand binding, ligand release, protein activation, and allostery. PMID:24224873

Molecular landscape of the interaction between the urease accessory proteins UreE and UreG.

PubMed

Merloni, Anna; Dobrovolska, Olena; Zambelli, Barbara; Agostini, Federico; Bazzani, Micaela; Musiani, Francesco; Ciurli, Stefano

2014-09-01

Urease, the most efficient enzyme so far discovered, depends on the presence of nickel ions in the catalytic site for its activity. The transformation of inactive apo-urease into active holo-urease requires the insertion of two Ni(II) ions in the substrate binding site, a process that involves the interaction of four accessory proteins named UreD, UreF, UreG and UreE. This study, carried out using calorimetric and NMR-based structural analysis, is focused on the interaction between UreE and UreG from Sporosarcina pasteurii, a highly ureolytic bacterium. Isothermal calorimetric protein-protein titrations revealed the occurrence of a binding event between SpUreE and SpUreG, entailing two independent steps with positive cooperativity (Kd1=42±9μM; Kd2=1.7±0.3μM). This was interpreted as indicating the formation of the (UreE)2(UreG)2 hetero-oligomer upon binding of two UreG monomers onto the pre-formed UreE dimer. The molecular details of this interaction were elucidated using high-resolution NMR spectroscopy. The occurrence of SpUreE chemical shift perturbations upon addition of SpUreG was investigated and analyzed to establish the protein-protein interaction site. The latter appears to involve the Ni(II) binding site as well as mobile portions on the C-terminal and the N-terminal domains. Docking calculations based on the information obtained from NMR provided a structural basis for the protein-protein contact site. The high sequence and structural similarity within these protein classes suggests a generality of the interaction mode among homologous proteins. The implications of these results on the molecular details of the urease activation process are considered and analyzed. Copyright © 2014 Elsevier B.V. All rights reserved.

Hologram stability evaluation for Microsoft HoloLens

NASA Astrophysics Data System (ADS)

Vassallo, Reid; Rankin, Adam; Chen, Elvis C. S.; Peters, Terry M.

2017-03-01

Augmented reality (AR) has an increasing presence in the world of image-guided interventions which is amplified by the availability of consumer-grade head-mounted display (HMD) technology. The Microsoft® HoloLensTM optical passthrough device is at the forefront of consumer technology, as it is the first un-tethered head mounted computer (HMC). It shows promise of effectiveness in guiding clinical interventions, however its accuracy and stability must still be evaluated for the clinical environment. We have developed an evaluative protocol for the HoloLensTM using an optical measurement device to digitize the perceived pose of the rendered hologram. This evaluates the ability of the HoloLensTM to maintain the hologram in its intended pose. The stability is measured when actions are performed that may cause a shift in the holograms' pose due to errors in its simultaneous localization and mapping. An emphasis is placed on actions that are more likely to be performed in a clinical setting. This will be used to determine the most applicable use cases for this technology in the future and how to minimize errors when in use. Our results show promise of this device's potential for intraoperative clinical use. Further analysis must be performed to evaluate other potential sources of hologram disruption.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramelot, Theresa A.; Rossi, Paolo M.; Forouhar, Farhad

The solution nuclear magnetic resonance (NMR) structures and backbone (15)N dynamics of the specialized acyl carrier protein (ACP), RpAcpXL, from Rhodopseudomonas palustris, in both the apo form and holo form modified by covalent attachment of 4'-phosphopantetheine at S37, are virtually identical, monomeric, and correspond to the closed conformation. The structures have an extra α-helix compared to the archetypical ACP from Escherichia coli, which has four helices, resulting in a larger opening to the hydrophobic cavity. Chemical shift differences between apo- and holo-RpAcpXL indicated some differences in the hinge region between α2 and α3 and in the hydrophobic cavity environment, butmore » corresponding changes in nuclear Overhauser effect cross-peak patterns were not detected. In contrast to the NMR structures, apo-RpAcpXL was observed in an open conformation in crystals that diffracted to 2.0 Å resolution, which resulted from movement of α3. On the basis of the crystal structure, the predicted biological assembly is a homodimer. Although the possible biological significance of dimerization is unknown, there is potential that the resulting large shared hydrophobic cavity could accommodate the very long-chain fatty acid (28-30 carbons) that this specialized ACP is known to synthesize and transfer to lipid A. These structures are the first representatives of the AcpXL family and the first to indicate that dimerization may be important for the function of these specialized ACPs.« less

The Effect of Salts in Promoting Specific and Competitive Interactions between Zinc Finger Proteins and Metals

NASA Astrophysics Data System (ADS)

Li, Gongyu; Yuan, Siming; Zheng, Shihui; Chen, Yuting; Zheng, Zhen; Liu, Yangzhong; Huang, Guangming

2017-12-01

Specific protein-metal interactions (PMIs) fulfill essential functions in cells and organic bodies, and activation of these functions in vivo are mostly modulated by the complex environmental factors, including pH value, small biomolecules, and salts. Specifically, the role of salts in promoting specific PMIs and their competition among various metals has remained untapped mainly due to the difficulty to distinguish nonspecific PMIs from specific PMIs by classic spectroscopic techniques. Herein, we report Hofmeister salts differentially promote the specific PMIs by combining nanoelectrospray ionization mass spectrometry and spectroscopic techniques (fluorescence measurement and circular dichroism). Furthermore, to explore the influence of salts in competitive binding between metalloproteins and various metals, we designed a series of competitive experiments and applied to a well-defined model system, the competitive binding of zinc (II) and arsenic (III) to holo-promyelocytic leukemia protein (PML). These experiments not only provided new insights at the molecular scale as complementary to previous NMR and spectroscopic results, but also deduced the relative binding ability between zinc finger proteins and metals at the molecular scale, which avoids the mass spectrometric titration-based determination of binding constants that is frequently affected and often degraded by variable solution conditions including salt contents. [Figure not available: see fulltext.

Mixed Reality with HoloLens: Where Virtual Reality Meets Augmented Reality in the Operating Room.

PubMed

Tepper, Oren M; Rudy, Hayeem L; Lefkowitz, Aaron; Weimer, Katie A; Marks, Shelby M; Stern, Carrie S; Garfein, Evan S

2017-11-01

Virtual reality and augmented reality devices have recently been described in the surgical literature. The authors have previously explored various iterations of these devices, and although they show promise, it has become clear that virtual reality and/or augmented reality devices alone do not adequately meet the demands of surgeons. The solution may lie in a hybrid technology known as mixed reality, which merges many virtual reality and augmented realty features. Microsoft's HoloLens, the first commercially available mixed reality device, provides surgeons intraoperative hands-free access to complex data, the real environment, and bidirectional communication. This report describes the use of HoloLens in the operating room to improve decision-making and surgical workflow. The pace of mixed reality-related technological development will undoubtedly be rapid in the coming years, and plastic surgeons are ideally suited to both lead and benefit from this advance.

[Preliminary use of HoloLens glasses in surgery of liver cancer].

PubMed

Shi, Lei; Luo, Tao; Zhang, Li; Kang, Zhongcheng; Chen, Jie; Wu, Feiyue; Luo, Jia

2018-05-28

To establish the preoperative three dimensional (3D) model of liver cancer, and to precisely match the preoperative planning with the target organs during the operation.
 Methods: The 3D model reconstruction based on magnetic resonance data, which was combined with virtual reality technology via HoloLens glasses, was applied in the operation of liver cancer to achieve preoperative 3D modeling and surgical planning, and to directly match it with the operative target organs during operation.
 Results: The 3D model reconstruction of liver cancer based on magnetic resonance data was completed. The exact match with the target organ was performed during the operation via HoloLens glasses leaded by the 3D model.
 Conclusion: Magnetic resonance data can be used for the 3D model reconstruction to improve preoperative assessment and accurate match during the operation.

Label free imaging of cell-substrate contacts by holographic total internal reflection microscopy.

PubMed

Mandracchia, Biagio; Gennari, Oriella; Marchesano, Valentina; Paturzo, Melania; Ferraro, Pietro

2017-09-01

The study of cell adhesion contacts is pivotal to understand cell mechanics and interaction at substrates or chemical and physical stimuli. We designed and built a HoloTIR microscope for label-free quantitative phase imaging of total internal reflection. Here we show for the first time that HoloTIR is a good choice for label-free study of focal contacts and of cell/substrate interaction as its sensitivity is enhanced in comparison with standard TIR microscopy. Finally, the simplicity of implementation and relative low cost, due to the requirement of less optical components, make HoloTIR a reasonable alternative, or even an addition, to TIRF microscopy for mapping cell/substratum topography. As a proof of concept, we studied the formation of focal contacts of fibroblasts on three substrates with different levels of affinity for cell adhesion. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Updateable 3D Display Using Large Area Photorefractive Polymer Devices

DTIC Science & Technology

2013-04-01

across the thin PR layer. The critical parameters for the buffer layer are: transparent over the range 400nm to 800nm, high dielectric strength , ease of...Holographic 3D Display for Telepresence". Holo- pack.Holo- print 2011, November 9th 2011, (Las-Vegas, NV).  J. Thomas et al., "Recent advances in...AFRL-OSR-VA-TR-2013-0199 Updateable 3D Display Using Large Area Photofractive Polymer Devics Pierre Alexandre Blanche TIPD

HoloMonitor M4: holographic imaging cytometer for real-time kinetic label-free live-cell analysis of adherent cells

NASA Astrophysics Data System (ADS)

Sebesta, Mikael; Egelberg, Peter J.; Langberg, Anders; Lindskov, Jens-Henrik; Alm, Kersti; Janicke, Birgit

2016-03-01

Live-cell imaging enables studying dynamic cellular processes that cannot be visualized in fixed-cell assays. An increasing number of scientists in academia and the pharmaceutical industry are choosing live-cell analysis over or in addition to traditional fixed-cell assays. We have developed a time-lapse label-free imaging cytometer HoloMonitorM4. HoloMonitor M4 assists researchers to overcome inherent disadvantages of fluorescent analysis, specifically effects of chemical labels or genetic modifications which can alter cellular behavior. Additionally, label-free analysis is simple and eliminates the costs associated with staining procedures. The underlying technology principle is based on digital off-axis holography. While multiple alternatives exist for this type of analysis, we prioritized our developments to achieve the following: a) All-inclusive system - hardware and sophisticated cytometric analysis software; b) Ease of use enabling utilization of instrumentation by expert- and entrylevel researchers alike; c) Validated quantitative assay end-points tracked over time such as optical path length shift, optical volume and multiple derived imaging parameters; d) Reliable digital autofocus; e) Robust long-term operation in the incubator environment; f) High throughput and walk-away capability; and finally g) Data management suitable for single- and multi-user networks. We provide examples of HoloMonitor applications of label-free cell viability measurements and monitoring of cell cycle phase distribution.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rashin, Alexander A., E-mail: alexander-rashin@hotmail.com; Iowa State University, 112 Office and Lab Bldg, Ames, IA 50011-3020; Domagalski, Marcin J.

Conformational differences between myoglobin structures are studied. Most structural differences in whale myoglobin beyond the uncertainty threshold can be correlated with a few specific structural factors. There are always exceptions and a search for additional factors is needed. The results might have serious implications for biological insights from conformational differences. Validation of general ideas about the origins of conformational differences in proteins is critical in order to arrive at meaningful functional insights. Here, principal component analysis (PCA) and distance difference matrices are used to validate some such ideas about the conformational differences between 291 myoglobin structures from sperm whale, horsemore » and pig. Almost all of the horse and pig structures form compact PCA clusters with only minor coordinate differences and outliers that are easily explained. The 222 whale structures form a few dense clusters with multiple outliers. A few whale outliers with a prominent distortion of the GH loop are very similar to the cluster of horse structures, which all have a similar GH-loop distortion apparently owing to intermolecular crystal lattice hydrogen bonds to the GH loop from residues near the distal histidine His64. The variations of the GH-loop coordinates in the whale structures are likely to be owing to the observed alternative intermolecular crystal lattice bond, with the change to the GH loop distorting bonds correlated with the binding of specific ‘unusual’ ligands. Such an alternative intermolecular bond is not observed in horse myoglobins, obliterating any correlation with the ligands. Intermolecular bonds do not usually cause significant coordinate differences and cannot be validated as their universal cause. Most of the native-like whale myoglobin structure outliers can be correlated with a few specific factors. However, these factors do not always lead to coordinate differences beyond the previously determined uncertainty thresholds. The binding of unusual ligands by myoglobin, leading to crystal-induced distortions, suggests that some of the conformational differences between the apo and holo structures might not be ‘functionally important’ but rather artifacts caused by the binding of ‘unusual’ substrate analogs. The causes of P6 symmetry in myoglobin crystals and the relationship between crystal and solution structures are also discussed.« less

Crystal structure of Escherichia coli diaminopropionate ammonia-lyase reveals mechanism of enzyme activation and catalysis.

PubMed

Bisht, Shveta; Rajaram, Venkatesan; Bharath, Sakshibeedu R; Kalyani, Josyula Nitya; Khan, Farida; Rao, Appaji N; Savithri, Handanahal S; Murthy, Mathur R N

2012-06-08

Pyridoxal 5'-phosphate (PLP)-dependent enzymes utilize the unique chemistry of a pyridine ring to carry out diverse reactions involving amino acids. Diaminopropionate (DAP) ammonia-lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that catalyzes the degradation of d- and l-forms of DAP to pyruvate and ammonia. Here, we report the first crystal structure of DAPAL from Escherichia coli (EcDAPAL) in tetragonal and monoclinic forms at 2.0 and 2.2 Å resolutions, respectively. Structures of EcDAPAL soaked with substrates were also determined. EcDAPAL has a typical fold type II PLP-dependent enzyme topology consisting of a large and a small domain with the active site at the interface of the two domains. The enzyme is a homodimer with a unique biological interface not observed earlier. Structure of the enzyme in the tetragonal form had PLP bound at the active site, whereas the monoclinic structure was in the apo-form. Analysis of the apo and holo structures revealed that the region around the active site undergoes transition from a disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. EcDAPAL soaked with dl-DAP revealed density at the active site appropriate for the reaction intermediate aminoacrylate, which is consistent with the observation that EcDAPAL has low activity under crystallization conditions. Based on the analysis of the structure and results of site-directed mutagenesis, a two-base mechanism of catalysis involving Asp(120) and Lys(77) is suggested.

Solution structure and backbone dynamics of an endopeptidase HycI from Escherichia coli: implications for mechanism of the [NiFe] hydrogenase maturation.

PubMed

Yang, Fan; Hu, Wei; Xu, Huimin; Li, Congmin; Xia, Bin; Jin, Changwen

2007-02-09

[NiFe] hydrogenases are metalloenzymes involved in many biological processes concerning the metabolism of hydrogen. The maturation of the large subunit of these hydrogenases requires the cleavage of a peptide at the C terminus by an endopeptidase before the final formation of the [NiFe] metallocenter. HycI is an endopeptidase of the M52 family and responsible for the C-terminal cleavage of the large subunit of hydrogenase 3 in Escherichia coli. Although extensive studies were performed, the molecular mechanism of recognition and cleavage of hydrogenase 3 remains elusive. Herein, we report the solution structure of E. coli HycI determined by high resolution nuclear magnetic resonance spectroscopy. This is the first solution structure of the apo form of endopeptidase of the M52 family reported thus far. The overall structure is similar to the crystal structure of holo-HybD in the same family. However, significant diversity was observed between the two structures. Especially, HycI shows an open conformation at the putative nickel-binding site, whereas HybD adopts a closed conformation. In addition, we performed backbone dynamic studies to probe the motional properties of the apo form of HycI. Furthermore, the metal ion titration experiments provide insightful information on the substrate recognition and cleavage processes. Taken together, our current structural, biochemical, and dynamic studies extend the knowledge of the M52 family proteins and provide novel insights into the biological function of HycI.

Paralogs of the C-Terminal Domain of the Cyanobacterial Orange Carotenoid Protein Are Carotenoid Donors to Helical Carotenoid Proteins1[OPEN

PubMed Central

Muzzopappa, Fernando; Wilson, Adjélé; Yogarajah, Vinosa; Cot, Sandrine; Perreau, François; Montigny, Cédric; Bourcier de Carbon, Céline

2017-01-01

The photoactive Orange Carotenoid Protein (OCP) photoprotects cyanobacteria cells by quenching singlet oxygen and excess excitation energy. Its N-terminal domain is the active part of the protein, and the C-terminal domain regulates the activity. Recently, the characteristics of a family of soluble carotenoid-binding proteins (Helical Carotenoid Proteins [HCPs]), paralogs of the N-terminal domain of OCP, were described. Bioinformatics studies also revealed the existence of genes coding for homologs of CTD. Here, we show that the latter genes encode carotenoid proteins (CTDHs). This family of proteins contains two subgroups with distinct characteristics. One CTDH of each clade was further characterized, and they proved to be very good singlet oxygen quenchers. When synthesized in Escherichia coli or Synechocystis PCC 6803, CTDHs formed dimers that share a carotenoid molecule and are able to transfer their carotenoid to apo-HCPs and apo-OCP. The CTDHs from clade 2 have a cysteine in position 103. A disulfide bond is easily formed between the monomers of the dimer preventing carotenoid transfer. This suggests that the transfer of the carotenoid could be redox regulated in clade 2 CTDH. We also demonstrate here that apo-OCPs and apo-CTDHs are able to take the carotenoid directly from membranes, while HCPs are unable to do so. HCPs need the presence of CTDH to become holo-proteins. We propose that, in cyanobacteria, the CTDHs are carotenoid donors to HCPs. PMID:28935842

Role of adapter function in oncoprotein-mediated activation of NF-kappaB. Human T-cell leukemia virus type I Tax interacts directly with IkappaB kinase gamma.

PubMed

Jin, D Y; Giordano, V; Kibler, K V; Nakano, H; Jeang, K T

1999-06-18

Mechanisms by which the human T-cell leukemia virus type I Tax oncoprotein activates NF-kappaB remain incompletely understood. Although others have described an interaction between Tax and a holo-IkappaB kinase (IKK) complex, the exact details of protein-protein contact are not fully defined. Here we show that Tax binds to neither IKK-alpha nor IKK-beta but instead complexes directly with IKK-gamma, a newly characterized component of the IKK complex. This direct interaction with IKK-gamma correlates with Tax-induced IkappaB-alpha phosphorylation and NF-kappaB activation. Thus, our findings establish IKK-gamma as a key molecule for adapting an oncoprotein-specific signaling to IKK-alpha and IKK-beta.

αRep A3: A Versatile Artificial Scaffold for Metalloenzyme Design.

PubMed

Di Meo, Thibault; Ghattas, Wadih; Herrero, Christian; Velours, Christophe; Minard, Philippe; Mahy, Jean-Pierre; Ricoux, Rémy; Urvoas, Agathe

2017-07-26

αRep refers to a new family of artificial proteins based on a thermostable α-helical repeated motif. One of its members, αRep A3, forms a stable homo-dimer with a wide cleft that is able to accommodate metal complexes and thus appears to be suitable for generating new artificial biocatalysts. Based on the crystal structure of αRep A3, two positions (F119 and Y26) were chosen, and independently changed into cysteine residues. A phenanthroline ligand was covalently attached to the unique cysteine residue of each protein variant, and the corresponding biohybrids were purified and characterized. Once mutated and coupled to phenanthroline, the protein remained folded and dimeric. Copper(II) was specifically bound by the two biohybrids with two different binding modes. Furthermore, the holo-biohybrid A3F119NPH was found to be capable of enantioselectively catalyzing Diels-Alder (D-A) cycloadditions with up to 62 % ee. This study validates the choice of the αRep A3 dimer as a protein scaffold and provides a promising new route for the design and production of new enantioselective biohybrids based on entirely artificial proteins obtained from a highly diverse library. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural Basis for "Flip-Flop" Action of Human Pyruvate Dehydrogenase

NASA Technical Reports Server (NTRS)

Ciszak, Ewa; Korotchkina, Lioubov; Dominiak, Paulina; Sidhu, Sukhdeep; Patel, Mulchand

2003-01-01

The derivative of vitamin B1, thiamin pyrophosphate is a cofactor of pyruvate dehydrogenase, a component enzyme of the mitochondrial pyruvate dehydrogenase multienzyme complex that plays a major role in directing energy metabolism in the cell. This cofactor is used to cleave the C(sup alpha)-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. In alpha(sub 2)beta(sub 2)-tetrameric human pyruvate dehydrogenase, there are two cofactor binding sites, each of them being a center of independently conducted, although highly coordinated enzymatic reactions. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites can now be understood based on the recently determined crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95A resolution. The structure of pyruvate dehydrogenase was determined using a combination of MAD phasing and molecular replacement followed by rounds of torsion-angles molecular-dynamics simulated-annealing refinement. The final pyruvate dehydrogenase structure included coordinates for all protein amino acids two cofactor molecules, two magnesium and two potassium ions, and 742 water molecules. The structure was refined to R = 0.202 and R(sub free) = 0.244. Our structural analysis of the enzyme folding and domain assembly identified a simple mechanism of this protein motion required for the conduct of catalytic action.

Identification and assessment of markers of biotin status in healthy adults

PubMed Central

Eng, Wei Kay; Giraud, David; Schlegel, Vicki L.; Wang, Dong; Lee, Bo Hyun; Zempleni, Janos

2016-01-01

Human biotin requirements are unknown and the identification of reliable markers of biotin status is necessary to fill this knowledge gap. Here, we used an outpatient feeding protocol to create states of biotin deficiency, sufficiency and supplementation in sixteen healthy men and women. A total of twenty possible markers of biotin status were assessed, including the abundance of biotinylated carboxylases in lymphocytes, the expression of genes from biotin metabolism and the urinary excretion of biotin and organic acids. Only the abundance of biotinylated 3-methylcrotonyl-CoA carboxylase (holo-MCC) and propionyl-CoA carboxylase (holo-PCC) allowed for distinguishing biotin-deficient and biotin-sufficient individuals. The urinary excretion of biotin reliably identified biotin-supplemented subjects, but did not distinguish between biotin-depleted and biotin-sufficient individuals. The urinary excretion of 3-hydroxyisovaleric acid detected some biotin-deficient subjects, but produced a meaningful number of false-negative results and did not distinguish between biotin-sufficient and biotin-supplemented individuals. None of the other organic acids that were tested were useful markers of biotin status. Likewise, the abundance of mRNA coding for biotin transporters, holocarboxylase synthetase and biotin-dependent carboxylases in lymphocytes were not different among the treatment groups. Generally, datasets were characterised by variations that exceeded those seen in studies in cell cultures. We conclude that holo-MCC and holo-PCC are the most reliable, single markers of biotin status tested in the present study. PMID:23302490

Innovative approaches to vitamin A assessment.

PubMed

Craft, N E

2001-05-01

The health and sight of millions of children are compromised each year as a consequence of vitamin A (VA) deficiency. Serum retinol is the most commonly used indicator of VA status. Unfortunately, its use is impractical for national surveys because it involves collection of venous blood, centrifugation and frozen storage before analysis. To make VA assessment more practical, we have developed approaches incorporating dried blood spots (DBS) or portable instrumentation. DBS have been used as a sample matrix to screen neonates for many biochemical compounds. Until recently, it was not thought that VA was stable in DBS. However, we demonstrated that the measure of DBS retinol correlates well with serum retinol in both healthy adults (r(2) = 0.88-0.90) and compromised populations (r(2) = 0.73-0.84). Compared with serum retinol, the sensitivity and specificity of detecting VA deficiency by DBS retinol range from 73 to 93% and from 90 to 100%, respectively. Although few data are available, retinol binding protein (RBP) can also be measured in DBS. RBP has been used as a surrogate marker for serum retinol. Correlations coefficients (r(2)) between serum RBP and serum retinol range from 0.4 to 0.8. In addition, work has been done to develop portable instrumentation to measure VA status in the field. A fluorometer has been optimized for VA fluorescence and is linear into the deficient range for the direct fluorimetric measurement of serum holo-RBP. Progress is being made to use the instrument to directly measure holo-RBP in a drop of whole blood.

Insight into cofactor recognition in arylamine N-acetyltransferase enzymes: structure of Mesorhizobium loti arylamine N-acetyltransferase in complex with coenzyme A.

PubMed

Xu, Ximing; Li de la Sierra-Gallay, Inés; Kubiak, Xavier; Duval, Romain; Chaffotte, Alain F; Dupret, Jean Marie; Haouz, Ahmed; Rodrigues-Lima, Fernando

2015-02-01

Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes that catalyze the acetyl-CoA-dependent acetylation of arylamines. To better understand the mode of binding of the cofactor by this family of enzymes, the structure of Mesorhizobium loti NAT1 [(RHILO)NAT1] was determined in complex with CoA. The F42W mutant of (RHILO)NAT1 was used as it is well expressed in Escherichia coli and displays enzymatic properties similar to those of the wild type. The apo and holo structures of (RHILO)NAT1 F42W were solved at 1.8 and 2 Å resolution, respectively. As observed in the Mycobacterium marinum NAT1-CoA complex, in (RHILO)NAT1 CoA binding induces slight structural rearrangements that are mostly confined to certain residues of its `P-loop'. Importantly, it was found that the mode of binding of CoA is highly similar to that of M. marinum NAT1 but different from the modes reported for Bacillus anthracis NAT1 and Homo sapiens NAT2. Therefore, in contrast to previous data, this study shows that different orthologous NATs can bind their cofactors in a similar way, suggesting that the mode of binding CoA in this family of enzymes is less diverse than previously thought. Moreover, it supports the notion that the presence of the `mammalian/eukaryotic insertion loop' in certain NAT enzymes impacts the mode of binding CoA by imposing structural constraints.

Entrapment of Carbon Dioxide in the Active Site of Carbonic Anhydrase II*♦

PubMed Central

Domsic, John F.; Avvaru, Balendu Sankara; Kim, Chae Un; Gruner, Sol M.; Agbandje-McKenna, Mavis; Silverman, David N.; McKenna, Robert

2008-01-01

The visualization at near atomic resolution of transient substrates in the active site of enzymes is fundamental to fully understanding their mechanism of action. Here we show the application of using CO2-pressurized, cryo-cooled crystals to capture the first step of CO2 hydration catalyzed by the zinc-metalloenzyme human carbonic anhydrase II, the binding of substrate CO2, for both the holo and the apo (without zinc) enzyme to 1.1Å resolution. Until now, the feasibility of such a study was thought to be technically too challenging because of the low solubility of CO2 and the fast turnover to bicarbonate by the enzyme (Liang, J. Y., and Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3675–3679). These structures provide insight into the long hypothesized binding of CO2 in a hydrophobic pocket at the active site and demonstrate that the zinc does not play a critical role in the binding or orientation of CO2. This method may also have a much broader implication for the study of other enzymes for which CO2 is a substrate or product and for the capturing of transient substrates and revealing hydrophobic pockets in proteins. PMID:18768466

Canadian Whole-Farm Model Holos - Development, Stakeholder Involvement, and Model Application

NASA Astrophysics Data System (ADS)

Kroebel, R.; Janzen, H.; Beauchemin, K. A.

2017-12-01

Agriculture and Agri-Food Canada's Holos model, based mostly on emission factors, aims to explore the effect of management on Canadian whole-farm greenhouse gas emissions. The model includes 27 commonly grown annual and perennial crops, summer fallow, grassland, and 8 types of tree plantings, along with beef, dairy, sheep, swine and other livestock or poultry operations. Model outputs encompass net emissions of CO2, CH4, and N2O (in CO2 equivalents), calculated for various farm components. Where possible, algorithms are drawn from peer-reviewed publications. For consistency, Holos is aligned with the Canadian sustainability indicator and national greenhouse gas inventory objectives. Although primarily an exploratory tool for research, the model's design makes it accessible and instructive also to agricultural producers, educators, and policy makers. Model development, therefore, proceeds iteratively, with extensive stakeholder feedback from training sessions or annual workshops. To make the model accessible to diverse users, the team developed a multi-layered interface, with general farming scenarios for general use, but giving access to detailed coefficients and assumptions to researchers. The model relies on extensive climate, soil, and agronomic databases to populate regionally-applicable default values thereby minimizing keyboard entries. In an initial application, the model was used to assess greenhouse gas emissions from the Canadian beef production system; it showed that enteric methane accounted for 63% of total GHG emissions and that 84% of emissions originated from the cow-calf herd. The model further showed that GHG emission intensity per kg beef, nationally, declined by 14% from 1981 to 2011, owing to gains in production efficiency. Holos is now being used to consider further potential advances through improved rations or other management options. We are now aiming to expand into questions of grazing management, and are developing a novel carbon modelling approach based on the ICBM model. Also under development are sub-models to predict ammonia volatilization and water budgets. Development of Holos is expected to continue, forging an interactive link between ongoing research and the interests of stakeholders in an ever-changing agricultural environment.

Influence of the [4Fe–4S] cluster coordinating cysteines on active site maturation and catalytic properties of C. reinhardtii [FeFe]-hydrogenase† †Electronic supplementary information (ESI) available: Tables showing the mutagenic primers used in this study, the iron content of apoHydA1 and holoHydA1 variants after 1 h and 30 h of in vitro maturation, and a summary of the relevant redox states. Figures showing spectroscopic characterization of apoHydA1 “A” and “D” variants, in vivo maturation and oxidation screening of holoHydA1 variants, annotated FTIR spectra of holoHydA1 variants, comparison of normalized FTIR spectra of holoHydA1 variants after 1 h and 30 h of in vitro maturation, in vitro maturation followed by FTIR over 30 h, and a comparison of cyclic voltammograms of holoHydA1 variants from pH 5 to pH 10. See DOI: 10.1039/c7sc03444j

PubMed Central

Kertess, Leonie; Adamska-Venkatesh, Agnieszka; Rodríguez-Maciá, Patricia; Rüdiger, Olaf; Lubitz, Wolfgang

2017-01-01

[FeFe]-Hydrogenases catalyze the evolution and oxidation of hydrogen using a characteristic cofactor, termed the H-cluster. This comprises an all cysteine coordinated [4Fe–4S] cluster and a unique [2Fe] moiety, coupled together via a single cysteine. The coordination of the [4Fe–4S] cluster in HydA1 from Chlamydomonas reinhardtii was altered by single exchange of each cysteine (C115, C170, C362, and C366) with alanine, aspartate, or serine using site-directed mutagenesis. In contrast to cysteine 115, the other three cysteines were found to be dispensable for stable [4Fe–4S] cluster incorporation based on iron determination, UV/vis spectroscopy and electron paramagnetic resonance. However, the presence of a preformed [4Fe–4S] cluster alone does not guarantee stable incorporation of the [2Fe] cluster. Only variants C170D, C170S, C362D, and C362S showed characteristic signals for an inserted [2Fe] cluster in Fourier-transform infrared spectroscopy. Hydrogen evolution and oxidation were observed for these variants in solution based assays and protein-film electrochemistry. Catalytic activity was lowered for all variants and the ability to operate in either direction was also influenced. PMID:29568461

S81L and G170R mutations causing Primary Hyperoxaluria type I in homozygosis and heterozygosis: an example of positive interallelic complementation

PubMed Central

Montioli, Riccardo; Roncador, Alessandro; Oppici, Elisa; Mandrile, Giorgia; Giachino, Daniela Francesca; Cellini, Barbara; Borri Voltattorni, Carla

2014-01-01

Primary Hyperoxaluria type I (PH1) is a rare disease due to the deficit of peroxisomal alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal-5′-phosphate (PLP) enzyme present in humans as major (Ma) and minor (Mi) allele. PH1-causing mutations are mostly missense identified in both homozygous and compound heterozygous patients. Until now, the pathogenesis of PH1 has been only studied by approaches mimicking homozygous patients, whereas the molecular aspects of the genotype-enzymatic-clinical phenotype relationship in compound heterozygous patients are completely unknown. Here, for the first time, we elucidate the enzymatic phenotype linked to the S81L mutation on AGT-Ma, relative to a PLP-binding residue, and how it changes when the most common mutation G170R on AGT-Mi, known to cause AGT mistargeting without affecting the enzyme functionality, is present in the second allele. By using a bicistronic eukaryotic expression vector, we demonstrate that (i) S81L-Ma is mainly in its apo-form and has a significant peroxisomal localization and (ii) S81L and G170R monomers interact giving rise to the G170R-Mi/S81L-Ma holo-form, which is imported into peroxisomes and exhibits an enhanced functionality with respect to the parental enzymes. These data, integrated with the biochemical features of the heterodimer and the homodimeric counterparts in their purified recombinant form, (i) highlight the molecular basis of the pathogenicity of S81L-Ma and (ii) provide evidence for a positive interallelic complementation between the S81L and G170R monomers. Our study represents a valid approach to investigate the molecular pathogenesis of PH1 in compound heterozygous patients. PMID:24990153

Effect of iron saturation level of lactoferrin on osteogenic activity in vitro and in vivo.

PubMed

Wang, X Y; Guo, H Y; Zhang, W; Wen, P C; Zhang, H; Guo, Z R; Ren, F Z

2013-01-01

We studied the effect of iron saturation level on the osteogenic activity of lactoferrin (LF) in vitro and in vivo. Different iron saturation levels of LF (1.0, 9.0, 38, 58, and 96%) were prepared as the following samples: apo-LF, LF-9, LF-38, LF-58, and holo-LF. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, we observed that the stimulating osteoblast proliferation activity of LF in vitro decreased with increasing iron saturation level at 100 and 1,000 μg/mL. In vivo, 4-wk-old ICR Swiss male mice were randomly divided into 4 groups: blank control (physiological saline), negative control (BSA), apo-LF, and holo-LF. Four groups of mice were injected subcutaneously with physiological saline, BSA, apo-LF, or holo-LF over the calvarial surface twice a day for 5 consecutive days at a dose of 4 mg/kg per day. Bone histomorphometry showed that new bone formation (assessed using tetracycline-HCl labels) tended to be stronger with apo-LF than with holo-LF. Using fluorescence spectroscopy and circular dichroism measurements, we found that exposure of tryptophan increased, α-helix content increased, but β-structure content decreased with increasing iron saturation level. These findings indicated that the osteogenic activity of LF decreases with increasing iron saturation level in vitro and in vivo, which may be related to conformational changes in LF. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The Holo-Transcriptome of the Zoantharian Protopalythoa variabilis (Cnidaria: Anthozoa): A Plentiful Source of Enzymes for Potential Application in Green Chemistry, Industrial and Pharmaceutical Biotechnology.

PubMed

R L Morlighem, Jean-Étienne; Huang, Chen; Liao, Qiwen; Braga Gomes, Paula; Daniel Pérez, Carlos; de Brandão Prieto-da-Silva, Álvaro Rossan; Ming-Yuen Lee, Simon; Rádis-Baptista, Gandhi

2018-06-13

Marine invertebrates, such as sponges, tunicates and cnidarians (zoantharians and scleractinian corals), form functional assemblages, known as holobionts, with numerous microbes. This type of species-specific symbiotic association can be a repository of myriad valuable low molecular weight organic compounds, bioactive peptides and enzymes. The zoantharian Protopalythoa variabilis (Cnidaria: Anthozoa) is one such example of a marine holobiont that inhabits the coastal reefs of the tropical Atlantic coast and is an interesting source of secondary metabolites and biologically active polypeptides. In the present study, we analyzed the entire holo-transcriptome of P. variabilis , looking for enzyme precursors expressed in the zoantharian-microbiota assemblage that are potentially useful as industrial biocatalysts and biopharmaceuticals. In addition to hundreds of predicted enzymes that fit into the classes of hydrolases, oxidoreductases and transferases that were found, novel enzyme precursors with multiple activities in single structures and enzymes with incomplete Enzyme Commission numbers were revealed. Our results indicated the predictive expression of thirteen multifunctional enzymes and 694 enzyme sequences with partially characterized activities, distributed in 23 sub-subclasses. These predicted enzyme structures and activities can prospectively be harnessed for applications in diverse areas of industrial and pharmaceutical biotechnology.

Double Tryptophan Exciton Probe to Gauge Proximal Side Chains in Proteins- Augmentation at Low Temperature

PubMed Central

Gasymov, Oktay K.; Abduragimov, Adil R.; Glasgow, Ben J.

2015-01-01

The circular dichroic (CD) exciton couplet between tryptophans and/or tyrosines offers the potential to probe distances within 10Å in proteins. The exciton effect has been used with native chromophores in critical positions in a few proteins. Here, site-directed mutagenesis created double tryptophan probes for key sites of a protein (tear lipocalin). For tear lipocalin the crystal and solution structures are concordant in both apo- and holo-forms. Double tryptophan substitutions were performed at sites that could probe conformation and were likely within 10 Å. Far-UV CD spectra of double Trp mutants were performed with controls that had non-interacting substituted tryptophans. Low temperature (77K) was tested for augmentation of the exciton signal. Exciton coupling appeared with tryptophan substitutions at positions within loop A-B (28 and 31, 33), between loop A-B (28) and strand G (103 and 105), as well as between the strands B (35) and C (56). The CD exciton couplet signals were amplified 3–5 fold at 77K. The results were concordant with close distances in crystal and solution structures. The exciton couplets had functional significance and correctly assigned the holo-conformation. The methodology creates an effective probe to identify proximal amino acids in a variety of motifs. PMID:25693116

Magnesium-dependent RNA binding to the PA endonuclease domain of the avian influenza polymerase.

PubMed

Xiao, Shiyan; Klein, Michael L; LeBard, David N; Levine, Benjamin G; Liang, Haojun; MacDermaid, Christopher M; Alfonso-Prieto, Mercedes

2014-01-30

Influenza A viruses are highly pathogenic and pose an unpredictable public health danger to humans. An attractive target for developing new antiviral drugs is the PA N-terminal domain (PAN) of influenza polymerase, which is responsible for the endonuclease activity and essential for viral replication. Recently, the crystal structures of the holo form of PAN as well as PAN bound to different inhibitors have been reported, but the potency and selectivity of these inhibitors still need to be improved. New drug design can be guided by a better understanding of the endonuclease activity of PAN. However, this requires the structure of PAN in complex with the host mRNA, which has not been determined yet. In particular, divalent metal ions are known to be essential for RNA cleavage, but it is not clear whether there is either one or two Mg ions in the PAN active site. In the present work, we have modeled the complex of the PAN endonuclease domain with the host mRNA in the presence of either one or two Mg(2+) by using all-atom molecular dynamics. These simulations identify crucial interactions between the enzyme and the nucleic acid. Moreover, they validate a previous hypothesis that a second metal ion binds in the presence of the RNA substrate and therefore support a two-metal ion mechanism, in which K134 decreases the pKa of the nucleophilic water. Nevertheless, at low Mg concentrations an alternative, one-metal ion mechanism is possible, with K137 as the catalytic lysine and H41 as the general base, rationalizing previous unexpected mutagenesis results. The RNA-enzyme interactions determined here could likely be used to design more specific endonuclease inhibitors to fight influenza viral infections.

Ground Based Operational Testing Of Holographic Scanning Lidars : The HOLO Experiments

NASA Technical Reports Server (NTRS)

Schwemmer, Geary K.; Wilkerson, Thomas D.; Sanders, Jason A.; Guerra, David V.; Miller, David O.; Moody, Stephen E.

2000-01-01

Two aerosol backscatter lidar measurement campaigns were conducted using two holographic scanning lidars and one zenith staring lidar for the purposes of reliability testing under field conditions three new lidar systems and to develop new scanning measurement techniques and applications. The first campaign took place near the campus of Utah State University in Logan Utah in March of 1999 and is called HOLO-1. HOLO-2 was conducted in June of 1999 on the campus of Saint Anselm College, near the city of Manchester, New Hampshire. Each campaign covered a period of approximately one week of nearly continuous observation of cloud and aerosol backscatter in the visible and near infrared by lidar, and wide field visible sky images by video camera in the daytime. The scanning capability coupled with a high rep-rate, high average power laser enables both high spatial and high temporal resolution observations that Particularly intriguing is the possibility of deriving atmospheric wind profiles from temporal analysis of aerosol backscatter spatial structure obtained by conical scan without the use of Doppler techniques.

Manipulating Protein-Protein Interactions in Nonribosomal Peptide Synthetase Type II Peptidyl Carrier Proteins.

PubMed

Jaremko, Matt J; Lee, D John; Patel, Ashay; Winslow, Victoria; Opella, Stanley J; McCammon, J Andrew; Burkart, Michael D

2017-10-10

In an effort to elucidate and engineer interactions in type II nonribosomal peptide synthetases, we analyzed biomolecular recognition between the essential peptidyl carrier proteins and adenylation domains using nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics, and mutational studies. Three peptidyl carrier proteins, PigG, PltL, and RedO, in addition to their cognate adenylation domains, PigI, PltF, and RedM, were investigated for their cross-species activity. Of the three peptidyl carrier proteins, only PigG showed substantial cross-pathway activity. Characterization of the novel NMR solution structure of holo-PigG and molecular dynamics simulations of holo-PltL and holo-PigG revealed differences in structures and dynamics of these carrier proteins. NMR titration experiments revealed perturbations of the chemical shifts of the loop 1 residues of these peptidyl carrier proteins upon their interaction with the adenylation domain. These experiments revealed a key region for the protein-protein interaction. Mutational studies supported the role of loop 1 in molecular recognition, as mutations to this region of the peptidyl carrier proteins significantly modulated their activities.

Apoenzyme of aspartate aminotransferase isozymes in serum and its diagnostic usefullness for hepatic diseases.

PubMed

Kamei, S; Ohkubo, A; Yamanaka, M

1979-08-15

Aspartate aminotransferase in the sera of normal subjects and of patients with hepatic diseases has been immunologically separated into two isoenzymes, cytosolic aspartate aminotransferase and mitochondrial aspartate aminotransferase. The activity of the isoenzymes was measured in three different buffer solutions with or without pyridoxal 5'-phosphate. To attain maximal activation, the apoenzyme of mitochondrial fraction must be preincubated with pyridoxal 5'-phosphate longer than that of the cytosolic fraction in either of the three reaction mixtures. In most sera the activity of both isoenzymes increased substantially in the presence of pyridoxal 5'-phosphate regardless of the type of buffer solutions. Both the apoenzymatic activity and the ratio of apo- to holo-enzymatic activity of each of the isoenzymes varied among samples from the patients with hepatic diseases. However, significantly high ratios of apo- to holo-enzymatic activity of both isoenzymes were observed in the patients with hepatoma in contrast with those with other hepatic diseases. These findings suggest that the simultaneous measurement of both apo- and holo-enzyme activities of aspartate aminotransferase isoenzymes may be useful in the clinical assessment of hepatic diseases.

Glycolipid acquisition by human glycolipid transfer protein dramatically alters intrinsic tryptophan fluorescence: insights into glycolipid binding affinity.

PubMed

Zhai, Xiuhong; Malakhova, Margarita L; Pike, Helen M; Benson, Linda M; Bergen, H Robert; Sugár, István P; Malinina, Lucy; Patel, Dinshaw J; Brown, Rhoderick E

2009-05-15

Glycolipid transfer proteins (GLTPs) are small, soluble proteins that selectively accelerate the intermembrane transfer of glycolipids. The GLTP fold is conformationally unique among lipid binding/transfer proteins and serves as the prototype and founding member of the new GLTP superfamily. In the present study, changes in human GLTP tryptophan fluorescence, induced by membrane vesicles containing glycolipid, are shown to reflect glycolipid binding when vesicle concentrations are low. Characterization of the glycolipid-induced "signature response," i.e. approximately 40% decrease in Trp intensity and approximately 12-nm blue shift in emission wavelength maximum, involved various modes of glycolipid presentation, i.e. microinjection/dilution of lipid-ethanol solutions or phosphatidylcholine vesicles, prepared by sonication or extrusion and containing embedded glycolipids. High resolution x-ray structures of apo- and holo-GLTP indicate that major conformational alterations are not responsible for the glycolipid-induced GLTP signature response. Instead, glycolipid binding alters the local environment of Trp-96, which accounts for approximately 70% of total emission intensity of three Trp residues in GLTP and provides a stacking platform that aids formation of a hydrogen bond network with the ceramide-linked sugar of the glycolipid headgroup. The changes in Trp signal were used to quantitatively assess human GLTP binding affinity for various lipids including glycolipids containing different sugar headgroups and homogenous acyl chains. The presence of the glycolipid acyl chain and at least one sugar were essential for achieving a low-to-submicromolar dissociation constant that was only slightly altered by increased sugar headgroup complexity.

ProBiS-CHARMMing: Web Interface for Prediction and Optimization of Ligands in Protein Binding Sites.

PubMed

Konc, Janez; Miller, Benjamin T; Štular, Tanja; Lešnik, Samo; Woodcock, H Lee; Brooks, Bernard R; Janežič, Dušanka

2015-11-23

Proteins often exist only as apo structures (unligated) in the Protein Data Bank, with their corresponding holo structures (with ligands) unavailable. However, apoproteins may not represent the amino-acid residue arrangement upon ligand binding well, which is especially problematic for molecular docking. We developed the ProBiS-CHARMMing web interface by connecting the ProBiS ( http://probis.cmm.ki.si ) and CHARMMing ( http://www.charmming.org ) web servers into one functional unit that enables prediction of protein-ligand complexes and allows for their geometry optimization and interaction energy calculation. The ProBiS web server predicts ligands (small compounds, proteins, nucleic acids, and single-atom ligands) that may bind to a query protein. This is achieved by comparing its surface structure against a nonredundant database of protein structures and finding those that have binding sites similar to that of the query protein. Existing ligands found in the similar binding sites are then transposed to the query according to predictions from ProBiS. The CHARMMing web server enables, among other things, minimization and potential energy calculation for a wide variety of biomolecular systems, and it is used here to optimize the geometry of the predicted protein-ligand complex structures using the CHARMM force field and to calculate their interaction energies with the corresponding query proteins. We show how ProBiS-CHARMMing can be used to predict ligands and their poses for a particular binding site, and minimize the predicted protein-ligand complexes to obtain representations of holoproteins. The ProBiS-CHARMMing web interface is freely available for academic users at http://probis.nih.gov.

Exploration of Multi-State Conformational Dynamics and Underlying Global Functional Landscape of Maltose Binding Protein

PubMed Central

Wang, Yong; Tang, Chun; Wang, Erkang; Wang, Jin

2012-01-01

An increasing number of biological machines have been revealed to have more than two macroscopic states. Quantifying the underlying multiple-basin functional landscape is essential for understanding their functions. However, the present models seem to be insufficient to describe such multiple-state systems. To meet this challenge, we have developed a coarse grained triple-basin structure-based model with implicit ligand. Based on our model, the constructed functional landscape is sufficiently sampled by the brute-force molecular dynamics simulation. We explored maltose-binding protein (MBP) which undergoes large-scale domain motion between open, apo-closed (partially closed) and holo-closed (fully closed) states responding to ligand binding. We revealed an underlying mechanism whereby major induced fit and minor population shift pathways co-exist by quantitative flux analysis. We found that the hinge regions play an important role in the functional dynamics as well as that increases in its flexibility promote population shifts. This finding provides a theoretical explanation of the mechanistic discrepancies in PBP protein family. We also found a functional “backtracking” behavior that favors conformational change. We further explored the underlying folding landscape in response to ligand binding. Consistent with earlier experimental findings, the presence of ligand increases the cooperativity and stability of MBP. This work provides the first study to explore the folding dynamics and functional dynamics under the same theoretical framework using our triple-basin functional model. PMID:22532792

Increased retinol-free RBP4 contributes to insulin resistance in gestational diabetes mellitus.

PubMed

Chen, Yanmin; Lv, Ping; Du, Mengkai; Liang, Zhaoxia; Zhou, Menglin; Chen, Danqing

2017-07-01

Retinol-binding protein 4 (RBP4) is a circulating retinol transporter that is strongly associated with insulin resistance. The aim of this study was to evaluate the RBP4 and retinol level in rat model of gestational diabetes mellitus and the relationship between retinol-free RBP4 (apo-RBP4), retinol-bound RBP4 (holo-RBP4) and insulin resistance. Pregnant rats were administered streptozotocin to induce diabetes. The RBP4 and retinol levels were evaluated in GDM and normal pregnant rats. After then, normal pregnant rats were divided into two groups to receive either apo-RBP4 or vehicle injection. The metabolic parameters and insulin signaling in adipose tissue, skeletal muscle and liver were determined in apo-RBP4 and control groups. Primary human adipocytes were cultured in vitro with different proportions of apo-RBP4 and holo-RBP4 for 24 h. The interaction between RBP4 and STRA6 was assessed by co-immunoprecipitation, and the expression of JAK-STAT pathway and insulin signaling were detected by Western blotting and immunofluorescence. We found increases in serum RBP4 levels and the RBP4:retinol ratio but not in the retinol levels in GDM rats. Exogenous apo-RBP4 injection attenuated insulin sensitivity in pregnant rats. In vitro, a prolonged interaction between RBP4 and STRA6 was observed when apo-RBP4 was present. In response to increased apo-RBP4 levels, cells showed elevated activation of the JAK2/STAT5 cascade and SOCS3 expression, decreased phosphorylation of IR and IRS1, and attenuated GLUT4 translocation and glucose uptake upon insulin stimulation. Apo-RBP4 is a ligand that activates the STRA6 signaling cascade, inducing insulin resistance in GDM.

Identification and screening of potent antimicrobial peptides in arthropod genomes.

PubMed

Duwadi, Deepesh; Shrestha, Anishma; Yilma, Binyam; Kozlovski, Itamar; Sa-Eed, Munaya; Dahal, Nikesh; Jukosky, James

2018-05-01

Using tBLASTn and BLASTp searches, we queried recently sequenced arthropod genomes and expressed sequence tags (ESTs) using a database of known arthropod cecropins, defensins, and attacins. We identified and synthesized 6 potential AMPs and screened them for antimicrobial activity. Using radial diffusion assays and microtiter antimicrobial assays, we assessed the in vitro antimicrobial effects of these peptides against several human pathogens including Gram-positive and Gram-negative bacteria and fungi. We also conducted hemolysis assays to examine the cytotoxicity of these peptides to mammalian cells. Four of the six peptides identified showed antimicrobial effects in these assays. We also created truncated versions of these four peptides to assay their antimicrobial activity. Two cecropins derived from the monarch butterfly genome (Danaus plexippus), DAN1 and DAN2, showed minimum inhibitory concentrations (MICs) in the range of 2-16 μg/ml when screened against Gram-negative bacteria. HOLO1 and LOUDEF1, two defensin-like peptides derived from red flour beetle (Tribolium castaneum) and human body louse (Pediculus humanus humanus), respectively, exhibited MICs in the range of 13-25 μg/ml against Gram-positive bacteria. Furthermore, HOLO1 showed an MIC less than 5 μg/ml against the fungal species Candida albicans. These peptides exhibited no hemolytic activity at concentrations up to 200 μg/ml. The truncated peptides derived from DAN2 and HOLO1 showed very little antimicrobial activity. Our experiments show that the peptides DAN1, DAN2, HOLO1, and LOUDEF1 showed potent antimicrobial activity in vitro against common human pathogens, did not lyse mammalian red blood cells, and indicates their potential as templates for novel therapeutic agents against microbial infection. Copyright © 2018 Elsevier Inc. All rights reserved.

Destination Mars Grand Opening

NASA Image and Video Library

2016-09-18

Apollo 11 astronaut Buzz Aldrin, left and Erisa Hines of NASA’s Jet Propulsion Laboratory (JPL) in Pasadena, California, try out Microsoft HoloLens mixed reality headset during a preview of the new Destination: Mars experience at the Kennedy Space Center Visitor Complex. Destination: Mars gives guests an opportunity to “visit” several sites on Mars using real imagery from NASA’s Curiosity Mars Rover. Based on OnSight, a tool created by NASA’s Jet Propulsion Laboratory in Pasadena, California, the experience brings guests together with a holographic version of Aldrin and Curiosity rover driver Hines as they are guided to Mars using Microsoft HoloLens mixed reality headset. Photo credit: NASA/Charles Babir

Positive Newborn Screen for Methylmalonic Aciduria Identifies the First Mutation in TCblR/CD320, the Gene for Cellular Uptake of Transcobalamin-bound Vitamin B12

PubMed Central

Quadros, Edward V.; Lai, Shao-Chiang; Nakayama, Yasumi; Sequeira, Jeffrey M.; Hannibal, Luciana; Wang, Sihe; Jacobsen, Donald W.; Fedosov, Sergey; Wright, Erica; Gallagher, Renata C.; Anastasio, Natascia; Watkins, David; Rosenblatt, David S.

2010-01-01

Elevated methylmalonic acid in five asymptomatic newborns whose fibroblasts showed decreased uptake of transcobalamin-bound cobalamin (holo-TC), suggested a defect in the cellular uptake of cobalamin. Analysis of TCblR/CD320, the gene for the receptor for cellular uptake of holo-TC, identified a homozygous single codon deletion, c.262_264GAG (p.E88del), resulting in the loss of a glutamic acid residue in the low-density lipoprotein receptor type A-like domain. Inserting the codon by site-directed mutagenesis fully restored TCblR function. PMID:20524213

Through the HoloLens™ looking glass: augmented reality for extremity reconstruction surgery using 3D vascular models with perforating vessels.

PubMed

Pratt, Philip; Ives, Matthew; Lawton, Graham; Simmons, Jonathan; Radev, Nasko; Spyropoulou, Liana; Amiras, Dimitri

2018-01-01

Precision and planning are key to reconstructive surgery. Augmented reality (AR) can bring the information within preoperative computed tomography angiography (CTA) imaging to life, allowing the surgeon to 'see through' the patient's skin and appreciate the underlying anatomy without making a single incision. This work has demonstrated that AR can assist the accurate identification, dissection and execution of vascular pedunculated flaps during reconstructive surgery. Separate volumes of osseous, vascular, skin, soft tissue structures and relevant vascular perforators were delineated from preoperative CTA scans to generate three-dimensional images using two complementary segmentation software packages. These were converted to polygonal models and rendered by means of a custom application within the HoloLens™ stereo head-mounted display. Intraoperatively, the models were registered manually to their respective subjects by the operating surgeon using a combination of tracked hand gestures and voice commands; AR was used to aid navigation and accurate dissection. Identification of the subsurface location of vascular perforators through AR overlay was compared to the positions obtained by audible Doppler ultrasound. Through a preliminary HoloLens-assisted case series, the operating surgeon was able to demonstrate precise and efficient localisation of perforating vessels.

Interaction between dimer interface residues of native and mutated SOD1 protein: a theoretical study.

PubMed

Keerthana, S P; Kolandaivel, P

2015-04-01

Cu-Zn superoxide dismutase 1 (SOD1) is a highly conserved bimetallic protein enzyme, used for the scavenging the superoxide radicals (O2 (-)) produced due to aerobic metabolism in the mitochondrial respiratory chain. Over 100 mutations have been identified and found to be in the homodimeric structure of SOD1. The enzyme has to be maintained in its dimeric state for the structural stability and enzymatic activity. From our investigation, we found that the mutations apart from the dimer interface residues are found to affect the dimer stability of protein and hence enhancing the aggregation and misfolding tendency of mutated protein. The homodimeric state of SOD1 is found to be held together by the non-covalent interactions. The molecular dynamics simulation has been used to study the hydrogen bond interactions between the dimer interface residues of the monomers in native and mutated forms of SOD1 in apo- and holo-states. The results obtained by this analysis reveal the fact that the loss of hydrogen bond interactions between the monomers of the dimer is responsible for the reduced stability of the apo- and holo-mutant forms of SOD1. The conformers with dimer interface residues in native and mutated protein obtained by the molecular dynamics simulation is subjected to quantum mechanical study using M052X/6-31G(d) level of theory. The charge transfer between N-H···O interactions in the dimer interface residues were studied. The weak interaction between the monomers of the dimer accounts for the reduced dimerization and enhanced deformation energy in the mutated SOD1 protein.

Biomarkers and Algorithms for the Diagnosis of Vitamin B12 Deficiency.

PubMed

Hannibal, Luciana; Lysne, Vegard; Bjørke-Monsen, Anne-Lise; Behringer, Sidney; Grünert, Sarah C; Spiekerkoetter, Ute; Jacobsen, Donald W; Blom, Henk J

2016-01-01

Vitamin B12 (cobalamin, Cbl, B12) is an indispensable water-soluble micronutrient that serves as a coenzyme for cytosolic methionine synthase (MS) and mitochondrial methylmalonyl-CoA mutase (MCM). Deficiency of Cbl, whether nutritional or due to inborn errors of Cbl metabolism, inactivate MS and MCM leading to the accumulation of homocysteine (Hcy) and methylmalonic acid (MMA), respectively. In conjunction with total B12 and its bioactive protein-bound form, holo-transcobalamin (holo-TC), Hcy, and MMA are the preferred serum biomarkers utilized to determine B12 status. Clinically, vitamin B12 deficiency leads to neurological deterioration and megaloblastic anemia, and, if left untreated, to death. Subclinical vitamin B12 deficiency (usually defined as a total serum B12 of <200 pmol/L) presents asymptomatically or with rather subtle generic symptoms that oftentimes are mistakenly ascribed to unrelated disorders. Numerous studies have now established that serum vitamin B12 has limited diagnostic value as a stand-alone marker. Low serum levels of vitamin B12 not always represent deficiency, and likewise, severe functional deficiency of the micronutrient has been documented in the presence of normal and even high levels of serum vitamin B12. This review discusses the usefulness and limitations of current biomarkers of B12 status in newborn screening, infant and adult diagnostics, the algorithms utilized to diagnose B12 deficiency and unusual findings of vitamin B12 status in various human disorders.

Cyclophilin40 isomerase activity is regulated by a temperature-dependent allosteric interaction with Hsp90.

PubMed

Blackburn, Elizabeth A; Wear, Martin A; Landré, Vivian; Narayan, Vikram; Ning, Jia; Erman, Burak; Ball, Kathryn L; Walkinshaw, Malcolm D

2015-09-01

Cyclophilin 40 (Cyp40) comprises an N-terminal cyclophilin domain with peptidyl-prolyl isomerase (PPIase) activity and a C-terminal tetratricopeptide repeat (TPR) domain that binds to the C-terminal-EEVD sequence common to both heat shock protein 70 (Hsp70) and Hsp90. We show in the present study that binding of peptides containing the MEEVD motif reduces the PPIase activity by ∼30%. CD and fluorescence assays show that the TPR domain is less stable than the cyclophilin domain and is stabilized by peptide binding. Isothermal titration calorimetry (ITC) shows that the affinity for the-MEEVD peptide is temperature sensitive in the physiological temperature range. Results from these biophysical studies fit with the MD simulations of the apo and holo (peptide-bound) structures which show a significant reduction in root mean square (RMS) fluctuation in both TPR and cyclophilin domains when-MEEVD is bound. The MD simulations of the apo-protein also highlight strong anti-correlated motions between residues around the PPIase-active site and a band of residues running across four of the seven helices in the TPR domain. Peptide binding leads to a distortion in the shape of the active site and a significant reduction in these strongly anti-correlated motions, providing an explanation for the allosteric effect of ligand binding and loss of PPIase activity. Together the experimental and MD results suggest that on heat shock, dissociation of Cyp40 from complexes mediated by the TPR domain leads to an increased pool of free Cyp40 capable of acting as an isomerase/chaperone in conditions of cellular stress. © 2015 Authors.

Cyclophilin40 isomerase activity is regulated by a temperature-dependent allosteric interaction with Hsp90

PubMed Central

Blackburn, Elizabeth A.; Wear, Martin A.; Landré, Vivian; Narayan, Vikram; Ning, Jia; Erman, Burak; Ball, Kathryn L.; Walkinshaw, Malcolm D.

2015-01-01

Cyclophilin 40 (Cyp40) comprises an N-terminal cyclophilin domain with peptidyl-prolyl isomerase (PPIase) activity and a C-terminal tetratricopeptide repeat (TPR) domain that binds to the C-terminal–EEVD sequence common to both heat shock protein 70 (Hsp70) and Hsp90. We show in the present study that binding of peptides containing the MEEVD motif reduces the PPIase activity by ∼30%. CD and fluorescence assays show that the TPR domain is less stable than the cyclophilin domain and is stabilized by peptide binding. Isothermal titration calorimetry (ITC) shows that the affinity for the–MEEVD peptide is temperature sensitive in the physiological temperature range. Results from these biophysical studies fit with the MD simulations of the apo and holo (peptide-bound) structures which show a significant reduction in root mean square (RMS) fluctuation in both TPR and cyclophilin domains when–MEEVD is bound. The MD simulations of the apo-protein also highlight strong anti-correlated motions between residues around the PPIase-active site and a band of residues running across four of the seven helices in the TPR domain. Peptide binding leads to a distortion in the shape of the active site and a significant reduction in these strongly anti-correlated motions, providing an explanation for the allosteric effect of ligand binding and loss of PPIase activity. Together the experimental and MD results suggest that on heat shock, dissociation of Cyp40 from complexes mediated by the TPR domain leads to an increased pool of free Cyp40 capable of acting as an isomerase/chaperone in conditions of cellular stress. PMID:26330616

Early biomarker response and patient preferences to oral and intramuscular vitamin B12 substitution in primary care: a randomised parallel-group trial.

PubMed

Metaxas, Corina; Mathis, Deborah; Jeger, Cyrill; Hersberger, Kurt Eduard; Arnet, Isabelle; Walter, Philipp

2017-04-19

Vitamin B12 (VB12) deficiency can be treated with oral high-dose substitution or intramuscular (i.m.) injection of VB12. Whenever alternative routes of administration exist, patient preferences should be considered when choosing the treatment. We aimed to assess outpatient preferences towards oral or IM VB12 substitution and confirm noninferiority of early biomarker response with oral treatment, in a typical primary care population. Prospective randomised nonblinded parallel-group trial. Patients were recruited by their general practitioner and randomly assigned to oral or IM treatment. Group O-oral was given 28 tablets of 1000 µg cyanocobalamin in a monthly punch card fitted with an electronic monitoring system. Group I-IM received four, weekly injections of 1000 µg hydroxocobalamin. Blood samples were drawn before the first administration and after 1, 2 and 4 weeks of treatment, and analysed for VB12, holotranscobalamin (HoloTc), homocysteine (Hcy) and methylmalonic acid (MMA). For group O-oral, treatment adher-ence and percentage of days with 2 dosing events were calcu-lated. Before and after 28 days of treatment, patients were asked to fill in a questionnaire about their preference for the therapy options and associated factors. Between November 2013 and December 2015, 37 patients (age: 49.5 ± 18.5 years; women: 60.5%) were recruited for oral (19) or IM (18) treatment. Baseline values with 95% confidence intervals for serum VB12, HoloTc, Hcy and MMA were 158 pmol/l [145-172], 49.0 pmol/l [40.4-57.5], 14.8 µmol/l [12.0-17.7] and 304 nmol/l [219-390], respective-ly, in group O-oral and 164 pmol/l [154-174], 50.1 pmol/l [38.7-61.6], 13.0 µmol/l [11.0-15.1] and 321 nmol/l [215-427], respectively, in group I-IM (not significant). After 1 month of treatment, levels of VB12 and HoloTc showed a significant increase compared with baseline (group O-oral: VB12 354 pmol/l [298-410] and HoloTc 156 pmol/l [116-196]; group I-IM: VB12 2796 pmol/l [1277-4314] and HoloTc 1269 pmol/l [103-2435]). Hcy and MMA levels showed a significant decrease compared with baseline (group O-oral: Hcy 13.8 µmol/l [10.7-16.8] and MMA 168 nmol/l [134-202]; group I-IM: Hcy 8.5 µmol/l [7.1-9.8] and MMA 156 nmol/l [121-190]). HoloTc and MMA levels were normalised in all patients after 4 weeks of treatment, whereas normalisation of VB12 and Hcy was reached by all patients in group I-IM only. Response of VB12, HoloTc and Hcy was more pronounced in group I-IM (p <0.01) and the primary hypothesis that oral VB12 treatment would be noninfe-rior to IM treatment was rejected. Average adherence to thera-py was 99.6 ± 1.1% and days with 2 dosing events reached 5.6%. Before randomisation, preference was in favour of oral treatment (45.9%, n = 17) over IM administration (21.6%, n = 8). Twelve patients (32.4%) had no preference. Nine (24.3%) patients changed their preference after treatment. Patients who obtained their preferred route of administration main-tained their preference in the case of oral treatment and changed their preference after IM treatment. Differences in VB12 levels between groups were higher than expected. Therefore, noninferiority of oral treat-ment had to be rejected. However, normalisation of HoloTc and MMA was reached by all patients after a 1-month treatment period. The clinical benefit of the exaggerated biomarker re-sponse after IM treatment within a typical primary care popula-tion is questionable. Midterm biomarker effects and patient preferences should be considered when a therapeutic scheme is chosen. Initial rating in favour of either IM or oral therapy can change over time and justifies repeated re-evaluation of patient preferences. (ClinicalTrials.gov ID NCT01832129).

MgATP hydrolysis destabilizes the interaction between subunit H and yeast V1-ATPase, highlighting H's role in V-ATPase regulation by reversible disassembly.

PubMed

Sharma, Stuti; Oot, Rebecca A; Wilkens, Stephan

2018-05-12

Vacuolar H+-ATPases (V-ATPases; V1Vo-ATPases) are rotary motor proton pumps that acidify intracellular compartments and in some tissues, the extracellular space. V-ATPase is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel sectors. An important player in V-ATPase regulation is subunit H, which binds at the interface of V1 and Vo. H is required for MgATPase activity in holo V-ATPase, but also for stabilizing the MgADP inhibited state in membrane detached V1. However, how H fulfills these two functions is poorly understood. To characterize the H-V1 interaction and its role in reversible disassembly, we determined binding affinities of full length H and its N-terminal domain (HNT) for an isolated heterodimer of subunits E and G (EG), the N-terminal domain of subunit a (aNT), and V1 lacking subunit H (V1ΔH). Using isothermal titration calorimetry (ITC) and biolayer interferometry (BLI), we show that HNT binds EG with moderate affinity, that full length H binds aNT weakly, and that both H and HNT bind V1ΔH with high affinity. We also found that only one molecule of HNT binds V1ΔH with high affinity, suggesting conformational asymmetry of the three EG heterodimers in V1ΔH. Moreover, MgATP hydrolysis-driven conformational changes in V1 destabilized the interaction of H, or HNT, with V1ΔH, suggesting an interplay between MgADP inhibition and subunit H. Our observation that H binding is affected by MgATP hydrolysis in V1 points to H's role in the mechanism of reversible disassembly. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Calcium-dependent disorder-to-order transitions are central to the secretion and folding of the CyaA toxin of Bordetella pertussis, the causative agent of whooping cough.

PubMed

O'Brien, Darragh P; Perez, Ana Cristina Sotomayor; Karst, Johanna; Cannella, Sara E; Enguéné, Véronique Yvette Ntsogo; Hessel, Audrey; Raoux-Barbot, Dorothée; Voegele, Alexis; Subrini, Orso; Davi, Marilyne; Guijarro, J Inaki; Raynal, Bertrand; Baron, Bruno; England, Patrick; Hernandez, Belen; Ghomi, Mahmoud; Hourdel, Véronique; Malosse, Christian; Chamot-Rooke, Julia; Vachette, Patrice; Durand, Dominique; Brier, Sébastien; Ladant, Daniel; Chenal, Alexandre

2018-07-01

The adenylate cyclase toxin (CyaA) plays an essential role in the early stages of respiratory tract colonization by Bordetella pertussis, the causative agent of whooping cough. Once secreted, CyaA invades eukaryotic cells, leading to cell death. The cell intoxication process involves a unique mechanism of translocation of the CyaA catalytic domain directly across the plasma membrane of the target cell. Herein, we review our recent results describing how calcium is involved in several steps of this intoxication process. In conditions mimicking the low calcium environment of the crowded bacterial cytosol, we show that the C-terminal, calcium-binding Repeat-in-ToXin (RTX) domain of CyaA, RD, is an extended, intrinsically disordered polypeptide chain with a significant level of local, secondary structure elements, appropriately sized for transport through the narrow channel of the secretion system. Upon secretion, the high calcium concentration in the extracellular milieu induces the refolding of RD, which likely acts as a scaffold to favor the refolding of the upstream domains of the full-length protein. Due to the presence of hydrophobic regions, CyaA is prone to aggregate into multimeric forms in vitro, in the absence of a chaotropic agent. We have recently defined the experimental conditions required for CyaA folding, comprising both calcium binding and molecular confinement. These parameters are critical for CyaA folding into a stable, monomeric and functional form. The monomeric, calcium-loaded (holo) toxin exhibits efficient liposome permeabilization and hemolytic activities in vitro, even in a fully calcium-free environment. By contrast, the toxin requires sub-millimolar calcium concentrations in solution to translocate its catalytic domain across the plasma membrane, indicating that free calcium in solution is actively involved in the CyaA toxin translocation process. Overall, this data demonstrates the remarkable adaptation of bacterial RTX toxins to the diversity of calcium concentrations it is exposed to in the successive environments encountered in the course of the intoxication process. Copyright © 2018 Elsevier Ltd. All rights reserved.

Glycolipid Acquisition by Human Glycolipid Transfer Protein Dramatically Alters Intrinsic Tryptophan Fluorescence

PubMed Central

Zhai, Xiuhong; Malakhova, Margarita L.; Pike, Helen M.; Benson, Linda M.; Bergen, H. Robert; Sugár, István P.; Malinina, Lucy; Patel, Dinshaw J.; Brown, Rhoderick E.

2009-01-01

Glycolipid transfer proteins (GLTPs) are small, soluble proteins that selectively accelerate the intermembrane transfer of glycolipids. The GLTP fold is conformationally unique among lipid binding/transfer proteins and serves as the prototype and founding member of the new GLTP superfamily. In the present study, changes in human GLTP tryptophan fluorescence, induced by membrane vesicles containing glycolipid, are shown to reflect glycolipid binding when vesicle concentrations are low. Characterization of the glycolipid-induced “signature response,” i.e. ∼40% decrease in Trp intensity and ∼12-nm blue shift in emission wavelength maximum, involved various modes of glycolipid presentation, i.e. microinjection/dilution of lipid-ethanol solutions or phosphatidylcholine vesicles, prepared by sonication or extrusion and containing embedded glycolipids. High resolution x-ray structures of apo- and holo-GLTP indicate that major conformational alterations are not responsible for the glycolipid-induced GLTP signature response. Instead, glycolipid binding alters the local environment of Trp-96, which accounts for ∼70% of total emission intensity of three Trp residues in GLTP and provides a stacking platform that aids formation of a hydrogen bond network with the ceramide-linked sugar of the glycolipid headgroup. The changes in Trp signal were used to quantitatively assess human GLTP binding affinity for various lipids including glycolipids containing different sugar headgroups and homogenous acyl chains. The presence of the glycolipid acyl chain and at least one sugar were essential for achieving a low-to-submicromolar dissociation constant that was only slightly altered by increased sugar headgroup complexity. PMID:19270338

Bacillus subtilis δ Factor Functions as a Transcriptional Regulator by Facilitating the Open Complex Formation.

PubMed

Prajapati, Ranjit Kumar; Sengupta, Shreya; Rudra, Paulami; Mukhopadhyay, Jayanta

2016-01-15

Most bacterial RNA polymerases (RNAP) contain five conserved subunits, viz. 2α, β, β', and ω. However, in many Gram-positive bacteria, especially in fermicutes, RNAP is associated with an additional factor, called δ. For over three decades since its identification, it had been thought that δ functioned as a subunit of RNAP to enhance the level of transcripts by recycling RNAP. In support of the previous observations, we also find that δ is involved in recycling of RNAP by releasing the RNA from the ternary complex. We further show that δ binds to RNA and is able to recycle RNAP when the length of the nascent RNA reaches a critical length. However, in this work we decipher a new function of δ. Performing biochemical and mutational analysis, we show that Bacillus subtilis δ binds to DNA immediately upstream of the promoter element at A-rich sequences on the abrB and rrnB1 promoters and facilitates open complex formation. As a result, δ facilitates RNAP to initiate transcription in the second scale, compared with minute scale in the absence of δ. Using transcription assay, we show that δ-mediated recycling of RNAP cannot be the sole reason for the enhancement of transcript yield. Our observation that δ does not bind to RNAP holo enzyme but is required to bind to DNA upstream of the -35 promoter element for transcription activation suggests that δ functions as a transcriptional regulator. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Botulinum Neurotoxins and Botulism: A Novel Therapeutic Approach

PubMed Central

Thanongsaksrikul, Jeeraphong; Chaicumpa, Wanpen

2011-01-01

Specific treatment is not available for human botulism. Current remedial mainstay is the passive administration of polyclonal antibody to botulinum neurotoxin (BoNT) derived from heterologous species (immunized animal or mouse hybridoma) together with supportive and symptomatic management. The antibody works extracellularly, probably by blocking the binding of receptor binding (R) domain to the neuronal receptors; thus inhibiting cellular entry of the holo-BoNT. The antibody cannot neutralize the intracellular toxin. Moreover, a conventional antibody with relatively large molecular size (150 kDa) is not accessible to the enzymatic groove and, thus, cannot directly inhibit the BoNT zinc metalloprotease activity. Recently, a 15–20 kDa single domain antibody (VHH) that binds specifically to light chain of BoNT serotype A was produced from a humanized-camel VH/VHH phage display library. The VHH has high sequence homology (>80%) to the human VH and could block the enzymatic activity of the BoNT. Molecular docking revealed not only the interface binding between the VHH and the toxin but also an insertion of the VHH CDR3 into the toxin enzymatic pocket. It is envisaged that, by molecular linking the VHH to a cell penetrating peptide (CPP), the CPP-VHH fusion protein would be able to traverse the hydrophobic cell membrane into the cytoplasm and inhibit the intracellular BoNT. This presents a novel and safe immunotherapeutic strategy for botulism by using a cell penetrating, humanized-single domain antibody that inhibits the BoNT by means of a direct blockade of the groove of the menace enzyme. PMID:22069720

The delivery of copper for thylakoid import observed by NMR

PubMed Central

Banci, Lucia; Bertini, Ivano; Ciofi-Baffoni, Simone; Kandias, Nikolaos G.; Robinson, Nigel J.; Spyroulias, Georgios A.; Su, Xun-Cheng; Tottey, Stephen; Vanarotti, Murugendra

2006-01-01

The thylakoid compartments of plant chloroplasts are a vital destination for copper. Copper is needed to form holo-plastocyanin, which must shuttle electrons between photosystems to convert light into biologically useful chemical energy. Copper can bind tightly to proteins, so it has been hypothesized that copper partitions onto ligand-exchange pathways to reach intracellular locations without inflicting damage en route. The copper metallochaperone Atx1 of chloroplast-related cyanobacteria (ScAtx1) engages in bacterial two-hybrid interactions with N-terminal domains of copper-transporting ATPases CtaA (cell import) and PacS (thylakoid import). Here we visualize copper delivery. The N-terminal domain PacSN has a ferredoxin-like fold that forms copper-dependent heterodimers with ScAtx1. Removal of copper, by the addition of the cuprous-ion chelator bathocuproine disulfonate, disrupts this heterodimer, as shown from a reduction of the overall tumbling rate of the protein mixture. The NMR spectral changes of the heterodimer versus the separate proteins reveal that loops 1, 3, and 5 (the carboxyl tail) of the ScAtx1 Cu(I) site switch to an apo-like configuration in the heterodimer. NMR data (2JNH couplings in the imidazole ring of 15N ScAtx1 His-61) also show that His-61, bound to copper(I) in [Cu(I)ScAtx1]2, is not coordinated to copper in the heterodimer. A model for the PacSN/Cu(I)/ScAtx1 complex is presented. Contact with PacSN induces change to the ScAtx1 copper-coordination sphere that drives copper release for thylakoid import. These data also elaborate on the mechanism to keep copper(I) out of the ZiaAN ATPase zinc sites. PMID:16707580

Spectral Analysis: From Additive Perspective to Multiplicative Perspective

NASA Astrophysics Data System (ADS)

Wu, Z.

2017-12-01

The early usage of trigonometric functions can be traced back to at least 17th century BC. It was Bhaskara II of the 12th century CE who first proved the mathematical equivalence between the sum of two trigonometric functions of any given angles and the product of two trigonometric functions of related angles, which has been taught these days in middle school classroom. The additive perspective of trigonometric functions led to the development of the Fourier transform that is used to express any functions as the sum of a set of trigonometric functions and opened a new mathematical field called harmonic analysis. Unfortunately, Fourier's sum cannot directly express nonlinear interactions between trigonometric components of different periods, and thereby lacking the capability of quantifying nonlinear interactions in dynamical systems. In this talk, the speaker will introduce the Huang transform and Holo-spectrum which were pioneered by Norden Huang and emphasizes the multiplicative perspective of trigonometric functions in expressing any function. Holo-spectrum is a multi-dimensional spectral expression of a time series that explicitly identifies the interactions among different scales and quantifies nonlinear interactions hidden in a time series. Along with this introduction, the developing concepts of physical, rather than mathematical, analysis of data will be explained. Various enlightening applications of Holo-spectrum analysis in atmospheric and climate studies will also be presented.

Update on the Native American Holo-Portrait series

NASA Astrophysics Data System (ADS)

Diamond, Mark C.

1995-02-01

In March of 1990 I filmed the first in the Native American Holo-Portrait series which I showed video of at the last Lake Forest Symposium. Since then I have done several additional film shoots with almost 40 different holo-portraits currently in the can. With around 500 Native American nations within the northern hemisphere the prospect of filming one hologram per nation looks like a multi-decade project. All 35 mm cine has been shot to date in color on extremely fine grain T formula emulsion stocks. Some of the footage has been shot using the rotating subject configuration and others with the laterally translating camera configuration. In the future I may film all subjects in both formats if time allows. Other images have been shot at reservations and pow-wows. All location work is accomplished with lighting and generators on hand because of questionable power availability at some of the sites. I have received a favorable response for potential support from the Smithsonian's Native American Museum now under construction on the mall next to the Air & Space Museum in Washington, D.C. This will be a fine display space for these portraits. However, I am planning to produce editions in several formats in order to allow for global distribution of these images.

Binding of Pseudomonas aeruginosa Apo-Bacterioferritin Associated Ferredoxin to Bacterioferritin B Promotes Heme Mediation of Electron Delivery and Mobilization of Core Mineral Iron†

PubMed Central

Weeratunga, Saroja K.; Gee, Casey E.; Lovell, Scott; Zeng, Yuhong; Woodin, Carrie L.; Rivera, Mario

2009-01-01

The bfrB gene from Pseudomonas aeruginosa was cloned and expressed in E. coli. The resultant protein (BfrB), which assembles into a 445.3 kDa complex0020from 24 identical subunits, binds 12 molecules of heme axially coordinated by two Met residues. BfrB, isolated with 5–10 iron atoms per protein molecule, was reconstituted with ferrous ions to prepare samples with a core mineral containing 600 ± 40 ferric ions per BfrB molecule and approximately one phosphate molecule per iron atom. In the presence of sodium dithionite or in the presence of P. aeruginosa ferredoxin NADP reductase (FPR) and NADPH the heme in BfrB remains oxidized and the core iron mineral is mobilized sluggishly. In stark contrast, addition of NADPH to a solution containing BfrB, FPR and the apo-form of P. aeruginosa bacterioferritin associated ferredoxin (apo-Bfd) results in rapid reduction of the heme in BfrB and in the efficient mobilization of the core iron mineral. Results from additional experimentation indicate that Bfd must bind to BfrB to promote heme mediation of electrons from the surface to the core to support the efficient mobilization of ferrous ions from BfrB. In this context, the thus far mysterious role of heme in bacterioferritins has been brought to the front by reconstituting BfrB with its physiological partner, apo-Bfd. These findings are discussed in the context of a model for the utilization of stored iron in which the significant upregulation of the bfd gene under low-iron conditions [Ochsner, U.A., Wilderman, P.J., Vasil, A.I., and Vasil, M.L. (2002) Mol. Microbiol. 45, 1277–1287] ensures sufficient concentrations of apo-Bfd to bind BfrB and unlock the iron stored in its core. Although these findings are in contrast to previous speculations suggesting redox mediation of electron transfer by holo-Bfd, the ability of apo-Bfd to promote iron mobilization is an economical strategy used by the cell because it obviates the need to further deplete cellular iron levels to assemble iron sulfur clusters in Bfd before the iron stored in BfrB can be mobilized and utilized. PMID:19575528

Vitamin B12 and folate levels in healthy Swiss senior citizens: a prospective study evaluating reference intervals and decision limits.

PubMed

Risch, Martin; Meier, Dominik W; Sakem, Benjamin; Medina Escobar, Pedro; Risch, Corina; Nydegger, Urs; Risch, Lorenz

2015-07-11

The vitamin B12 and folate status in nonanaemic healthy older persons needs attention the more so as decrease in levels may be anticipated from reduced haematinic provision and/or impaired intestinal uptake. A total of 1143 subjectively healthy Swiss midlands participants (637 females and 506 males), ≥60 years of age were included in this study. Levels of vitamin B12, holotranscobalamin (holoTC), methylmalonic acid (MMA), homocysteine (Hcy), serum folate, red blood cell (RBC) folate were measured. Further, Fedosov's wellness score was determined. Associations of age, gender, and cystatin C/creatinine-based estimated kidney function, with the investigated parameters were assessed. Reference intervals were calculated. Further, ROC analysis was done to assess accuracy of the individual parameters in recognizing a deficient vitamin B12 status. Finally, decision limits for sensitive, specific and optimal recognition of vitamin B12 status with individual parameters were derived. Three age groups: 60-69, 70-79 and ≥ 80 had median B12 (pmol/L) levels of 237, 228 and 231 respectively (p = 0.22), holoTC (pmol/L) of 52, 546 and 52 (p = 0.60) but Hcy (μmol/L) 12, 15 and 16 (p < 0.001), MMA (nmol/L) 207, 221 and 244 (p < 0.001). Hcy and MMA (both p < 0.001), but not holoTC (p = 0.12) and vitamin B12 (p = 0.44) were found to be affected by kidney function. In a linear regression model Fedosov's wellness score was independently associated with kidney function (p < 0.001) but not with age. Total serum folate and red blood cell (RBC) folate drift apart with increasing age: whereas the former decreases (p = 0.01) RBC folate remains in the same bandwidth across all age groups (p = 0.12) A common reference interval combining age and gender strata can be obtained for vitamin B12 and holoTC, whereas a more differentiated approach seems warranted for serum folate and RBC folate. Whereas the vitamin B12 and holoTC levels remain steady after 60 years of age, we observed a significant increment in MMA levels accompanied by increments in Hcy; this is better explained by age-related reduced kidney function than by vitamin B12 insufficiency. Total serum folate levels but not RBC folate levels decreased with progressing age.

Biomarkers and Algorithms for the Diagnosis of Vitamin B12 Deficiency

PubMed Central

Hannibal, Luciana; Lysne, Vegard; Bjørke-Monsen, Anne-Lise; Behringer, Sidney; Grünert, Sarah C.; Spiekerkoetter, Ute; Jacobsen, Donald W.; Blom, Henk J.

2016-01-01

Vitamin B12 (cobalamin, Cbl, B12) is an indispensable water-soluble micronutrient that serves as a coenzyme for cytosolic methionine synthase (MS) and mitochondrial methylmalonyl-CoA mutase (MCM). Deficiency of Cbl, whether nutritional or due to inborn errors of Cbl metabolism, inactivate MS and MCM leading to the accumulation of homocysteine (Hcy) and methylmalonic acid (MMA), respectively. In conjunction with total B12 and its bioactive protein-bound form, holo-transcobalamin (holo-TC), Hcy, and MMA are the preferred serum biomarkers utilized to determine B12 status. Clinically, vitamin B12 deficiency leads to neurological deterioration and megaloblastic anemia, and, if left untreated, to death. Subclinical vitamin B12 deficiency (usually defined as a total serum B12 of <200 pmol/L) presents asymptomatically or with rather subtle generic symptoms that oftentimes are mistakenly ascribed to unrelated disorders. Numerous studies have now established that serum vitamin B12 has limited diagnostic value as a stand-alone marker. Low serum levels of vitamin B12 not always represent deficiency, and likewise, severe functional deficiency of the micronutrient has been documented in the presence of normal and even high levels of serum vitamin B12. This review discusses the usefulness and limitations of current biomarkers of B12 status in newborn screening, infant and adult diagnostics, the algorithms utilized to diagnose B12 deficiency and unusual findings of vitamin B12 status in various human disorders. PMID:27446930

Structure at 1.3 A resolution of Rhodothermus marinus caa(3) cytochrome c domain.

PubMed

Srinivasan, Vasundara; Rajendran, Chitra; Sousa, Filipa L; Melo, Ana M P; Saraiva, Lígia M; Pereira, Manuela M; Santana, Margarida; Teixeira, Miguel; Michel, Hartmut

2005-02-04

The cytochrome c domain of subunit II from the Rhodothermus marinus caa(3) HiPIP:oxygen oxidoreductase, a member of the superfamily of heme-copper-containing terminal oxidases, was produced in Escherichia coli and characterised. The recombinant protein, which shows the same optical absorption and redox properties as the corresponding domain in the holo enzyme, was crystallized and its structure was determined to a resolution of 1.3 A by the multiwavelength anomalous dispersion (MAD) technique using the anomalous dispersion of the heme iron atom. The model was refined to final R(cryst) and R(free) values of 13.9% and 16.7%, respectively. The structure reveals the insertion of two short antiparallel beta-strands forming a small beta-sheet, an interesting variation of the classical all alpha-helical cytochrome c fold. This modification appears to be common to all known caa(3)-type terminal oxidases, as judged by comparative modelling and by analyses of the available amino acid sequences for these enzymes. This is the first high-resolution crystal structure reported for a cytochrome c domain of a caa(3)-type terminal oxidase. The R.marinus caa(3) uses HiPIP as the redox partner. The calculation of the electrostatic potential at the molecular surface of this extra C-terminal domain provides insights into the binding to its redox partner on one side and its interaction with the remaining subunit II on the other side.

Structural Model for the Flip-Flop Action in Thiamin Pyrophosphate-Dependent Human Pyruvate Dehydrogenase

NASA Technical Reports Server (NTRS)

Ciszak, Ewa; Dominiak, Paulina

2003-01-01

The derivative of vitamin B1 thiamin pyrophosphate (TPP) is a cofactor of enzymes performing catalysis in pathways of energy production, including (i) decarboxylation of alpha-keto acids followed by (ii) transketolation. These enzymes have shown a common mechanism of TPP activation by imposing an active V-conformation of this coenzyme that brings the N4 atom of the aminopyrimidine ring to the distance required for the intramolecular C-H N hydrogen-bonding with the C2- atom of the thiazolium ring. The reactive C2 atom of TPP is the nucleophile that attacks the carbonyl carbon of different substrates used by the TPP-dependent enzymes. The structure of the heterotetrameric human pyruvate dehydrogenase (Elp) recently determined in our laboratory (1) revealed the association pattern of the subunits and the specifics of two chemically equivalent cofactor binding sites. Dynamic nonequivalence of these two cofactor sites directs the flip-flop action of this enzyme, depending upon which two active sites effect each other (2). The crystal structure derived from the holo-form of Elp provided the basis for the model of the flip-flop action of Elp in which different steps of the catalytic reaction are performed in each of the two cofactor sites at any given moment, where these steps are governed by the concerted shuttle-like motion of the subunits. It is further proposed that balancing a hydrogen-bond network and related cofactor geometry determine the continuity of catalytic events.

Role of Dynamics in the Autoinhibition and Activation of the Hyperpolarization-activated Cyclic Nucleotide-modulated (HCN) Ion Channels*♦

PubMed Central

VanSchouwen, Bryan; Akimoto, Madoka; Sayadi, Maryam; Fogolari, Federico; Melacini, Giuseppe

2015-01-01

The hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels control rhythmicity in neurons and cardiomyocytes. Cyclic AMP allosterically modulates HCN through the cAMP-dependent formation of a tetrameric gating ring spanning the intracellular region (IR) of HCN, to which cAMP binds. Although the apo versus holo conformational changes of the cAMP-binding domain (CBD) have been previously mapped, only limited information is currently available on the HCN IR dynamics, which have been hypothesized to play a critical role in the cAMP-dependent gating of HCN. Here, using molecular dynamics simulations validated and complemented by experimental NMR and CD data, we comparatively analyze HCN IR dynamics in the four states of the thermodynamic cycle arising from the coupling between cAMP binding and tetramerization equilibria. This extensive set of molecular dynamics trajectories captures the active-to-inactive transition that had remained elusive for other CBDs, and it provides unprecedented insight on the role of IR dynamics in HCN autoinhibition and its release by cAMP. Specifically, the IR tetramerization domain becomes more flexible in the monomeric states, removing steric clashes that the apo-CDB structure would otherwise impose. Furthermore, the simulations reveal that the active/inactive structural transition for the apo-monomeric CBD occurs through a manifold of pathways that are more divergent than previously anticipated. Upon cAMP binding, these pathways become disallowed, pre-confining the CBD conformational ensemble to a tetramer-compatible state. This conformational confinement primes the IR for tetramerization and thus provides a model of how cAMP controls HCN channel gating. PMID:25944904

Interaction between NADH and electron-transferring flavoprotein from Megasphaera elsdenii.

PubMed

Sato, Kyosuke; Nishina, Yasuzo; Shiga, Kiyoshi

2013-06-01

Electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii is a heterodimer containing two FAD cofactors. Isolated ETF contains only one FAD molecule, FAD-1, because the other, FAD-2, is lost during purification. FAD-2 is recovered by adding FAD to the isolated ETF. The two FAD molecules in holoETF were characterized using NADH. Spectrophotometric titration of isolated ETF with NADH showed a two-electron reduction of FAD-1 according to a monophasic profile indicating that FAD-1 receives electrons from NADH without involvement of FAD-2. When holoETF was titrated with NADH, FAD-2 was reduced to an anionic semiquinone and then was fully reduced before the reduction of FAD-1. The midpoint potential values at pH 7 were +81, -136 and -279 mV for the reduction of oxidized FAD-2 to semiquinone, semiquinone to the fully reduced FAD-2 and the two-electron reduction of FAD-1, respectively. Both FAD-1 and FAD-2 in holoETF were reduced by excess NADH very rapidly. The reduction of FAD-2 was slowed by replacement of FAD-1 with 8-cyano-FAD indicating that FAD-2 receives electrons from FAD-1 but not from NADH directly. The present results suggest that FAD-2 is the counterpart of the FAD in human ETF, which contains one FAD and one AMP.

Characterization of protein phosphatase 2A acting on phosphorylated plasma membrane aquaporin of tulip petals.

PubMed

Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

2004-05-01

A protein phosphatase holo-type enzyme (38, 65, and 75 kDa) preparation and a free catalytic subunit (38 kDa) purified from tulip petals were characterized as protein phosphatase 2A (PP2A) by immunological and biochemical approaches. The plasma membrane containing the putative plasma membrane aquaporin (PM-AQP) was prepared from tulip petals, phosphorylated in vitro, and used as the substrate for both of the purified PP2A preparations. Although both preparations dephosphorylated the phosphorylated PM-AQP at 20 degrees C, only the holo-type enzyme preparation acted at 5 degrees C on the phosphorylated PM-AQP with higher substrate specificity, suggesting that regulatory subunits are required for low temperature-dependent dephosphorylation of PM-AQP in tulip petals.

Ga3+ as a mechanistic probe in Fe3+ transport: characterization of Ga3+ interaction with FbpA.

PubMed

Weaver, Katherine D; Heymann, Jared J; Mehta, Arnav; Roulhac, Petra L; Anderson, Damon S; Nowalk, Andrew J; Adhikari, Pratima; Mietzner, Timothy A; Fitzgerald, Michael C; Crumbliss, Alvin L

2008-08-01

The obligate human pathogens Haemophilus influenzae, Neisseria gonorrhoeae, and N. meningitidis utilize a highly conserved, three-protein ATP-binding cassette transporter (FbpABC) to shuttle free Fe(3+) from the periplasm and across the cytoplasmic membrane. The periplasmic binding protein, ferric binding protein (FbpA), is capable of transporting other trivalent cations, including Ga(3+), which, unlike Fe(3+), is not redox-active. Because of a similar size and charge as Fe(3+), Ga(3+) is widely used as a non-redox-active Fe(3+) substitute for studying metal complexation in proteins and bacterial populations. The investigations reported here elucidate the similarities and differences in FbpA sequestration of Ga(3+) and Fe(3+), focusing on metal selectivity and the resulting transport function. The thermodynamic binding constant for Ga(3+) complexed with FbpA at pH 6.5, in 50 mM 4-morpholineethanesulfonic acid, 200 mM KCl, 5 mM KH(2)PO(4) was determined by UV-difference spectroscopy as log K'eff=13.7+/-0.6. This represents a 10(5)-fold weaker binding relative to Fe(3+) at identical conditions. The unfolding/refolding behavior of Ga(3+) and Fe(3+) holo-FbpA were also studied using a matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy technique, stability of unpurified proteins from rates of H/D exchange (SUPREX). This analysis indicates significant differences between Fe(3+) and Ga(3+) sequestration with regard to protein folding behavior. A series of kinetic experiments established the lability of the Ga(3+)FbpA-PO(4) assembly, and the similarities/differences of stepwise loading of Fe(3+) into apo- or Ga(3+)-loaded FbpA. These biophysical characterization data are used to interpret FbpA-mediated Ga(3+) transport and toxicity in cell culture studies.

P2X4 Receptor in Silico and Electrophysiological Approaches Reveal Insights of Ivermectin and Zinc Allosteric Modulation

PubMed Central

Latapiat, Verónica; Rodríguez, Felipe E.; Godoy, Francisca; Montenegro, Felipe A.; Barrera, Nelson P.; Huidobro-Toro, Juan P.

2017-01-01

Protein allosteric modulation is a pillar of metabolic regulatory mechanisms; this concept has been extended to include ion channel regulation. P2XRs are ligand-gated channels activated by extracellular ATP, sensitive to trace metals and other chemicals. By combining in silico calculations with electrophysiological recordings, we investigated the molecular basis of P2X4R modulation by Zn(II) and ivermectin, an antiparasite drug currently used in veterinary medicine. To this aim, docking studies, molecular dynamics simulations and non-bonded energy calculations for the P2X4R in the apo and holo states or in the presence of ivermectin and/or Zn(II) were accomplished. Based on the crystallized Danio rerio P2X4R, the rat P2X4R, P2X2R, and P2X7R structures were modeled, to determine ivermectin binding localization. Calculations revealed that its allosteric site is restricted to transmembrane domains of the P2X4R; the role of Y42 and W46 plus S341 and non-polar residues were revealed as essential, and are not present in the homologous P2X2R or P2X7R transmembrane domains. This finding was confirmed by preferential binding conformations and electrophysiological data, revealing P2X4R modulator specificity. Zn(II) acts in the P2X4R extracellular domain neighboring the SS3 bridge. Molecular dynamics in the different P2X4R states revealed allosterism-induced stability. Pore and lateral fenestration measurements of the P2X4R showed conformational changes in the presence of both modulators compatible with a larger opening of the extracellular vestibule. Electrophysiological studies demonstrated additive effects in the ATP-gated currents by joint applications of ivermectin plus Zn(II). The C132A P2X4R mutant was insensitive to Zn(II); but IVM caused a 4.9 ± 0.7-fold increase in the ATP-evoked currents. Likewise, the simultaneous application of both modulators elicited a 7.1 ± 1.7-fold increase in the ATP-gated current. Moreover, the C126A P2X4R mutant evoked similar ATP-gated currents comparable to those of wild-type P2X4R. Finally, a P2X4/2R chimera did not respond to IVM but Zn(II) elicited a 2.7 ± 0.6-fold increase in the ATP-gated current. The application of IVM plus Zn(II) evoked a 2.7 ± 0.9-fold increase in the ATP-gated currents. In summary, allosteric modulators caused additive ATP-gated currents; consistent with lateral fenestration enlargement. Energy calculations demonstrated a favorable transition of the holo receptor state following both allosteric modulators binding, as expected for allosteric interactions. PMID:29326590

Visualizing UAS-collected imagery using augmented reality

NASA Astrophysics Data System (ADS)

Conover, Damon M.; Beidleman, Brittany; McAlinden, Ryan; Borel-Donohue, Christoph C.

2017-05-01

One of the areas where augmented reality will have an impact is in the visualization of 3-D data. 3-D data has traditionally been viewed on a 2-D screen, which has limited its utility. Augmented reality head-mounted displays, such as the Microsoft HoloLens, make it possible to view 3-D data overlaid on the real world. This allows a user to view and interact with the data in ways similar to how they would interact with a physical 3-D object, such as moving, rotating, or walking around it. A type of 3-D data that is particularly useful for military applications is geo-specific 3-D terrain data, and the visualization of this data is critical for training, mission planning, intelligence, and improved situational awareness. Advances in Unmanned Aerial Systems (UAS), photogrammetry software, and rendering hardware have drastically reduced the technological and financial obstacles in collecting aerial imagery and in generating 3-D terrain maps from that imagery. Because of this, there is an increased need to develop new tools for the exploitation of 3-D data. We will demonstrate how the HoloLens can be used as a tool for visualizing 3-D terrain data. We will describe: 1) how UAScollected imagery is used to create 3-D terrain maps, 2) how those maps are deployed to the HoloLens, 3) how a user can view and manipulate the maps, and 4) how multiple users can view the same virtual 3-D object at the same time.

Tracking solvent and protein movement during CO2 release in carbonic anhydrase II crystals

PubMed Central

Kim, Chae Un; Song, HyoJin; Avvaru, Balendu Sankara; Gruner, Sol M.; Park, SangYoun; McKenna, Robert

2016-01-01

Carbonic anhydrases are mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO2/HCO3−. Previously, the X-ray crystal structures of CO2-bound holo (zinc-bound) and apo (zinc-free) human carbonic anhydrase IIs (hCA IIs) were captured at high resolution. Here, we present sequential timeframe structures of holo- [T = 0 s (CO2-bound), 50 s, 3 min, 10 min, 25 min, and 1 h] and apo-hCA IIs [T = 0 s, 50 s, 3 min, and 10 min] during the “slow” release of CO2. Two active site waters, WDW (deep water) and WDW′ (this study), replace the vacated space created on CO2 release, and another water, WI (intermediate water), is seen to translocate to the proton wire position W1. In addition, on the rim of the active site pocket, a water W2′ (this study), in close proximity to residue His64 and W2, gradually exits the active site, whereas His64 concurrently rotates from pointing away (“out”) to pointing toward (“in”) active site rotameric conformation. This study provides for the first time, to our knowledge, structural “snapshots” of hCA II intermediate states during the formation of the His64-mediated proton wire that is induced as CO2 is released. Comparison of the holo- and apo-hCA II structures shows that the solvent network rearrangements require the presence of the zinc ion. PMID:27114542

Augmented Reality as a Telemedicine Platform for Remote Procedural Training.

PubMed

Wang, Shiyao; Parsons, Michael; Stone-McLean, Jordan; Rogers, Peter; Boyd, Sarah; Hoover, Kristopher; Meruvia-Pastor, Oscar; Gong, Minglun; Smith, Andrew

2017-10-10

Traditionally, rural areas in many countries are limited by a lack of access to health care due to the inherent challenges associated with recruitment and retention of healthcare professionals. Telemedicine, which uses communication technology to deliver medical services over distance, is an economical and potentially effective way to address this problem. In this research, we develop a new telepresence application using an Augmented Reality (AR) system. We explore the use of the Microsoft HoloLens to facilitate and enhance remote medical training. Intrinsic advantages of AR systems enable remote learners to perform complex medical procedures such as Point of Care Ultrasound (PoCUS) without visual interference. This research uses the HoloLens to capture the first-person view of a simulated rural emergency room (ER) through mixed reality capture (MRC) and serves as a novel telemedicine platform with remote pointing capabilities. The mentor's hand gestures are captured using a Leap Motion and virtually displayed in the AR space of the HoloLens. To explore the feasibility of the developed platform, twelve novice medical trainees were guided by a mentor through a simulated ultrasound exploration in a trauma scenario, as part of a pilot user study. The study explores the utility of the system from the trainees, mentor, and objective observers' perspectives and compares the findings to that of a more traditional multi-camera telemedicine solution. The results obtained provide valuable insight and guidance for the development of an AR-supported telemedicine platform.

Augmented Reality as a Telemedicine Platform for Remote Procedural Training

PubMed Central

Wang, Shiyao; Parsons, Michael; Stone-McLean, Jordan; Rogers, Peter; Boyd, Sarah; Hoover, Kristopher; Meruvia-Pastor, Oscar; Gong, Minglun; Smith, Andrew

2017-01-01

Traditionally, rural areas in many countries are limited by a lack of access to health care due to the inherent challenges associated with recruitment and retention of healthcare professionals. Telemedicine, which uses communication technology to deliver medical services over distance, is an economical and potentially effective way to address this problem. In this research, we develop a new telepresence application using an Augmented Reality (AR) system. We explore the use of the Microsoft HoloLens to facilitate and enhance remote medical training. Intrinsic advantages of AR systems enable remote learners to perform complex medical procedures such as Point of Care Ultrasound (PoCUS) without visual interference. This research uses the HoloLens to capture the first-person view of a simulated rural emergency room (ER) through mixed reality capture (MRC) and serves as a novel telemedicine platform with remote pointing capabilities. The mentor’s hand gestures are captured using a Leap Motion and virtually displayed in the AR space of the HoloLens. To explore the feasibility of the developed platform, twelve novice medical trainees were guided by a mentor through a simulated ultrasound exploration in a trauma scenario, as part of a pilot user study. The study explores the utility of the system from the trainees, mentor, and objective observers’ perspectives and compares the findings to that of a more traditional multi-camera telemedicine solution. The results obtained provide valuable insight and guidance for the development of an AR-supported telemedicine platform. PMID:28994720

HoloNetwork: communicating science through holography

NASA Astrophysics Data System (ADS)

Pombo, Pedro; Santos, Emanuel; Magalhães, Carolina

2017-03-01

Since 1997 a program dedicated to holography has been developed and implemented in Portugal. This program started with focus on schools and science education. The HoloNetwork was created and it has been spread at a National level, involving a group of thirty schools and hundreds of students and teachers. In 2009 this network started to work to achieve a new target, the general public. With this goal, a larger program was developed with focus on science and society and on science communication through holography. For the implementation of this new program, special holography outreach activities were built, dedicated to informal learning and seven Science Centers around Portugal were add into the HoloNetwork. During last years, we have been working on holography, based on two main branches, one dedicated to schools and with the aimed to promote physics teaching and to teach how to make holograms, and another dedicated to society and with the aimed to promote holography and to increase scientific literacy. This paper would analyze the educational program, all holography outreach activities, exhibitions or events, all equipments, materials and setups used and it would present the holographic techniques explored with students or with the public. Finally, the results obtained in this work would be present and explored, with focus on students impact and outcomes, taking into account the public engagement on holography and its effect into scientific culture and analyzing the quality of holograms made by students and by the general public. subject.

Trends in the Diversity, Distribution and Life History Strategy of Arctic Hydrozoa (Cnidaria)

PubMed Central

Ronowicz, Marta; Kukliński, Piotr; Mapstone, Gillian M.

2015-01-01

This is the first attempt to compile a comprehensive and updated species list for Hydrozoa in the Arctic, encompassing both hydroid and medusa stages and including Siphonophorae. We address the hypothesis that the presence of a pelagic stage (holo- or meroplanktonic) was not necessary to successfully recolonize the Arctic by Hydrozoa after the Last Glacial Maximum. Presence-absence data of Hydrozoa in the Arctic were prepared on the basis of historical and present-day literature. The Arctic was divided into ecoregions. Species were grouped into distributional categories according to their worldwide occurrences. Each species was classified according to life history strategy. The similarity of species composition among regions was calculated with the Bray-Curtis index. Average and variation in taxonomic distinctness were used to measure diversity at the taxonomic level. A total of 268 species were recorded. Arctic-boreal species were the most common and dominated each studied region. Nineteen percent of species were restricted to the Arctic. There was a predominance of benthic species over holo- and meroplanktonic species. Arctic, Arctic-Boreal and Boreal species were mostly benthic, while widely distributed species more frequently possessed a pelagic stage. Our results support hypothesis that the presence of a pelagic stage (holo- or meroplanktonic) was not necessary to successfully recolonize the Arctic. The predominance of benthic Hydrozoa suggests that the Arctic could have been colonised after the Last Glacial Maximum by hydroids rafting on floating substrata or recolonising from glacial refugia. PMID:25793294

Trends in the diversity, distribution and life history strategy of Arctic Hydrozoa (Cnidaria).

PubMed

Ronowicz, Marta; Kukliński, Piotr; Mapstone, Gillian M

2015-01-01

This is the first attempt to compile a comprehensive and updated species list for Hydrozoa in the Arctic, encompassing both hydroid and medusa stages and including Siphonophorae. We address the hypothesis that the presence of a pelagic stage (holo- or meroplanktonic) was not necessary to successfully recolonize the Arctic by Hydrozoa after the Last Glacial Maximum. Presence-absence data of Hydrozoa in the Arctic were prepared on the basis of historical and present-day literature. The Arctic was divided into ecoregions. Species were grouped into distributional categories according to their worldwide occurrences. Each species was classified according to life history strategy. The similarity of species composition among regions was calculated with the Bray-Curtis index. Average and variation in taxonomic distinctness were used to measure diversity at the taxonomic level. A total of 268 species were recorded. Arctic-boreal species were the most common and dominated each studied region. Nineteen percent of species were restricted to the Arctic. There was a predominance of benthic species over holo- and meroplanktonic species. Arctic, Arctic-Boreal and Boreal species were mostly benthic, while widely distributed species more frequently possessed a pelagic stage. Our results support hypothesis that the presence of a pelagic stage (holo- or meroplanktonic) was not necessary to successfully recolonize the Arctic. The predominance of benthic Hydrozoa suggests that the Arctic could have been colonised after the Last Glacial Maximum by hydroids rafting on floating substrata or recolonising from glacial refugia.

Understanding cAMP-dependent allostery by NMR spectroscopy: comparative analysis of the EPAC1 cAMP-binding domain in its apo and cAMP-bound states.

PubMed

Mazhab-Jafari, Mohammad T; Das, Rahul; Fotheringham, Steven A; SilDas, Soumita; Chowdhury, Somenath; Melacini, Giuseppe

2007-11-21

cAMP (adenosine 3',5'-cyclic monophosphate) is a ubiquitous second messenger that activates a multitude of essential cellular responses. Two key receptors for cAMP in eukaryotes are protein kinase A (PKA) and the exchange protein directly activated by cAMP (EPAC), which is a recently discovered guanine nucleotide exchange factor (GEF) for the small GTPases Rap1 and Rap2. Previous attempts to investigate the mechanism of allosteric activation of eukaryotic cAMP-binding domains (CBDs) at atomic or residue resolution have been hampered by the instability of the apo form, which requires the use of mixed apo/holo systems, that have provided only a partial picture of the CBD apo state and of the allosteric networks controlled by cAMP. Here, we show that, unlike other eukaryotic CBDs, both apo and cAMP-bound states of the EPAC1 CBD are stable under our experimental conditions, providing a unique opportunity to define at an unprecedented level of detail the allosteric interactions linking two critical functional sites of this CBD. These are the phosphate binding cassette (PBC), where cAMP binds, and the N-terminal helical bundle (NTHB), which is the site of the inhibitory interactions between the regulatory and catalytic regions of EPAC. Specifically, the combined analysis of the cAMP-dependent changes in chemical shifts, 2 degrees structure probabilities, hydrogen/hydrogen exchange (H/H) and hydrogen/deuterium exchange (H/D) protection factors reveals that the long-range communication between the PBC and the NTHB is implemented by two distinct intramolecular cAMP-signaling pathways, respectively, mediated by the beta2-beta3 loop and the alpha6 helix. Docking of cAMP into the PBC perturbs the NTHB inner core packing and the helical probabilities of selected NTHB residues. The proposed model is consistent with the allosteric role previously hypothesized for L273 and F300 based on site-directed mutagenesis; however, our data show that such a contact is part of a significantly more extended allosteric network that, unlike PKA, involves a tight coupling between the alpha- and beta-subdomains of the EPAC CBD. The proposed mechanism of allosteric activation will serve as a basis to understand agonism and antagonism in the EPAC system and provides also a general paradigm for how small ligands control protein-protein interfaces.

Ensemble pharmacophore meets ensemble docking: a novel screening strategy for the identification of RIPK1 inhibitors

NASA Astrophysics Data System (ADS)

Fayaz, S. M.; Rajanikant, G. K.

2014-07-01

Programmed cell death has been a fascinating area of research since it throws new challenges and questions in spite of the tremendous ongoing research in this field. Recently, necroptosis, a programmed form of necrotic cell death, has been implicated in many diseases including neurological disorders. Receptor interacting serine/threonine protein kinase 1 (RIPK1) is an important regulatory protein involved in the necroptosis and inhibition of this protein is essential to stop necroptotic process and eventually cell death. Current structure-based virtual screening methods involve a wide range of strategies and recently, considering the multiple protein structures for pharmacophore extraction has been emphasized as a way to improve the outcome. However, using the pharmacophoric information completely during docking is very important. Further, in such methods, using the appropriate protein structures for docking is desirable. If not, potential compound hits, obtained through pharmacophore-based screening, may not have correct ranks and scores after docking. Therefore, a comprehensive integration of different ensemble methods is essential, which may provide better virtual screening results. In this study, dual ensemble screening, a novel computational strategy was used to identify diverse and potent inhibitors against RIPK1. All the pharmacophore features present in the binding site were captured using both the apo and holo protein structures and an ensemble pharmacophore was built by combining these features. This ensemble pharmacophore was employed in pharmacophore-based screening of ZINC database. The compound hits, thus obtained, were subjected to ensemble docking. The leads acquired through docking were further validated through feature evaluation and molecular dynamics simulation.

Crystal structure of FabZ-ACP complex reveals a dynamic seesaw-like catalytic mechanism of dehydratase in fatty acid biosynthesis.

PubMed

Zhang, Lin; Xiao, Jianfeng; Xu, Jianrong; Fu, Tianran; Cao, Zhiwei; Zhu, Liang; Chen, Hong-Zhuan; Shen, Xu; Jiang, Hualiang; Zhang, Liang

2016-12-01

Fatty acid biosynthesis (FAS) is a vital process in cells. Fatty acids are essential for cell assembly and cellular metabolism. Abnormal FAS directly correlates with cell growth delay and human diseases, such as metabolic syndromes and various cancers. The FAS system utilizes an acyl carrier protein (ACP) as a transporter to stabilize and shuttle the growing fatty acid chain throughout enzymatic modules for stepwise catalysis. Studying the interactions between enzymatic modules and ACP is, therefore, critical for understanding the biological function of the FAS system. However, the information remains unclear due to the high flexibility of ACP and its weak interaction with enzymatic modules. We present here a 2.55 Å crystal structure of type II FAS dehydratase FabZ in complex with holo-ACP, which exhibits a highly symmetrical FabZ hexamer-ACP 3 stoichiometry with each ACP binding to a FabZ dimer subunit. Further structural analysis, together with biophysical and computational results, reveals a novel dynamic seesaw-like ACP binding and catalysis mechanism for the dehydratase module in the FAS system, which is regulated by a critical gatekeeper residue (Tyr100 in FabZ) that manipulates the movements of the β-sheet layer. These findings improve the general understanding of the dehydration process in the FAS system and will potentially facilitate drug and therapeutic design for diseases associated with abnormalities in FAS.

Disparity in holoprotein/apoprotein ratios of different standards used for immunoquantification of hepatic cytochrome P450 enzymes.

PubMed

Perrett, H F; Barter, Z E; Jones, B C; Yamazaki, H; Tucker, G T; Rostami-Hodjegan, A

2007-10-01

An analysis of reported hepatic abundances of CYP3A4 and 3A5 indicated that values determined by immunoquantification using commercially available, unpurified recombinant enzymes as standards are significantly lower than those determined using purified enzymes or human liver microsomes characterized with lysosomal peptides (CYP3A4: mean 45 versus 121 pmol/mg protein, p < 0.01; CYP3A5: mean 28 versus 83 pmol/mg protein, p < 0.05). When immunoquantifying cytochromes P450 (P450s), it is assumed that the holoprotein (holo)/apoprotein ratio is the same in the samples and the standard. Estimates of holo/apoprotein ratios from data reported for a range of P450s purified from human liver and non-commercial recombinant systems indicated less than complete and variable heme coupling dependent on enzyme and system.

Holo Structure and Steady State Kinetics of the Thiazolinyl Imine Reductases for Siderophore Biosynthesis

PubMed Central

Meneely, Kathleen M.; Ronnebaum, Trey A.; Riley, Andrew P.; Prisinzano, Thomas E.; Lamb, Audrey L.

2016-01-01

Thiazolinyl imine reductases catalyze the NADPH-dependent reduction of a thiazoline to a thiazolidine, a required step in the formation of the siderophores yersiniabactin (Yersinia spp.) and pyochelin (Pseudomonas aeruginosa). These stand-alone nonribosomal peptide tailoring domains are structural homologues of sugar oxidoreductases. Two closed structures of the thiazolinyl imine reductase from Yersinia enterocolitica (Irp3) are presented here: an NADP+-bound structure to 1.45 Å resolution and a holo structure to 1.28 Å resolution with NADP+ and a substrate analogue bound. Michaelis—Menten kinetics were measured using the same substrate analogue and the homologue from P. aeruginosa, PchG. The data presented here support the hypothesis that tyrosine 128 is the likely general acid residue for catalysis and also highlight the phosphopantetheine tunnel for tethering of the substrate to the nonribosomal peptide synthetase module during assembly line biosynthesis of the siderophore. PMID:27601130

Selected Images of the Effects of the October 15, 2006, Kiholo Bay-Mahukona, Hawai'i, Earthquakes and Recovery Efforts

USGS Publications Warehouse

Takahashi, Taeko Jane; Ikeda, Nancy A.; Okubo, Paul G.; Sako, Maurice K.; Dow, David C.; Priester, Anna M.; Steiner, Nolan A.

2011-01-01

Although the vast majority of earthquakes in the State of Hawaii are closely related to the active volcanism associated with the southeastern part of the Island of Hawai‘i, the October 2006 Kīholo Bay and Māhukona earthquakes clearly suggest the devastating potential of deeper lithospheric earthquakes. Large earthquakes thought to be nearly M7 have struck near the islands of Lāna‘i (1871) and Maui (1938). It is thought that these, like the 2006 earthquakes, were deep lithospheric flexure earthquakes (Wyss and Koyanagi, 1992; Klein and others, 2001). Thus, it is important to recognize the potential seismic hazard posed by such earthquakes beneath the older Hawaiian Islands. The data and observations afforded by the 2006 earthquakes promise to improve probabilistic seismic hazards modeling in Hawai‘i. The effects of the October 15, 2006, Kīholo Bay-Māhukona earthquakes are shown in images taken from the coastal route along the northern half of the Island of Hawai‘i, where damage was the most concentrated. The direction of presentation is counter-clockwise, from Pa‘auilo on the eastern or windward (Hāmākua) side to Kealakekua Bay on the western or leeward (Kona) side. A list of sites, their locations, coordinates, and distance from the epicenter at Kīholo Bay are given in table 1. A Google Earth map (fig. 7) and a topographic map (fig. 8) pinpoint the 36 sites where damage was documented and digital images were compiled for this collection.

Kā-HOLO Project: a protocol for a randomized controlled trial of a native cultural dance program for cardiovascular disease prevention in Native Hawaiians.

PubMed

Kaholokula, Joseph Keawe'aimoku; Look, Mele A; Wills, Thomas A; de Silva, Māpuana; Mabellos, Tricia; Seto, Todd B; Ahn, Hyeong Jun; Sinclair, Ka'imi A; Buchwald, Dedra

2017-04-17

As a major risk factor for cardiovascular and cerebrovascular disease (CVD), hypertension affects 33% of U.S. adults. Relative to other US races and ethnicities, Native Hawaiians have a high prevalence of hypertension and are 3 to 4 times more likely to have CVD. Effective, culturally-relevant interventions are needed to address CVD risk in this population. Investigators of the Kā-HOLO Project developed a study design to test the efficacy of an intervention that uses hula, a traditional Hawaiian dance, to increase physical activity and reduce CVD risk. A 2-arm randomized controlled trial with a wait-list control design will be implemented to test a 6-month intervention based on hula to manage blood pressure and reduce CVD risk in 250 adult Native Hawaiians with diagnosed hypertension. Half of the sample will be randomized to each arm, stratified across multiple study sites. Primary outcomes are reduction in systolic blood pressure and improvement in CVD risk as measured by the Framingham Risk Score. Other psychosocial and sociocultural measures will be included to determine mediators of intervention effects on primary outcomes. Assessments will be conducted at baseline, 3 months, and 6 months for all participants, and at 12 months for intervention participants only. This trial will elucidate the efficacy of a novel hypertension management program designed to reduce CVD risk in an indigenous population by using a cultural dance form as its physical activity component. The results of this culturally-based intervention will have implications for other indigenous populations globally and will offer a sustainable, culturally-relevant means of addressing CVD disparities. ClinicalTrials.gov: NCT02620709 , registration date November 23, 2015.

Protein-membrane interaction and fatty acid transfer from intestinal fatty acid-binding protein to membranes. Support for a multistep process.

PubMed

Falomir-Lockhart, Lisandro J; Laborde, Lisandro; Kahn, Peter C; Storch, Judith; Córsico, Betina

2006-05-19

Fatty acid transfer from intestinal fatty acid-binding protein (IFABP) to phospholipid membranes occurs during protein-membrane collisions. Electrostatic interactions involving the alpha-helical "portal" region of the protein have been shown to be of great importance. In the present study, the role of specific lysine residues in the alpha-helical region of IFABP was directly examined. A series of point mutants in rat IFABP was engineered in which the lysine positive charges in this domain were eliminated or reversed. Using a fluorescence resonance energy transfer assay, we analyzed the rates and mechanism of fatty acid transfer from wild type and mutant proteins to acceptor membranes. Most of the alpha-helical domain mutants showed slower absolute fatty acid transfer rates to zwitterionic membranes, with substitution of one of the lysines of the alpha2 helix, Lys27, resulting in a particularly dramatic decrease in the fatty acid transfer rate. Sensitivity to negatively charged phospholipid membranes was also reduced, with charge reversal mutants in the alpha2 helix the most affected. The results support the hypothesis that the portal region undergoes a conformational change during protein-membrane interaction, which leads to release of the bound fatty acid to the membrane and that the alpha2 segment is of particular importance in the establishment of charge-charge interactions between IFABP and membranes. Cross-linking experiments with a phospholipid-photoactivable reagent underscored the importance of charge-charge interactions, showing that the physical interaction between wild-type intestinal fatty acid-binding protein and phospholipid membranes is enhanced by electrostatic interactions. Protein-membrane interactions were also found to be enhanced by the presence of ligand, suggesting different collisional complex structures for holo- and apo-IFABP.

Replacing Arginine 33 for Alanine in the Hemophore HasA from Pseudomonas aeruginosa Causes Closure of the H32 Loop in the Apo-Protein

PubMed Central

Kumar, Ritesh; Qi, Yifei; Matsumura, Hirotoshi; Lovell, Scott; Yao, Huili; Battaile, Kevin P.; Im, Wonpil; Moënne-Loccoz, Pierre; Rivera, Mario

2017-01-01

Previous characterization of hemophores from Serratia marcescens (HasAs), Pseudomonas aeruginosa (HasAp) and Yersinia pestis (HasAyp) showed that hemin binds between two loops, where it is axially coordinated by H32 and Y75. The Y75 loop is structurally conserved in all three hemophores and harbors conserved ligand Y75. The other loop contains H32 in HasAs and HasAp, but a noncoordinating Q32 in HasAyp. The H32 loop in apo-HasAs and apo-HasAp is in an open conformation, which places H32 about 30 Å from the hemin-binding site. Hence, hemin binding onto the Y75 loop of HasAs or HasAp triggers a large relocation of the H32 loop from an open- to a closed-loop conformation and enables coordination of the hemin-iron by H32. In comparison, the Q32 loop in apo-HasAyp is in the closed conformation and hemin binding occurs with minimal reorganization and without coordinative interactions with the Q32 loop. Studies in crystallo and in solution have established that the open H32 loop in apo-HasAp and apo-HasAs is well structured and minimally affected by conformational dynamics. In this study we address the intriguing issue of the stability of the H32 loop in apo-HasAp and how hemin binding triggers its relocation. We address this question with a combination of NMR spectroscopy, X-ray crystallography, and molecular dynamics simulations and find that R33 is critical to the stability of the open H32 loop. Replacing R33 with A causes the H32 loop in R33A apo-HasAp to adopt a conformation similar to that of holo-HasAp. Finally, stopped-flow absorption and resonance Raman analyses of hemin binding to apo-R33A HasAp indicates that the closed H32 loop slows down the insertion of the heme inside the binding pocket, presumably as it obstructs access to the hydrophobic platform on the Y75 loop, but accelerate the completion of the heme iron coordination. PMID:27074415

Comparison of optical see-through head-mounted displays for surgical interventions with object-anchored 2D-display

PubMed Central

Barthel, Alexander; Johnson, Alex; Osgood, Greg; Kazanzides, Peter; Navab, Nassir; Fuerst, Bernhard

2018-01-01

Purpose Optical see-through head-mounted displays (OST-HMD) feature an unhindered and instantaneous view of the surgery site and can enable a mixed reality experience for surgeons during procedures. In this paper, we present a systematic approach to identify the criteria for evaluation of OST-HMD technologies for specific clinical scenarios, which benefit from using an object-anchored 2D-display visualizing medical information. Methods Criteria for evaluating the performance of OST-HMDs for visualization of medical information and its usage are identified and proposed. These include text readability, contrast perception, task load, frame rate, and system lag. We choose to compare three commercially available OST-HMDs, which are representatives of currently available head-mounted display technologies. A multi-user study and an offline experiment are conducted to evaluate their performance. Results Statistical analysis demonstrates that Microsoft HoloLens performs best among the three tested OST-HMDs, in terms of contrast perception, task load, and frame rate, while ODG R-7 offers similar text readability. The integration of indoor localization and fiducial tracking on the HoloLens provides significantly less system lag in a relatively motionless scenario. Conclusions With ever more OST-HMDs appearing on the market, the proposed criteria could be used in the evaluation of their suitability for mixed reality surgical intervention. Currently, Microsoft HoloLens may be more suitable than ODG R-7 and Epson Moverio BT-200 for clinical usability in terms of the evaluated criteria. To the best of our knowledge, this is the first paper that presents a methodology and conducts experiments to evaluate and compare OST-HMDs for their use as object-anchored 2D-display during interventions. PMID:28343301

On Holo-Hilbert Spectral Analysis: A Full Informational Spectral Representation for Nonlinear and Non-Stationary Data

NASA Technical Reports Server (NTRS)

Huang, Norden E.; Hu, Kun; Yang, Albert C. C.; Chang, Hsing-Chih; Jia, Deng; Liang, Wei-Kuang; Yeh, Jia Rong; Kao, Chu-Lan; Juan, Chi-Huang; Peng, Chung Kang;

Developing an Augmented Reality Environment for Earth Science Education

NASA Astrophysics Data System (ADS)

Pratt, M. J.; Skemer, P. A.; Arvidson, R. E.

2017-12-01

The emerging field of augmented reality (AR) provides new and exciting ways to explore geologic phenomena for research and education. The primary advantage of AR is that it allows users to physically explore complex three-dimensional structures that were previously inaccessible, for example a remote geologic outcrop or a mineral structure at the atomic scale. It is used, for example, with OnSight software during tactical operations to plan the Mars Curiosity rover's traverses by providing virtual views to walk through terrain and the rover at true scales. This mode of physical exploration allows users more freedom to investigate and understand the 3D structure than is possible on a flat computer screen, or within a static PowerPoint presentation during a classroom lecture. The Microsoft HoloLens headset provides the most-advanced, mobile AR platform currently available to developers. The Fossett Laboratory for Virtual Planetary Exploration at Washington University in St. Louis has applied this technology, coupled with photogrammetric software and the Unity 3D gaming engine, to develop photorealistic environments of 3D geologic outcrops from around the world. The untethered HoloLens provides an ideal platform for a classroom setting as it allows for shared experiences of the holograms of interest, projecting them in the same location for all users to explore. Furthermore, the HoloLens allows for face-to-face communication during use that is important in teaching, a feature that virtual reality does not allow. Our development of an AR application includes the design of an online database of photogrammetric outcrop models curated for the current limitations of AR technology. This database will be accessible to both those wishing to submit models, and is free to those wishing to use the application for teaching, outreach or research purposes.

On Holo-Hilbert spectral analysis: a full informational spectral representation for nonlinear and non-stationary data

PubMed Central

Huang, Norden E.; Hu, Kun; Yang, Albert C. C.; Chang, Hsing-Chih; Jia, Deng; Liang, Wei-Kuang; Yeh, Jia Rong; Kao, Chu-Lan; Juan, Chi-Hung; Peng, Chung Kang; Meijer, Johanna H.; Wang, Yung-Hung; Long, Steven R.; Wu, Zhauhua

2016-01-01

The Holo-Hilbert spectral analysis (HHSA) method is introduced to cure the deficiencies of traditional spectral analysis and to give a full informational representation of nonlinear and non-stationary data. It uses a nested empirical mode decomposition and Hilbert–Huang transform (HHT) approach to identify intrinsic amplitude and frequency modulations often present in nonlinear systems. Comparisons are first made with traditional spectrum analysis, which usually achieved its results through convolutional integral transforms based on additive expansions of an a priori determined basis, mostly under linear and stationary assumptions. Thus, for non-stationary processes, the best one could do historically was to use the time–frequency representations, in which the amplitude (or energy density) variation is still represented in terms of time. For nonlinear processes, the data can have both amplitude and frequency modulations (intra-mode and inter-mode) generated by two different mechanisms: linear additive or nonlinear multiplicative processes. As all existing spectral analysis methods are based on additive expansions, either a priori or adaptive, none of them could possibly represent the multiplicative processes. While the earlier adaptive HHT spectral analysis approach could accommodate the intra-wave nonlinearity quite remarkably, it remained that any inter-wave nonlinear multiplicative mechanisms that include cross-scale coupling and phase-lock modulations were left untreated. To resolve the multiplicative processes issue, additional dimensions in the spectrum result are needed to account for the variations in both the amplitude and frequency modulations simultaneously. HHSA accommodates all the processes: additive and multiplicative, intra-mode and inter-mode, stationary and non-stationary, linear and nonlinear interactions. The Holo prefix in HHSA denotes a multiple dimensional representation with both additive and multiplicative capabilities. PMID:26953180

Cobalamin status and its relation with depression, cognition and neuropathy in patients with type 2 diabetes mellitus using metformin.

PubMed

Biemans, Elke; Hart, Huberta E; Rutten, Guy E H M; Cuellar Renteria, Viviana G; Kooijman-Buiting, Antoinette M J; Beulens, Joline W J

2015-04-01

To investigate the associations of vitamin B12 (cobalamin and holotranscobalamin) status with depression, cognition and neuropathy in patients with type 2 diabetes using metformin. In an observational study, among 550 type 2 diabetes patients using metformin, cobalamin and holotranscobalamin (holoTCII) levels were measured at the annual diabetes checkup, and deficiencies were defined as <148 and <21 pmol/L, respectively. Depression and cognitive function were assessed with corresponding International Classification of Primary Care codes and questionnaires; neuropathy with medical record data and a questionnaire. Confounding variables were retrieved from medical records. Multivariable logistic and linear regressions were used with cobalamin status as independent variable; depression, cognition and neuropathy as dependent variables. The mean duration of diabetes was 8.4 years (±5.8); mean duration of metformin use was 64.1 months (±43.2), with a mean metformin dose of 1,306 mg/day. A sufficient cobalamin level was independently associated with a decreased risk of depression (OR 0.42; 95 % CI 0.23-0.78) and better cognitive performance (β = 1.79; 95 % CI 0.07-3.52) adjusted for confounders. This indicates that cobalamin-deficient patients had a 2.4 times higher chance of depression and a 1.79 point lower cognitive performance score. HoloTCII was not associated with any outcome. Cobalamin deficiency was associated with an increased risk of depression and worse cognitive performance, while holoTCII was not. Screening for cobalamin deficiency may be warranted in diabetes patients using metformin. Physicians should consider a cobalamin deficiency in diabetes patients using metformin with a depression or cognitive decline.

Comparison of optical see-through head-mounted displays for surgical interventions with object-anchored 2D-display.

PubMed

Qian, Long; Barthel, Alexander; Johnson, Alex; Osgood, Greg; Kazanzides, Peter; Navab, Nassir; Fuerst, Bernhard

2017-06-01

Optical see-through head-mounted displays (OST-HMD) feature an unhindered and instantaneous view of the surgery site and can enable a mixed reality experience for surgeons during procedures. In this paper, we present a systematic approach to identify the criteria for evaluation of OST-HMD technologies for specific clinical scenarios, which benefit from using an object-anchored 2D-display visualizing medical information. Criteria for evaluating the performance of OST-HMDs for visualization of medical information and its usage are identified and proposed. These include text readability, contrast perception, task load, frame rate, and system lag. We choose to compare three commercially available OST-HMDs, which are representatives of currently available head-mounted display technologies. A multi-user study and an offline experiment are conducted to evaluate their performance. Statistical analysis demonstrates that Microsoft HoloLens performs best among the three tested OST-HMDs, in terms of contrast perception, task load, and frame rate, while ODG R-7 offers similar text readability. The integration of indoor localization and fiducial tracking on the HoloLens provides significantly less system lag in a relatively motionless scenario. With ever more OST-HMDs appearing on the market, the proposed criteria could be used in the evaluation of their suitability for mixed reality surgical intervention. Currently, Microsoft HoloLens may be more suitable than ODG R-7 and Epson Moverio BT-200 for clinical usability in terms of the evaluated criteria. To the best of our knowledge, this is the first paper that presents a methodology and conducts experiments to evaluate and compare OST-HMDs for their use as object-anchored 2D-display during interventions.

The R117A variant of the Escherichia coli transacylase FabD synthesizes novel acyl-(acyl carrier proteins).

PubMed

Marcella, Aaron M; Barb, Adam W

2017-12-01

The commercial impact of fermentation systems producing novel and biorenewable chemicals will flourish with the expansion of enzymes engineered to synthesize new molecules. Though a small degree of natural variability exists in fatty acid biosynthesis, the molecular space accessible through enzyme engineering is fundamentally limitless. Prokaryotic fatty acid biosynthesis enzymes build carbon chains on a functionalized acyl carrier protein (ACP) that provides solubility, stability, and a scaffold for interactions with the synthetic enzymes. Here, we identify the malonyl-coenzyme A (CoA)/holo-ACP transacylase (FabD) from Escherichia coli as a platform enzyme for engineering to diversify microbial fatty acid biosynthesis. The FabD R117A variant produced novel ACP-based primer and extender units for fatty acid biosynthesis. Unlike the wild-type enzyme that is highly specific for malonyl-CoA to produce malonyl-ACP, the R117A variant synthesized acetyl-ACP, succinyl-ACP, isobutyryl-ACP, 2-butenoyl-ACP, and β-hydroxybutyryl-ACP among others from holo-ACP and the corresponding acyl-CoAs with specific activities from 3.7 to 120 nmol min -1  mg -1 . FabD R117A maintained K M values for holo-ACP (~ 40 μM) and displayed small changes in K M for acetoacetyl-CoA (110 ± 30 μM) and acetyl-CoA (200 ± 70 μM) when compared to malonyl-CoA (80 ± 30 μM). FabD R117A represents a novel catalyst that synthesizes a broad range of acyl-acyl-ACPs.

Modeling the interactions of the nucleotide excision repair UvrA(2) dimer with DNA.

PubMed

Gantchev, Tsvetan G; Hunting, Darel J

2010-12-28

The UvrA protein initiates the DNA damage recognition process by the bacterial nucleotide excision repair (NER) system. Recently, crystallographic structures of holo-UvrA(2) dimers from two different microorganisms have been released (Protein Data Bank entries 2r6f , 2vf7 , and 2vf8 ). However, the details of the DNA binding by UvrA(2) and other peculiarities involved in the damage recognition process remain unknown. We have undertaken a molecular modeling approach to appraise the possible modes of DNA-UvrA(2) interaction using molecular docking and short-scale guided molecular dynamics [continuum field, constrained, and/or unrestricted simulated annealing (SA)], taking into account the three-dimensional location of a series of mutation-identified UvrA residues implicated in DNA binding. The molecular docking was based on the assumptions that the UvrA(2) dimer is preformed prior to DNA binding and that no major protein conformational rearrangements, except moderate domain reorientations, are required for binding of undamaged DNA. As a first approximation, DNA was treated as a rigid ligand. From the electrostatic relief of the ventral surface of UvrA(2), we initially identified three, noncollinear DNA binding paths. Each of the three resulting nucleoprotein complexes (C1, C2, and C3) was analyzed separately, including calculation of binding energies, the number and type of interaction residues (including mutated ones), and the predominant mode of translational and rotational motion of specific protein domains after SA to ensure improved DNA binding. The UvrA(2) dimer can accommodate DNA in all three orientations, albeit with different binding strengths. One of the UvrA(2)-DNA complexes (C1) fulfilled most of the requirements (high interaction energy, proximity of DNA to mutated residues, etc.) expected for a natural, high-affinity DNA binding site. This nucleoprotein presents a structural organization that is designed to clamp and bend double-stranded DNA. We examined the binding site in more detail by docking DNAs of significantly different (AT- vs CG-enriched) sequences and by submitting the complexes to DNA-unrestricted SA. It was found that in a manner independent of the DNA sequence and applied MD protocols, UvrA(2) favors binding of a bent and unwound undamaged DNA, with a kink positioned in the proximity of the Zn3 hairpins, anticollinearly aligned at the bottom of the ventral protein surface. It is further hypothesized that the Zn3 modules play an essential role in the damage recognition process and that the apparent existence of a family of DNA binding sites might be biologically relevant. Our data should prove to be useful in rational (structure-based) mutation studies.

Xanthomonas TAL effectors hijack host basal transcription factor IIA α and γ subunits for invasion.

PubMed

Ma, Ling; Wang, Qiang; Yuan, Meng; Zou, Tingting; Yin, Ping; Wang, Shiping

2018-02-05

The Xanthomonas genus includes Gram-negative plant-pathogenic bacteria, which infect a broad range of crops and wild plant species, cause symptoms with leaf blights, streaks, spots, stripes, necrosis, wilt, cankers and gummosis on leaves, stems and fruits in a wide variety of plants via injecting their effector proteins into the host cell during infection. Among these virulent effectors, transcription activator-like effectors (TALEs) interact with the γ subunit of host transcription factor IIA (TFIIAγ) to activate the transcription of host disease susceptibility genes. Functional TFIIA is a ternary complex comprising α, β and γ subunits. However, whether TALEs recruit TFIIAα, TFIIAβ, or both remains unknown. The underlying molecular mechanisms by which TALEs mediate host susceptibility gene activation require full elucidation. Here, we show that TALEs interact with the α+γ binary subcomplex but not the α+β+γ ternary complex of rice TFIIA (holo-OsTFIIA). The transcription factor binding (TFB) regions of TALEs, which are highly conserved in Xanthomonas species, have a dominant role in these interactions. Furthermore, the interaction between TALEs and the α+γ complex exhibits robust DNA binding activity in vitro. These results collectively demonstrate that TALE-carrying pathogens hijack the host basal transcription factors TFIIAα and TFIIAγ, but not TFIIAβ, to enhance host susceptibility during pathogen infection. The uncovered mechanism widens new insights on host-microbe interaction and provide an applicable strategy to breed high-resistance crop varieties. Copyright © 2018 Elsevier Inc. All rights reserved.

The Bordetella Adenylate Cyclase Repeat-in-Toxin (RTX) Domain Is Immunodominant and Elicits Neutralizing Antibodies*

PubMed Central

Wang, Xianzhe; Maynard, Jennifer A.

2015-01-01

The adenylate cyclase toxin (ACT) is a multifunctional virulence factor secreted by Bordetella species. Upon interaction of its C-terminal hemolysin moiety with the cell surface receptor αMβ2 integrin, the N-terminal cyclase domain translocates into the host cell cytosol where it rapidly generates supraphysiological cAMP concentrations, which inhibit host cell anti-bacterial activities. Although ACT has been shown to induce protective immunity in mice, it is not included in any current acellular pertussis vaccines due to protein stability issues and a poor understanding of its role as a protective antigen. Here, we aimed to determine whether any single domain could recapitulate the antibody responses induced by the holo-toxin and to characterize the dominant neutralizing antibody response. We first immunized mice with ACT and screened antibody phage display libraries for binding to purified ACT. The vast majority of unique antibodies identified bound the C-terminal repeat-in-toxin (RTX) domain. Representative antibodies binding two nonoverlapping, neutralizing epitopes in the RTX domain prevented ACT association with J774A.1 macrophages and soluble αMβ2 integrin, suggesting that these antibodies inhibit the ACT-receptor interaction. Sera from mice immunized with the RTX domain showed similar neutralizing activity as ACT-immunized mice, indicating that this domain induced an antibody response similar to that induced by ACT. These data demonstrate that RTX can elicit neutralizing antibodies and suggest it may present an alternative to ACT. PMID:25505186

Structural and biochemical characterization of MCAT from photosynthetic microorganism Synechocystis sp. PCC 6803 reveal its stepwise catalytic mechanism.

PubMed

Liu, Yinghui; Feng, Yanbin; Wang, Yayue; Li, Xia; Cao, Xupeng; Xue, Song

2015-02-13

Malonyl-coenzyme A: acyl-carrier protein transacylase (MCAT) catalyzes the transfer of malonyl group from malonyl-CoA to the holo-acyl carrier protein (Holo-ACP), yielding malonyl-ACP. The overall reaction has been extensively studied in heterotrophic microorganisms, while its mechanism in photosynthetic autotrophs as well as the stepwise reaction information remains unclear. Here the 2.42 Å crystal structure of MCAT from photosynthetic microorganism Synechocystis sp. PCC 6803 is presented. It demonstrates that Arg113, Ser88 and His188 constitute catalytic triad. The second step involved ACP-MCAT-malonyl intermediate is speed-limited instead of the malonyl-CoA-MCAT intermediate in the first step. Therefore His87, Arg113 and Ser88 render different contributions for the two intermediates. Additionally, S88T mutant initializes the reaction by H87 deprotonating S88T which is different from the wild type. Copyright © 2015 Elsevier Inc. All rights reserved.

Computer Tomography and Hybrid Optical/Digital Methods for Aerodynamic Measurements.

DTIC Science & Technology

1987-12-28

Industrial Applications of Corn- on Axisymnnietric Flame ’Iempnlw res Measured by Holo- puted Tornographv arid NMI? Imiaging (Optical Society of graphic...Pontificia Universidad Catolica de Chile. Escuela de Ingenieria . Santiago, equal. The optical path length difference (OPD) be- Chile. tween the two rays

International Year of Light in Michigan: outdoor holography

NASA Astrophysics Data System (ADS)

Nees, John; Aku-Leh, Cynthia; Jones, Tim; Sala, Anca L.; Smith, Arlene; Dreyer, Elizabeth F. C.

2016-09-01

Outdoor holography is an activity created by the Michigan Light Project during the International Year of Light. Traditional holography is done in dark and quiet rooms. Using a kit from LitiHolo.com, we designed a way to make simple holograms outside in a noisy festival environment.

Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

NASA Technical Reports Server (NTRS)

Hong, Y. S.; Ciszak, Ewa; Patel, Mulchand

2000-01-01

Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For crystallization, E3 samples were prepared with and without His-tag. To minimize the aggregation of E3, apo- and holo- forms of E3s were tested, as well as a mutated E3. Dynamic light scattering measurements revealed that the E3 preparations without His-tag and substrate are highly monodispersive with regard to homodimers. Consequent crystallization trials of this E3 preparation led to single crystals of E3 grown by the vapor diffusion method. Crystals were obtained within a few days from solution containing poly (ethylene glycol) monomethyl ether 5000 as a precipitant. Autoindexing and integration of the X-ray diffraction data showed that E3 crystals belong to an orthorhombic system with unit cell parameters a-- 123. 1, b= 165.3 and c=214.3A. Further optimization of protein preparation and crystallization experiments for the structural determination will be discussed.

Flying under the radar: The non-canonical biochemistry and molecular biology of petrobactin from Bacillus anthracis.

PubMed

Hagan, A K; Carlson, P E; Hanna, P C

2016-10-01

The dramatic, rapid growth of Bacillus anthracis that occurs during systemic anthrax implies a crucial requirement for the efficient acquisition of iron. While recent advances in our understanding of B. anthracis iron acquisition systems indicate the use of strategies similar to other pathogens, this review focuses on unique features of the major siderophore system, petrobactin. Ways that petrobactin differs from other siderophores include: A. unique ferric iron binding moieties that allow petrobactin to evade host immune proteins; B. a biosynthetic operon that encodes enzymes from both major siderophore biosynthesis classes; C. redundancy in membrane transport systems for acquisition of Fe-petrobactin holo-complexes; and, D. regulation that appears to be controlled predominately by sensing the host-like environmental signals of temperature, CO 2 levels and oxidative stress, as opposed to canonical sensing of intracellular iron levels. We argue that these differences contribute in meaningful ways to B. anthracis pathogenesis. This review will also outline current major gaps in our understanding of the petrobactin iron acquisition system, some projected means for exploiting current knowledge, and potential future research directions. © 2016 John Wiley & Sons Ltd.

Multiplex image-based autophagy RNAi screening identifies SMCR8 as ULK1 kinase activity and gene expression regulator

PubMed Central

Jung, Jennifer; Nayak, Arnab; Schaeffer, Véronique; Starzetz, Tatjana; Kirsch, Achim K; Müller, Stefan; Dikic, Ivan; Mittelbronn, Michel; Behrends, Christian

2017-01-01

Autophagy is an intracellular recycling and degradation pathway that depends on membrane trafficking. Rab GTPases are central for autophagy but their regulation especially through the activity of Rab GEFs remains largely elusive. We employed a RNAi screen simultaneously monitoring different populations of autophagosomes and identified 34 out of 186 Rab GTPase, GAP and GEF family members as potential autophagy regulators, amongst them SMCR8. SMCR8 uses overlapping binding regions to associate with C9ORF72 or with a C9ORF72-ULK1 kinase complex holo-assembly, which function in maturation and formation of autophagosomes, respectively. While focusing on the role of SMCR8 during autophagy initiation, we found that kinase activity and gene expression of ULK1 are increased upon SMCR8 depletion. The latter phenotype involved association of SMCR8 with the ULK1 gene locus. Global mRNA expression analysis revealed that SMCR8 regulates transcription of several other autophagy genes including WIPI2. Collectively, we established SMCR8 as multifaceted negative autophagy regulator. DOI: http://dx.doi.org/10.7554/eLife.23063.001 PMID:28195531

Connecting Active-Site Loop Conformations and Catalysis in Triosephosphate Isomerase: Insights from a Rare Variation at Residue 96 in the Plasmodial Enzyme.

PubMed

Pareek, Vidhi; Samanta, Moumita; Joshi, Niranjan V; Balaram, Hemalatha; Murthy, Mathur R N; Balaram, Padmanabhan

2016-04-01

Despite extensive research into triosephosphate isomerases (TIMs), there exists a gap in understanding of the remarkable conjunction between catalytic loop-6 (residues 166-176) movement and the conformational flip of Glu165 (catalytic base) upon substrate binding that primes the active site for efficient catalysis. The overwhelming occurrence of serine at position 96 (98% of the 6277 unique TIM sequences), spatially proximal to E165 and the loop-6 residues, raises questions about its role in catalysis. Notably, Plasmodium falciparum TIM has an extremely rare residue--phenylalanine--at this position whereas, curiously, the mutant F96S was catalytically defective. We have obtained insights into the influence of residue 96 on the loop-6 conformational flip and E165 positioning by combining kinetic and structural studies on the PfTIM F96 mutants F96Y, F96A, F96S/S73A, and F96S/L167V with sequence conservation analysis and comparative analysis of the available apo and holo structures of the enzyme from diverse organisms. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Optimal affinity ranking for automated virtual screening validated in prospective D3R grand challenges

NASA Astrophysics Data System (ADS)

Wingert, Bentley M.; Oerlemans, Rick; Camacho, Carlos J.

2018-01-01

The goal of virtual screening is to generate a substantially reduced and enriched subset of compounds from a large virtual chemistry space. Critical in these efforts are methods to properly rank the binding affinity of compounds. Prospective evaluations of ranking strategies in the D3R grand challenges show that for targets with deep pockets the best correlations (Spearman ρ 0.5) were obtained by our submissions that docked compounds to the holo-receptors with the most chemically similar ligand. On the other hand, for targets with open pockets using multiple receptor structures is not a good strategy. Instead, docking to a single optimal receptor led to the best correlations (Spearman ρ 0.5), and overall performs better than any other method. Yet, choosing a suboptimal receptor for crossdocking can significantly undermine the affinity rankings. Our submissions that evaluated the free energy of congeneric compounds were also among the best in the community experiment. Error bars of around 1 kcal/mol are still too large to significantly improve the overall rankings. Collectively, our top of the line predictions show that automated virtual screening with rigid receptors perform better than flexible docking and other more complex methods.

Effects of protein-pheromone complexation on correlated chemical shift modulations.

PubMed

Perazzolo, Chiara; Wist, Julien; Loth, Karine; Poggi, Luisa; Homans, Steve; Bodenhausen, Geoffrey

2005-12-01

Major urinary protein (MUP) is a pheromone-carrying protein of the lipocalin family. Previous studies by isothermal titration calorimetry (ITC) show that the affinity of MUP for the pheromone 2-methoxy-3-isobutylpyrazine (IBMP) is mainly driven by enthalpy, with a small unfavourable entropic contribution. Entropic terms can be attributed in part to changes in internal motions of the protein upon binding. Slow internal motions can lead to correlated or anti-correlated modulations of the isotropic chemical shifts of carbonyl C' and amide N nuclei. Correlated chemical shift modulations (CSM/CSM) in MUP have been determined by measuring differences of the transverse relaxation rates of zero- and double-quantum coherences ZQC{C'N} and DQC{C'N}, and by accounting for the effects of correlated fluctuations of dipole-dipole couplings (DD/DD) and chemical shift anisotropies (CSA/CSA). The latter can be predicted from tensor parameters of C' and N nuclei that have been determined in earlier work. The effects of complexation on slow time-scale protein dynamics can be determined by comparing the temperature dependence of the relaxation rates of APO-MUP (i.e., without ligand) and HOLO-MUP (i.e., with IBMP as a ligand).

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bolding, Simon R.; Cleveland, Mathew Allen; Morel, Jim E.

In this paper, we have implemented a new high-order low-order (HOLO) algorithm for solving thermal radiative transfer problems. The low-order (LO) system is based on the spatial and angular moments of the transport equation and a linear-discontinuous finite-element spatial representation, producing equations similar to the standard S 2 equations. The LO solver is fully implicit in time and efficiently resolves the nonlinear temperature dependence at each time step. The high-order (HO) solver utilizes exponentially convergent Monte Carlo (ECMC) to give a globally accurate solution for the angular intensity to a fixed-source pure-absorber transport problem. This global solution is used tomore » compute consistency terms, which require the HO and LO solutions to converge toward the same solution. The use of ECMC allows for the efficient reduction of statistical noise in the Monte Carlo solution, reducing inaccuracies introduced through the LO consistency terms. Finally, we compare results with an implicit Monte Carlo code for one-dimensional gray test problems and demonstrate the efficiency of ECMC over standard Monte Carlo in this HOLO algorithm.« less

A High-Order Low-Order Algorithm with Exponentially Convergent Monte Carlo for Thermal Radiative Transfer

DOE PAGES

Bolding, Simon R.; Cleveland, Mathew Allen; Morel, Jim E.

2016-10-21

In this paper, we have implemented a new high-order low-order (HOLO) algorithm for solving thermal radiative transfer problems. The low-order (LO) system is based on the spatial and angular moments of the transport equation and a linear-discontinuous finite-element spatial representation, producing equations similar to the standard S 2 equations. The LO solver is fully implicit in time and efficiently resolves the nonlinear temperature dependence at each time step. The high-order (HO) solver utilizes exponentially convergent Monte Carlo (ECMC) to give a globally accurate solution for the angular intensity to a fixed-source pure-absorber transport problem. This global solution is used tomore » compute consistency terms, which require the HO and LO solutions to converge toward the same solution. The use of ECMC allows for the efficient reduction of statistical noise in the Monte Carlo solution, reducing inaccuracies introduced through the LO consistency terms. Finally, we compare results with an implicit Monte Carlo code for one-dimensional gray test problems and demonstrate the efficiency of ECMC over standard Monte Carlo in this HOLO algorithm.« less

Oral treatment with Cu(II)(atsm) increases mutant SOD1 in vivo but protects motor neurons and improves the phenotype of a transgenic mouse model of amyotrophic lateral sclerosis.

PubMed

Roberts, Blaine R; Lim, Nastasia K H; McAllum, Erin J; Donnelly, Paul S; Hare, Dominic J; Doble, Philip A; Turner, Bradley J; Price, Katherine A; Lim, Sin Chun; Paterson, Brett M; Hickey, James L; Rhoads, Timothy W; Williams, Jared R; Kanninen, Katja M; Hung, Lin W; Liddell, Jeffrey R; Grubman, Alexandra; Monty, Jean-Francois; Llanos, Roxana M; Kramer, David R; Mercer, Julian F B; Bush, Ashley I; Masters, Colin L; Duce, James A; Li, Qiao-Xin; Beckman, Joseph S; Barnham, Kevin J; White, Anthony R; Crouch, Peter J

2014-06-04

Mutations in the metallo-protein Cu/Zn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) in humans and an expression level-dependent phenotype in transgenic rodents. We show that oral treatment with the therapeutic agent diacetyl-bis(4-methylthiosemicarbazonato)copper(II) [Cu(II)(atsm)] increased the concentration of mutant SOD1 (SOD1G37R) in ALS model mice, but paradoxically improved locomotor function and survival of the mice. To determine why the mice with increased levels of mutant SOD1 had an improved phenotype, we analyzed tissues by mass spectrometry. These analyses revealed most SOD1 in the spinal cord tissue of the SOD1G37R mice was Cu deficient. Treating with Cu(II)(atsm) decreased the pool of Cu-deficient SOD1 and increased the pool of fully metallated (holo) SOD1. Tracking isotopically enriched (65)Cu(II)(atsm) confirmed the increase in holo-SOD1 involved transfer of Cu from Cu(II)(atsm) to SOD1, suggesting the improved locomotor function and survival of the Cu(II)(atsm)-treated SOD1G37R mice involved, at least in part, the ability of the compound to improve the Cu content of the mutant SOD1. This was supported by improved survival of SOD1G37R mice that expressed the human gene for the Cu uptake protein CTR1. Improving the metal content of mutant SOD1 in vivo with Cu(II)(atsm) did not decrease levels of misfolded SOD1. These outcomes indicate the metal content of SOD1 may be a greater determinant of the toxicity of the protein in mutant SOD1-associated forms of ALS than the mutations themselves. Improving the metal content of SOD1 therefore represents a valid therapeutic strategy for treating ALS caused by SOD1. Copyright © 2014 the authors 0270-6474/14/348021-11$15.00/0.

X-ray-induced catalytic active-site reduction of a multicopper oxidase: structural insights into the proton-relay mechanism and O 2 -reduction states

DOE Office of Scientific and Technical Information (OSTI.GOV)

Serrano-Posada, Hugo; Centeno-Leija, Sara; Rojas-Trejo, Sonia Patricia

2015-11-26

During X-ray data collection from a multicopper oxidase (MCO) crystal, electrons and protons are mainly released into the system by the radiolysis of water molecules, leading to the X-ray-induced reduction of O 2 to 2H 2O at the trinuclear copper cluster (TNC) of the enzyme. In this work, 12 crystallographic structures of Thermus thermophilus HB27 multicopper oxidase (Tth-MCO) in holo, apo and Hg-bound forms and with different X-ray absorbed doses have been determined. In holo Tth -MCO structures with four Cu atoms, the proton-donor residue Glu451 involved in O 2 reduction was found in a double conformation: Glu451a (~7 Åmore » from the TNC) and Glu451b (~4.5 Å from the TNC). A positive peak of electron density above 3.5σ in anF o-F c map for Glu451a O ε2 indicates the presence of a carboxyl functional group at the side chain, while its significant absence in Glu451b strongly suggests a carboxylate functional group. In contrast, for apo Tth -MCO and in Hg-bound structures neither the positive peak nor double conformations were observed. Together, these observations provide the first structural evidence for a proton-relay mechanism in the MCO family and also support previous studies indicating that Asp106 does not provide protons for this mechanism. In addition, eight composite structures (Tth -MCO-C1–8) with different X-ray-absorbed doses allowed the observation of different O 2-reduction states, and a total depletion of T2Cu at doses higher than 0.2 MGy showed the high susceptibility of this Cu atom to radiation damage, highlighting the importance of taking radiation effects into account in biochemical interpretations of an MCO structure.« less

Vitamin B-12 supplementation of rural Mexican women changes biochemical vitamin B-12 status indicators but does not affect hematology or a bone turnover marker.

PubMed

Shahab-Ferdows, Setareh; Anaya-Loyola, Miriam A; Vergara-Castañeda, Haydé; Rosado, Jorge L; Keyes, William R; Newman, John W; Miller, Joshua W; Allen, Lindsay H

2012-10-01

A high prevalence of low serum vitamin B-12 concentrations has been reported in studies and surveys in Latin America including Mexico, but the functional consequences are unknown. This randomized controlled trial assessed the response to a high-dose vitamin B-12 supplementation of women in rural Querétaro, Mexico. Participants aged 20-59 y were stratified at baseline to deficient, marginal, and adequate status groups (serum vitamin B-12, 75-148, 149-220, and >220 pmol/L, respectively), and each group was randomized to vitamin B-12 treatment (single dose of 1 mg i.m. then 500 μg/d orally for 3 mo, n = 70) or placebo (n = 62). Measures at baseline and 3 mo included: complete blood count, serum vitamin B-12, holotranscobalamin (holoTC), folate, ferritin, C-reactive protein (CRP), bone alkaline phosphatase, and methylmalonic acid (MMA) and plasma total homocysteine (tHcy). At baseline, 11% of the women were vitamin B-12 deficient and 22% had marginal status. HoloTC was low (<35 pmol/L) in 23% and correlated with serum vitamin B-12 (r = 0.7; P < 0.001). Elevated MMA (>271 nmol/L) and tHcy (>12 μmol/L) occurred in 21 and 31%, respectively, and correlated with serum vitamin B-12 (r = -0.28, P < 0.0007 and r = -0.20, P < 0.01, respectively). Supplementation increased serum vitamin B-12 and holoTC and lowered MMA and tHcy, normalizing all values except for elevated tHcy in 21% of the women. Supplementation did not affect hematology or bone-specific alkaline phosphatase. Vitamin B-12 supplementation normalized biochemical indicators of vitamin B-12 status in the treatment group but did not affect the functional outcomes measured.

Miniaturized multiwavelength digital holography sensor for extensive in-machine tool measurement

NASA Astrophysics Data System (ADS)

Seyler, Tobias; Fratz, Markus; Beckmann, Tobias; Bertz, Alexander; Carl, Daniel

2017-06-01

In this paper we present a miniaturized digital holographic sensor (HoloCut) for operation inside a machine tool. With state-of-the-art 3D measurement systems, short-range structures such as tool marks cannot be resolved inside a machine tool chamber. Up to now, measurements had to be conducted outside the machine tool and thus processing data are generated offline. The sensor presented here uses digital multiwavelength holography to get 3D-shape-information of the machined sample. By using three wavelengths, we get a large artificial wavelength with a large unambiguous measurement range of 0.5mm and achieve micron repeatability even in the presence of laser speckles on rough surfaces. In addition, a digital refocusing algorithm based on phase noise is implemented to extend the measurement range beyond the limits of the artificial wavelength and geometrical depth-of-focus. With complex wave field propagation, the focus plane can be shifted after the camera images have been taken and a sharp image with extended depth of focus is constructed consequently. With 20mm x 20mm field of view the sensor enables measurement of both macro- and micro-structure (such as tool marks) with an axial resolution of 1 µm, lateral resolution of 7 µm and consequently allows processing data to be generated online which in turn qualifies it as a machine tool control. To make HoloCut compact enough for operation inside a machining center, the beams are arranged in two planes: The beams are split into reference beam and object beam in the bottom plane and combined onto the camera in the top plane later on. Using a mechanical standard interface according to DIN 69893 and having a very compact size of 235mm x 140mm x 215mm (WxHxD) and a weight of 7.5 kg, HoloCut can be easily integrated into different machine tools and extends no more in height than a typical processing tool.

Tertiary structural changes and iron release from human serum transferrin.

PubMed

Mecklenburg, S L; Donohoe, R J; Olah, G A

1997-08-01

Iron release from human serum transferrin was investigated by comparison of the extent of bound iron, measured by charge transfer absorption band intensity (465 nm), with changes observed by small-angle solution X-ray scattering (SAXS) for a series of equilibrated samples between pH 5.69 and 7.77. The phosphate buffers used in this study promote iron release at relatively high pH values, with an empirical pK of 6.9 for the convolved release from the two sites. The spectral data reveal that the N-lobe release is nearly complete by pH 7.0, while the C-lobe remains primarily metal-laden. Conversely, the radius of gyration, Rg, determined from the SAXS data remains constant between pH 7.77 and 7.05, and the evolution of Rg between its value observed for the diferric protein at pH 7.77 (31.2+/-0.2 A) and that of the apo protein at pH 5.69 (33.9+/-0.4 A) exhibits an empirical pK of 6.6. While Rg is effectively constant in the pH range associated with iron release from the N-lobe, the radius of gyration of cross-section, Rc, increases from 16.9+/-0.2 A to 17.6+/-0.2 A. Model simulations suggest that two different rotations of the NII domain relative to the NI domain about a hinge deep in the iron-binding cleft of the N-lobe, one parallel with and one perpendicular to the plane of the iron-binding site, can be significantly advanced relative to their holo protein positions while yielding constant Rg and increased Rc values consistent with the scattering data. Rotation of the CII domain parallel with the C-lobe iron-binding site plane can partially account for the increased Rg values measured at low pH; however, no reasonable combined repositioning of the NII and CII domains yields the experimentally observed increase in Rg.

Proceedings of the Annual Gaseous Electronics Conference (40th) Held in Atlanta Georgia on 13-16 October 1987

DTIC Science & Technology

1988-07-01

BURNING PLASMA ARC BY A COMBINATION OF HOLOGRAPHIC INTERFEROMETRY AND EMISSION SPECTROSCOPY A. Shah, M. S. Dassanayake and K. Etemadi 5:03 - 5:16 NB...Free- burning Plasma Arc by a Combination of Holo- graphic Interferometry and Emission Spectros- copy, A. SHAH, M. S. DASSANAYAKE,AND K. ETEMA- DI

Effect of configuration and some processing variables on the properties of wood fiber-polyethylene composites

Treesearch

Chin-Yin Hwang; Chung-Yun Hse; Elvin T. Choong

1999-01-01

Chemical compositions and fiber measurement of virgin and recycled fibers from three sources were determined. Results revealed that virgin southern pine fiber had highest alcohol-benzene extractive and lignin contents and lowest holo- and alpha-cellulose content among the three fiber types. Fiber length distribution of virgin fiber was less sensitive to disintegration...

Augmenting the Thermal Flux Experiment: A Mixed Reality Approach with the HoloLens

ERIC Educational Resources Information Center

Strzys, M. P.; Kapp, S.; Thees, M.; Kuhn, J.; Lukowicz, P.; Knierim, P.; Schmidt, A.

2017-01-01

In the field of Virtual Reality (VR) and Augmented Reality (AR), technologies have made huge progress during the last years and also reached the field of education. The virtuality continuum, ranging from pure virtuality on one side to the real world on the other, has been successfully covered by the use of immersive technologies like head-mounted…

Proteasome dynamics between proliferation and quiescence stages of Saccharomyces cerevisiae.

PubMed

Yedidi, Ravikiran S; Fatehi, Amatullah K; Enenkel, Cordula

The ubiquitin-proteasome system (UPS) plays a critical role in cellular protein homeostasis and is required for the turnover of short-lived and unwanted proteins, which are targeted by poly-ubiquitination for degradation. Proteasome is the key protease of UPS and consists of multiple subunits, which are organized into a catalytic core particle (CP) and a regulatory particle (RP). In Saccharomyces cerevisiae, proteasome holo-enzymes are engaged in degrading poly-ubiquitinated substrates and are mostly localized in the nucleus during cell proliferation. While in quiescence, the RP and CP are sequestered into motile and reversible storage granules in the cytoplasm, called proteasome storage granules (PSGs). The reversible nature of PSGs allows the proteasomes to be transported back into the nucleus upon exit from quiescence. Nuclear import of RP and CP through nuclear pores occurs via the canonical pathway that includes the importin-αβ heterodimer and takes advantage of the Ran-GTP gradient across the nuclear membrane. Dependent on the growth stage, either inactive precursor complexes or mature holo-enzymes are imported into the nucleus. The present review discusses the dynamics of proteasomes including their assembly, nucleo-cytoplasmic transport during proliferation and the sequestration of proteasomes into PSGs during quiescence. [Formula: see text].

Localization and Function of the Membrane-bound Riboflavin in the Na+-translocating NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae*

PubMed Central

Casutt, Marco S.; Huber, Tamara; Brunisholz, René; Tao, Minli; Fritz, Günter; Steuber, Julia

2010-01-01

The sodium ion-translocating NADH:quinone oxidoreductase (Na+-NQR) from the human pathogen Vibrio cholerae is a respiratory membrane protein complex that couples the oxidation of NADH to the transport of Na+ across the bacterial membrane. The Na+-NQR comprises the six subunits NqrABCDEF, but the stoichiometry and arrangement of these subunits are unknown. Redox-active cofactors are FAD and a 2Fe-2S cluster on NqrF, covalently attached FMNs on NqrB and NqrC, and riboflavin and ubiquinone-8 with unknown localization in the complex. By analyzing the cofactor content and NADH oxidation activity of subcomplexes of the Na+-NQR lacking individual subunits, the riboflavin cofactor was unequivocally assigned to the membrane-bound NqrB subunit. Quantitative analysis of the N-terminal amino acids of the holo-complex revealed that NqrB is present in a single copy in the holo-complex. It is concluded that the hydrophobic NqrB harbors one riboflavin in addition to its covalently attached FMN. The catalytic role of two flavins in subunit NqrB during the reduction of ubiquinone to ubiquinol by the Na+-NQR is discussed. PMID:20558724

Joint neutron crystallographic and NMR solution studies of Tyr residue ionization and hydrogen bonding: Implications for enzyme-mediated proton transfer

PubMed Central

Michalczyk, Ryszard; Unkefer, Clifford J.; Bacik, John-Paul; Schrader, Tobias E.; Ostermann, Andreas; Kovalevsky, Andrey Y.; McKenna, Robert; Fisher, Suzanne Zoë

2015-01-01

Human carbonic anhydrase II (HCA II) uses a Zn-bound OH−/H2O mechanism to catalyze the reversible hydration of CO2. This catalysis also involves a separate proton transfer step, mediated by an ordered solvent network coordinated by hydrophilic residues. One of these residues, Tyr7, was previously shown to be deprotonated in the neutron crystal structure at pH 10. This observation indicated that Tyr7 has a perturbed pKa compared with free tyrosine. To further probe the pKa of this residue, NMR spectroscopic measurements of [13C]Tyr-labeled holo HCA II (with active-site Zn present) were preformed to titrate all Tyr residues between pH 5.4–11.0. In addition, neutron studies of apo HCA II (with Zn removed from the active site) at pH 7.5 and holo HCA II at pH 6 were conducted. This detailed interrogation of tyrosines in HCA II by NMR and neutron crystallography revealed a significantly lowered pKa of Tyr7 and how pH and Tyr proximity to Zn affect hydrogen-bonding interactions. PMID:25902526

Inhibition of HSV cell-to-cell spread by lactoferrin and lactoferricin.

PubMed

Jenssen, Håvard; Sandvik, Kjersti; Andersen, Jeanette H; Hancock, Robert E W; Gutteberg, Tore J

2008-09-01

The milk protein lactoferrin (Lf) has multiple functions, including immune stimulation and antiviral activity towards herpes simplex virus 1 and 2 (HSV-1 and HSV-2); antiviral activity has also been reported for the N-terminal pepsin-derived fragment lactoferricin (Lfcin). The anti-HSV mode of action of Lf and Lfcin is assumed to involve, in part, their interaction with the cell surface glycosaminoglycan heparan sulfate, thereby blocking of viral entry. In this study we investigated the ability of human and bovine Lf and Lfcin to inhibit viral cell-to-cell spread as well as the involvement of cell surface glycosaminoglycans during viral cell-to-cell spread. Lf and Lfcin from both human and bovine origin, inhibited cell-to-cell spread of both HSV-1 and HSV-2. Inhibition of cell-to-cell spread by bovine Lfcin involved cell surface chondroitin sulfate. Based on transmission electron microscopy studies, human Lfcin, like bovine Lfcin, was randomly distributed intracellularly, thus differences in their antiviral activity could not be explained by differences in their distribution. In contrast, the cellular localization of iron-saturated (holo)-Lf appeared to differ from that of apo-Lf, indicating that holo- and apo-Lf may exhibit different antiviral mechanisms.

Preparation of holo- and malonyl-[acyl-carrier-protein] in a manner suitable for analog development.

PubMed

Marcella, Aaron M; Jing, Fuyuan; Barb, Adam W

2015-11-01

The fatty acid biosynthetic pathway generates highly reduced carbon based molecules. For this reason fatty acid synthesis is a target of pathway engineering to produce novel specialty or commodity chemicals using renewable techniques to supplant molecules currently derived from petroleum. Malonyl-[acyl carrier protein] (malonyl-ACP) is a key metabolite in the fatty acid pathway and donates two carbon units to the growing fatty acid chain during each step of biosynthesis. Attempts to test engineered fatty acid biosynthesis enzymes in vitro will require malonyl-ACP or malonyl-ACP analogs. Malonyl-ACP is challenging to prepare due to the instability of the carboxylate leaving group and the multiple steps of post-translational modification required to activate ACP. Here we report the expression and purification of holo- and malonyl-ACP from Escherichia coli with high yields (>15 mg per L of expression). The malonyl-ACP is efficiently recognized by the E. coli keto-acyl synthase enzyme, FabH. A FabH assay using malonyl-ACP and a coumarin-based fluorescent reagent is described that provides a high throughput alternative to reported radioactive assays. Copyright © 2015 Elsevier Inc. All rights reserved.

Free-energy landscape, principal component analysis, and structural clustering to identify representative conformations from molecular dynamics simulations: the myoglobin case.

PubMed

Papaleo, Elena; Mereghetti, Paolo; Fantucci, Piercarlo; Grandori, Rita; De Gioia, Luca

2009-01-01

Several molecular dynamics (MD) simulations were used to sample conformations in the neighborhood of the native structure of holo-myoglobin (holo-Mb), collecting trajectories spanning 0.22 micros at 300 K. Principal component (PCA) and free-energy landscape (FEL) analyses, integrated by cluster analysis, which was performed considering the position and structures of the individual helices of the globin fold, were carried out. The coherence between the different structural clusters and the basins of the FEL, together with the convergence of parameters derived by PCA indicates that an accurate description of the Mb conformational space around the native state was achieved by multiple MD trajectories spanning at least 0.14 micros. The integration of FEL, PCA, and structural clustering was shown to be a very useful approach to gain an overall view of the conformational landscape accessible to a protein and to identify representative protein substates. This method could be also used to investigate the conformational and dynamical properties of Mb apo-, mutant, or delete versions, in which greater conformational variability is expected and, therefore identification of representative substates from the simulations is relevant to disclose structure-function relationship.

Allosteric ligands for the pharmacologically important Flavivirus target (NS5) from ZINC database based on pharmacophoric points, free energy calculations and dynamics correlation.

PubMed

Khan, Abbas; Saleem, Shoaib; Idrees, Muhammad; Ali, Syed Shujait; Junaid, Muhammad; Chandra Kaushik, Aman; Wei, Dong-Qing

2018-04-11

Dengue virus belongs to a group of human pathogens, which causes different diseases, dengue hemorrhagic fever and dengue shock syndrome in humans. It possesses RNA as a genetic material and is replicated with the aid of NS5 protein. RNA-dependent RNA polymerase (RdRp) is an important domain of NS5 in the replication of that virus. The catalytic process activity of RdRp is making it an important target for antiviral chemical therapy. To date, No FDA drug has been approved and marketed for the treatment of diseases caused by Dengue virus. So, there is a dire need to advance an area of active antiviral inhibitors that is safe, less expensive and widely available. An experimentally validated complex of Dengue NS5 and compound 27 (6LS) were used as pharmacophoric input and hits were identified. We also used Molecular dynamics (MD) simulations alongside free energy and dynamics of the internal residues of the apo and holo systems to understand the binding mechanism. Our analysis resulted that the three inhibitors (ZINC72070002, ZINC6551486, and ZINC39588257) greatly affected the interior dynamics and residual signaling to dysfunction the replicative role of NS5. The interaction of these inhibitors caused the loss of the correlated motion of NS5 near to the N terminus and helped the stability of the binding complex. This investigation provided a methodological route to discover allosteric inhibitors against the epidemics of this Flaviviruses. Allosteric inhibitors are important and major assets in considering the development of the competitive and robust antiviral agents such as against Dengue viral infection. Copyright © 2018 Elsevier Inc. All rights reserved.

N-terminus determines activity and specificity of styrene monooxygenase reductases.

PubMed

Heine, Thomas; Scholtissek, Anika; Westphal, Adrie H; van Berkel, Willem J H; Tischler, Dirk

2017-12-01

Styrene monooxygenases (SMOs) are two-enzyme systems that catalyze the enantioselective epoxidation of styrene to (S)-styrene oxide. The FADH 2 co-substrate of the epoxidase component (StyA) is supplied by an NADH-dependent flavin reductase (StyB). The genome of Rhodococcus opacus 1CP encodes two SMO systems. One system, which we define as E1-type, displays homology to the SMO from Pseudomonas taiwanensis VLB120. The other system, originally reported as a fused system (RoStyA2B), is defined as E2-type. Here we found that E1-type RoStyB is inhibited by FMN, while RoStyA2B is known to be active with FMN. To rationalize the observed specificity of RoStyB for FAD, we generated an artificial reductase, designated as RoStyBart, in which the first 22 amino acid residues of RoStyB were joined to the reductase part of RoStyA2B, while the oxygenase part (A2) was removed. RoStyBart mainly purified as apo-protein and mimicked RoStyB in being inhibited by FMN. Pre-incubation with FAD yielded a turnover number at 30°C of 133.9±3.5s -1 , one of the highest rates observed for StyB reductases. RoStyBart holo-enzyme switches to a ping-pong mechanism and fluorescence analysis indicated for unproductive binding of FMN to the second (co-substrate) binding site. In summary, it is shown for the first time that optimization of the N-termini of StyB reductases allows the evolution of their activity and specificity. Copyright © 2017 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jha, Ramesh K.; Kern, Teresa L.; Kim, Youngchang

A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires access to an expanded repertoire of TFs. Using homology modeling and ligand docking for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to ‘sense’ a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 107 variants of PobR, four were active when dosed with pNP, with two mutants showing a specificity switch frommore » the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production from hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show the fluorescence of the cells correlated with the catalytic efficiency of the PTE variant expressed in each cell. High selectivity between similar molecules (4HB versus pNP), high sensitivity for pNP detection (~2 μM) and agreement of apo- and holo-structures of PobR scaffold with predetermined computational models are other significant results presented in this work.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jha, Ramesh K.; Kern, Theresa L.; Kim, Youngchang

A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires an access to an expanded repertoire of TFs. Using ligand docked homology models for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to ‘sense’ a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 10 7 variants of PobR, four were active when pNP was added as an inducer, with two mutants showingmore » a specificity switch from the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production and detect hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show that the fluorescence of the cells correlated with the catalytic efficiency of PTE variants, each cell expressed. High selectivity for similar molecules (4HB vs pNP), high sensitivity for pNP detection (~2 μM) and agreement of apo- and holo- structures of PobR scaffold with computational models are notable successes presented in this work.« less

Human bactericidal/permeability-increasing protein and a recombinant NH2-terminal fragment cause killing of serum-resistant gram-negative bacteria in whole blood and inhibit tumor necrosis factor release induced by the bacteria.

PubMed Central

Weiss, J; Elsbach, P; Shu, C; Castillo, J; Grinna, L; Horwitz, A; Theofan, G

1992-01-01

The bactericidal/permeability-increasing protein (BPI) of neutrophils and BPI fragments neutralize the effects of isolated Gram-negative bacterial lipopolysaccharides both in vitro and in vivo. Since endotoxin most commonly enters the host as constituents of invading Gram-negative bacteria, we raised the question: Can BPI and its bioactive fragments also protect against whole bacteria? To determine whether the bactericidal and endotoxin-neutralizing activities of BPI/fragments are expressed when Gram-negative bacteria are introduced to the complex environment of whole blood we examined the effects of added BPI and proteolytically prepared and recombinant NH2-terminal fragments on: (a) the fate of serum-resistant encapsulated Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa that survive the antibacterial actions of whole blood and (b) the ability of these bacteria to trigger cytokine release. Added BPI in nanomolar concentrations killed each of three encapsulated strains of E. coli and in closely parallel fashion inhibited tumor necrosis factor (TNF) release. Holo-BPI and its NH2-terminal fragment were equipotent toward a rough LPS chemotype K1-encapsulated strain, but the fragment was substantially more potent than holo-BPI toward two encapsulated smooth LPS chemotype strains. TNF release induced by K. pneumoniae and P. aeruginosa was also inhibited by both holo-BPI and fragment but, at the protein concentrations tested, P. aeruginosa was killed only by the fragment and K. pneumoniae was not killed by either protein. The bactericidal action of BPI/fragment toward E. coli is inhibited by C7-depleted serum, but accelerated by normal serum, indicating that BPI, acting in synergy with late complement components, enhances extracellular killing of serum-resistant bacteria. Thus, BPI and an even more potent NH2-terminal fragment may protect against Gram-negative bacteria in the host by blocking bacterial proliferation as well as endotoxin-mediated effects, not only as components of the intracellular antibacterial arsenal of the neutrophil, but also as potentially therapeutic extracellular agents. PMID:1522221

Human bactericidal/permeability-increasing protein and a recombinant NH2-terminal fragment cause killing of serum-resistant gram-negative bacteria in whole blood and inhibit tumor necrosis factor release induced by the bacteria.

PubMed

Weiss, J; Elsbach, P; Shu, C; Castillo, J; Grinna, L; Horwitz, A; Theofan, G

1992-09-01

The bactericidal/permeability-increasing protein (BPI) of neutrophils and BPI fragments neutralize the effects of isolated Gram-negative bacterial lipopolysaccharides both in vitro and in vivo. Since endotoxin most commonly enters the host as constituents of invading Gram-negative bacteria, we raised the question: Can BPI and its bioactive fragments also protect against whole bacteria? To determine whether the bactericidal and endotoxin-neutralizing activities of BPI/fragments are expressed when Gram-negative bacteria are introduced to the complex environment of whole blood we examined the effects of added BPI and proteolytically prepared and recombinant NH2-terminal fragments on: (a) the fate of serum-resistant encapsulated Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa that survive the antibacterial actions of whole blood and (b) the ability of these bacteria to trigger cytokine release. Added BPI in nanomolar concentrations killed each of three encapsulated strains of E. coli and in closely parallel fashion inhibited tumor necrosis factor (TNF) release. Holo-BPI and its NH2-terminal fragment were equipotent toward a rough LPS chemotype K1-encapsulated strain, but the fragment was substantially more potent than holo-BPI toward two encapsulated smooth LPS chemotype strains. TNF release induced by K. pneumoniae and P. aeruginosa was also inhibited by both holo-BPI and fragment but, at the protein concentrations tested, P. aeruginosa was killed only by the fragment and K. pneumoniae was not killed by either protein. The bactericidal action of BPI/fragment toward E. coli is inhibited by C7-depleted serum, but accelerated by normal serum, indicating that BPI, acting in synergy with late complement components, enhances extracellular killing of serum-resistant bacteria. Thus, BPI and an even more potent NH2-terminal fragment may protect against Gram-negative bacteria in the host by blocking bacterial proliferation as well as endotoxin-mediated effects, not only as components of the intracellular antibacterial arsenal of the neutrophil, but also as potentially therapeutic extracellular agents.

Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus.

PubMed

Halavaty, Andrei S; Rich, Rebecca L; Chen, Chao; Joo, Jeong Chan; Minasov, George; Dubrovska, Ievgeniia; Winsor, James R; Myszka, David G; Duban, Mark; Shuvalova, Ludmilla; Yakunin, Alexander F; Anderson, Wayne F

2015-05-01

When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL (SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NAD(+)) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD(+), NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme.

X-ray structural characterization of imidazolylcobalamin and histidinylcobalamin: cobalamin models for aquacobalamin bound to the B12 transporter protein transcobalamin.

PubMed

Hannibal, Luciana; Bunge, Scott D; van Eldik, Rudi; Jacobsen, Donald W; Kratky, Christoph; Gruber, Karl; Brasch, Nicola E

2007-04-30

The X-ray structures of imidazolylcobalamin (ImCbl) and histidinylcobalamin (HisCbl) are reported. These structures are of interest given that the recent structures of human and bovine transcobalamin prepared in their holo forms from aquacobalamin show a histidine residue of the metalloprotein bound at the beta-axial site of the cobalamin (Wuerges, J. et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 4386-4391). The beta-axial Co-N bond distances for ImCbl and HisCbl are 1.94(1) and 1.951(7) A, respectively. The alpha-axial Co-N bond distances to the 5,6-dimethylbenzimidazole are 2.01(1) and 1.979(8) A for ImCbl and HisCbl, respectively, and are typical for cobalamins with weak sigma-donor ligands at the beta-axial site. The corrin fold angles of 11.8(3) degrees (ImCbl) and 12.0(3) degrees (HisCbl) are smaller than those typically observed for cobalamins.

A comparison of acceleration methods for solving the neutron transport k-eigenvalue problem

NASA Astrophysics Data System (ADS)

Willert, Jeffrey; Park, H.; Knoll, D. A.

2014-10-01

Over the past several years a number of papers have been written describing modern techniques for numerically computing the dominant eigenvalue of the neutron transport criticality problem. These methods fall into two distinct categories. The first category of methods rewrite the multi-group k-eigenvalue problem as a nonlinear system of equations and solve the resulting system using either a Jacobian-Free Newton-Krylov (JFNK) method or Nonlinear Krylov Acceleration (NKA), a variant of Anderson Acceleration. These methods are generally successful in significantly reducing the number of transport sweeps required to compute the dominant eigenvalue. The second category of methods utilize Moment-Based Acceleration (or High-Order/Low-Order (HOLO) Acceleration). These methods solve a sequence of modified diffusion eigenvalue problems whose solutions converge to the solution of the original transport eigenvalue problem. This second class of methods is, in our experience, always superior to the first, as most of the computational work is eliminated by the acceleration from the LO diffusion system. In this paper, we review each of these methods. Our computational results support our claim that the choice of which nonlinear solver to use, JFNK or NKA, should be secondary. The primary computational savings result from the implementation of a HOLO algorithm. We display computational results for a series of challenging multi-dimensional test problems.

Durability and Damage Tolerance of Bismaleimide Composites. Volume 1. Technical Report

DTIC Science & Technology

1988-06-01

Test Setup Figure 1-14, Failed Loaded Holo Static Spoifton 106 mru FaJIus Stma. FuMr Materal Spwima anT VPIU h Kok Fo Faflurep t PldN Stromsym EVIM hm...Environrnental Chamber tot ETW TesI $ 1 36 12.5 x lOft.Ibs x 7ft-lbsI- __ 8.5 14ft-bs Figure 146. Outer Mold Une Impact Locations and Eery -Levela 5.3.5

NADH/NADPH bi-cofactor-utilizing and thermoactive ketol-acid reductoisomerase from Sulfolobus acidocaldarius.

PubMed

Chen, Chin-Yu; Ko, Tzu-Ping; Lin, Kuan-Fu; Lin, Bo-Lin; Huang, Chun-Hsiang; Chiang, Cheng-Hung; Horng, Jia-Cherng

2018-05-08

Ketol-acid reductoisomerase (KARI) is a bifunctional enzyme in the second step of branched-chain amino acids biosynthetic pathway. Most KARIs prefer NADPH as a cofactor. However, KARI with a preference for NADH is desirable in industrial applications including anaerobic fermentation for the production of branched-chain amino acids or biofuels. Here, we characterize a thermoacidophilic archaeal Sac-KARI from Sulfolobus acidocaldarius and present its crystal structure at a 1.75-Å resolution. By comparison with other holo-KARI structures, one sulphate ion is observed in each binding site for the 2'-phosphate of NADPH, implicating its NADPH preference. Sac-KARI has very high affinity for NADPH and NADH, with K M values of 0.4 μM for NADPH and 6.0 μM for NADH, suggesting that both are good cofactors at low concentrations although NADPH is favoured over NADH. Furthermore, Sac-KARI can catalyze 2(S)-acetolactate (2S-AL) with either cofactor from 25 to 60 °C, but the enzyme has higher activity by using NADPH. In addition, the catalytic activity of Sac-KARI increases significantly with elevated temperatures and reaches an optimum at 60 °C. Bi-cofactor utilization and the thermoactivity of Sac-KARI make it a potential candidate for use in metabolic engineering or industrial applications under anaerobic or harsh conditions.

Levels of plasma ceruloplasmin protein are markedly lower following dietary copper deficiency in rodents

PubMed Central

Broderius, Margaret; Mostad, Elise; Wendroth, Krista; Prohaska, Joseph R.

2010-01-01

Ceruloplasmin (Cp) is a multicopper oxidase and the most abundant copper binding protein in vertebrate plasma. Loss of function mutations in humans or experimental deletion in mice result in iron overload consistent with a putative ferroxidase function. Prior work suggested plasma may contain multiple ferroxidases. Studies were conducted in Holtzman rats (Rattus novegicus), albino mice (Mus musculus), Cp -/- mice, and adult humans (Homo sapiens) to investigate the copper-iron interaction. Dietary copper-deficient (CuD) rats and mice were produced using a modified AIN-76A diet. Results confirmed that o-dianisidine is a better substrate than paraphenylene diamine (PPD) for assessing diamine oxidase activity of Cp. Plasma from CuD rat dams and pups, and CuD and Cp -/- mice contained no detectable Cp diamine oxidase activity. Importantly, no ferroxidase activity was detectable for CuD rats, mice, or Cp -/- mice compared to robust activity for copper-adequate (CuA) rodent controls using western membrane assay. Immunoblot protocols detected major reductions (60-90%) in Cp protein in plasma of CuD rodents but no alteration in liver mRNA levels by qRT-PCR. Data are consistent with apo-Cp being less stable than holo-Cp. Further research is needed to explain normal plasma iron in CuD mice. Reduction in Cp is a sensitive biomarker for copper deficiency. PMID:20170749

A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor

DOE PAGES

Jha, Ramesh K.; Kern, Theresa L.; Kim, Youngchang; ...

2016-08-30

A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires an access to an expanded repertoire of TFs. Using ligand docked homology models for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to ‘sense’ a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 10 7 variants of PobR, four were active when pNP was added as an inducer, with two mutants showingmore » a specificity switch from the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production and detect hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show that the fluorescence of the cells correlated with the catalytic efficiency of PTE variants, each cell expressed. High selectivity for similar molecules (4HB vs pNP), high sensitivity for pNP detection (~2 μM) and agreement of apo- and holo- structures of PobR scaffold with computational models are notable successes presented in this work.« less

Isolation of thylakoid membrane complexes from rice by a new double-strips BN/SDS-PAGE and bioinformatics prediction of stromal ridge subunits interaction.

PubMed

Shao, Jinzhen; Zhang, Yubo; Yu, Jianlan; Guo, Lin; Ding, Yi

2011-01-01

Thylakoid membrane complexes of rice (Oryza sativa L.) play crucial roles in growth and crop production. Understanding of protein interactions within the complex would provide new insights into photosynthesis. Here, a new "Double-Strips BN/SDS-PAGE" method was employed to separate thylakoid membrane complexes in order to increase the protein abundance on 2D-gels and to facilitate the identification of hydrophobic transmembrane proteins. A total of 58 protein spots could be observed and subunit constitution of these complexes exhibited on 2D-gels. The generality of this new approach was confirmed using thylakoid membrane from spinach (Spinacia oleracea) and pumpkin (Cucurita spp). Furthermore, the proteins separated from rice thylakoid membrane were identified by the mass spectrometry (MS). The stromal ridge proteins PsaD and PsaE were identified both in the holo- and core- PSI complexes of rice. Using molecular dynamics simulation to explore the recognition mechanism of these subunits, we showed that salt bridge interactions between residues R19 of PsaC and E168 of PasD as well as R75 of PsaC and E91 of PsaD played important roles in the stability of the complex. This stromal ridge subunits interaction was also supported by the subsequent analysis of the binding free energy, the intramolecular distances and the intramolecular energy.

Binding of polarity-sensitive hydrophobic ligands to erythroid and nonerythroid spectrin: fluorescence and molecular modeling studies.

PubMed

Patra, Malay; Mitra, Madhurima; Chakrabarti, Abhijit; Mukhopadhyay, Chaitali

2014-01-01

We have used three polarity-sensitive fluorescence probes, 6-propionyl 2-(N,N-dimethyl-amino) naphthalene (Prodan), pyrene and 8-anilino 1-naphthalene sulphonic acid, to study their binding with erythroid and nonerythroid spectrin, using fluorescence spectroscopy. We have found that both bind to prodan and pyrene with high affinities with apparent dissociation constants (Kd) of .50 and .17 μM, for prodan, and .04 and .02 μM, for pyrene, respectively. The most striking aspect of these bindings have been that the binding stoichiometry have been equal to 1 in erythroid spectrin, both in dimeric and tetrameric form, and in tetrameric nonerythroid spectrin. From an estimate of apparent dielectric constants, the polarity of the binding site in both erythroid and nonerythroid forms have been found to be extremely hydrophobic. Thermodynamic parameters associated with such binding revealed that the binding is favored by positive change in entropy. Molecular docking studies alone indicate that both prodan and pyrene bind to the four major structural domains, following the order in the strength of binding to the Ankyrin binding domain > SH3 domain > Self-association domain > N-terminal domain of α-spectrin of both forms of spectrin. The binding experiments, particularly with the tetrameric nonerythroid spectrin, however, indicate more toward the self association domain in offering the unique binding site, since the binding stoichiometry have been 1 in all forms of dimeric and tetrameric spectrin, so far studied by us. Further studies are needed to characterize the hydrophobic binding sites in both forms of spectrin.

Interaction of small molecules with double-stranded RNA: spectroscopic, viscometric, and calorimetric study of hoechst and proflavine binding to PolyCG structures.

PubMed

Sinha, Rangana; Hossain, Maidul; Kumar, Gopinatha Suresh

2009-04-01

Design and synthesis of new small molecules binding to double-stranded RNA necessitate complete understanding of the molecular aspects of the binding of many existing molecules. Toward this goal, in this work we evaluated the biophysical aspects of the interaction of a DNA intercalator (proflavine) and a minor groove binder (hoechst 33258) with two polymorphic forms of polyCG, namely, the right-handed Watson-Crick base paired A-form and the left-handed Hoogsteen base paired H(L)-form, by absorption, fluorescence, and viscometry experiments. The energetics of the interaction of these molecules with the RNA structures has also been elucidated by isothermal titration calorimetry (ITC). Results suggest that proflavine strongly intercalates in both forms of polyCG, whereas hoechst shows mainly groove-binding modes. The binding of both drugs to both forms of RNA resulted in significant conformational change to the RNA structure with the bound molecules being placed in the chiral RNA helix. ITC profiles for both proflavine and hoechst show two binding sites. Binding of proflavine to both forms of RNA is endothermic and entropy driven in the first site and exothermic and enthalpy driven in the second site, whereas hoechst binding to both forms of RNA is exothermic and enthalpy driven in the first site and endothermic and entropy driven in the second site. This study suggests that the binding affinity characteristics and energetics of interaction of these DNA binding molecules with the RNA conformations are significantly different and may serve as data for future development of effective structure-selective RNA-based drugs.

Functional map of arrestin binding to phosphorylated opsin, with and without agonist.

PubMed

Peterhans, Christian; Lally, Ciara C M; Ostermaier, Martin K; Sommer, Martha E; Standfuss, Jörg

2016-06-28

Arrestins desensitize G protein-coupled receptors (GPCRs) and act as mediators of signalling. Here we investigated the interactions of arrestin-1 with two functionally distinct forms of the dim-light photoreceptor rhodopsin. Using unbiased scanning mutagenesis we probed the individual contribution of each arrestin residue to the interaction with the phosphorylated apo-receptor (Ops-P) and the agonist-bound form (Meta II-P). Disruption of the polar core or displacement of the C-tail strengthened binding to both receptor forms. In contrast, mutations of phosphate-binding residues (phosphosensors) suggest the phosphorylated receptor C-terminus binds arrestin differently for Meta II-P and Ops-P. Likewise, mutations within the inter-domain interface, variations in the receptor-binding loops and the C-edge of arrestin reveal different binding modes. In summary, our results indicate that arrestin-1 binding to Meta II-P and Ops-P is similarly dependent on arrestin activation, although the complexes formed with these two receptor forms are structurally distinct.

HoloHands: games console interface for controlling holographic optical manipulation

NASA Astrophysics Data System (ADS)

McDonald, C.; McPherson, M.; McDougall, C.; McGloin, D.

2012-10-01

The increased application of holographic optical manipulation techniques within the life sciences has sparked the development of accessible interfaces for control of holographic optical tweezers. Of particular interest are those that employ familiar, commercially available technologies. Here we present the use of a low cost games console interface, the Microsoft Kinect for the control of holographic optical tweezers and a study into the effect of using such a system upon the quality of trap generated.

Structural basis of nonribosomal peptide macrocyclization in fungi.

PubMed

Zhang, Jinru; Liu, Nicholas; Cacho, Ralph A; Gong, Zhou; Liu, Zhu; Qin, Wenming; Tang, Chun; Tang, Yi; Zhou, Jiahai

2016-12-01

Nonribosomal peptide synthetases (NRPSs) in fungi biosynthesize important pharmaceutical compounds, including penicillin, cyclosporine and echinocandin. To understand the fungal strategy of forging the macrocyclic peptide linkage, we determined the crystal structures of the terminal condensation-like (C T ) domain and the holo thiolation (T)-C T complex of Penicillium aethiopicum TqaA. The first, to our knowledge, structural depiction of the terminal module in a fungal NRPS provides a molecular blueprint for generating new macrocyclic peptide natural products.

Mixed Reality Meets Pharmaceutical Development.

PubMed

Forrest, William P; Mackey, Megan A; Shah, Vivek M; Hassell, Kerry M; Shah, Prashant; Wylie, Jennifer L; Gopinath, Janakiraman; Balderhaar, Henning; Li, Li; Wuelfing, W Peter; Helmy, Roy

2017-12-01

As science evolves, the need for more efficient and innovative knowledge transfer capabilities becomes evident. Advances in drug discovery and delivery sciences have directly impacted the pharmaceutical industry, though the added complexities have not shortened the development process. These added complexities also make it difficult for scientists to rapidly and effectively transfer knowledge to offset the lengthened drug development timelines. While webcams, camera phones, and iPads have been explored as potential new methods of real-time information sharing, the non-"hands-free" nature and lack of viewer and observer point-of-view render them unsuitable for the R&D laboratory or manufacturing setting. As an alternative solution, the Microsoft HoloLens mixed-reality headset was evaluated as a more efficient, hands-free method of knowledge transfer and information sharing. After completing a traditional method transfer between 3 R&D sites (Rahway, NJ; West Point, PA and Schnachen, Switzerland), a retrospective analysis of efficiency gain was performed through the comparison of a mock method transfer between NJ and PA sites using the HoloLens. The results demonstrated a minimum 10-fold gain in efficiency, weighing in from a savings in time, cost, and the ability to have real-time data analysis and discussion. In addition, other use cases were evaluated involving vendor and contract research/manufacturing organizations. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Yeast silencing factor Sir4 and a subset of nucleoporins form a complex distinct from nuclear pore complexes

PubMed Central

Ptak, Christopher; Roesner, Ulyss K.

2017-01-01

Interactions occurring at the nuclear envelope (NE)–chromatin interface influence both NE structure and chromatin organization. Insights into the functions of NE–chromatin interactions have come from the study of yeast subtelomeric chromatin and its association with the NE, including the identification of various proteins necessary for tethering subtelomeric chromatin to the NE and the silencing of resident genes. Here we show that four of these proteins—the silencing factor Sir4, NE-associated Esc1, the SUMO E3 ligase Siz2, and the nuclear pore complex (NPC) protein Nup170—physically and functionally interact with one another and a subset of NPC components (nucleoporins or Nups). Importantly, this group of Nups is largely restricted to members of the inner and outer NPC rings, but it lacks numerous others including cytoplasmically and nucleoplasmically positioned Nups. We propose that this Sir4-associated Nup complex is distinct from holo-NPCs and that it plays a role in subtelomeric chromatin organization and NE tethering. PMID:28883038

Yeast silencing factor Sir4 and a subset of nucleoporins form a complex distinct from nuclear pore complexes.

PubMed

Lapetina, Diego L; Ptak, Christopher; Roesner, Ulyss K; Wozniak, Richard W

2017-10-02

Interactions occurring at the nuclear envelope (NE)-chromatin interface influence both NE structure and chromatin organization. Insights into the functions of NE-chromatin interactions have come from the study of yeast subtelomeric chromatin and its association with the NE, including the identification of various proteins necessary for tethering subtelomeric chromatin to the NE and the silencing of resident genes. Here we show that four of these proteins-the silencing factor Sir4, NE-associated Esc1, the SUMO E3 ligase Siz2, and the nuclear pore complex (NPC) protein Nup170-physically and functionally interact with one another and a subset of NPC components (nucleoporins or Nups). Importantly, this group of Nups is largely restricted to members of the inner and outer NPC rings, but it lacks numerous others including cytoplasmically and nucleoplasmically positioned Nups. We propose that this Sir4-associated Nup complex is distinct from holo-NPCs and that it plays a role in subtelomeric chromatin organization and NE tethering. © 2017 Lapetina et al.

Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Halavaty, Andrei S.; Rich, Rebecca L.; Chen, Chao

When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL ( SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NADmore » +) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD +, NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Finally, five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme.« less

Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus

DOE PAGES

Halavaty, Andrei S.; Rich, Rebecca L.; Chen, Chao; ...

2015-04-25

When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL ( SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NADmore » +) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD +, NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Finally, five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme.« less

Switch on the engine: how the eukaryotic replicative helicase MCM2-7 becomes activated.

PubMed

Tognetti, Silvia; Riera, Alberto; Speck, Christian

2015-03-01

A crucial step during eukaryotic initiation of DNA replication is the correct loading and activation of the replicative DNA helicase, which ensures that each replication origin fires only once. Unregulated DNA helicase loading and activation, as it occurs in cancer, can cause severe DNA damage and genomic instability. The essential mini-chromosome maintenance proteins 2-7 (MCM2-7) represent the core of the eukaryotic replicative helicase that is loaded at DNA replication origins during G1-phase of the cell cycle. The MCM2-7 helicase activity, however, is only triggered during S-phase once the holo-helicase Cdc45-MCM2-7-GINS (CMG) has been formed. A large number of factors and several kinases interact and contribute to CMG formation and helicase activation, though the exact mechanisms remain unclear. Crucially, upon DNA damage, this reaction is temporarily halted to ensure genome integrity. Here, we review the current understanding of helicase activation; we focus on protein interactions during CMG formation, discuss structural changes during helicase activation, and outline similarities and differences of the prokaryotic and eukaryotic helicase activation process.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cody, V.; Pace, J.; Rosowsky, A.

It has been shown that 2,4-diamino-6-arylmethylpteridines and 2,4-diamino-5-arylmethylpyrimidines containing an O-carboxylalkyloxy group in the aryl moiety are potent and selective inhibitors of the dihydrofolate reductase (DHFR) from opportunistic pathogens such as Pneumocystis carinii, the causative agent of Pneumocystis pneumonia in HIV/AIDS patients. In order to understand the structure-activity profile observed for a series of substituted dibenz[b,f]azepine antifolates, the crystal structures of mouse DHFR (mDHFR; a mammalian homologue) holo and ternary complexes with NADPH and the inhibitor 2,4-diamino-6-(2{prime}-hydroxydibenz[b,f]azepin-5-yl)methylpteridine were determined to 1.9 and 1.4 A resolution, respectively. Structural data for the ternary complex with the potent O-(3-carboxypropyl) inhibitor PT684 revealed nomore » electron density for the O-carboxylalkyloxy side chain. The side chain was either cleaved or completely disordered. The electron density fitted the less potent hydroxyl compound PT684a. Additionally, cocrystallization of mDHFR with NADPH and the less potent 2{prime}-(4-carboxybenzyl) inhibitor PT682 showed no electron density for the inhibitor and resulted in the first report of a holoenzyme complex despite several attempts at crystallization of a ternary complex. Modeling data of PT682 in the active site of mDHFR and P. carinii DHFR (pcDHFR) indicate that binding would require ligand-induced conformational changes to the enzyme for the inhibitor to fit into the active site or that the inhibitor side chain would have to adopt an alternative binding mode to that observed for other carboxyalkyloxy inhibitors. These data also show that the mDHFR complexes have a decreased active-site volume as reflected in the relative shift of helix C (residues 59-64) by 0.6 A compared with pcDHFR ternary complexes. These data are consistent with the greater inhibitory potency against pcDHFR.« less

Temperature-induced conformational change at the catalytic site of Sulfolobus solfataricus alcohol dehydrogenase highlighted by Asn249Tyr substitution. A hydrogen/deuterium exchange, kinetic, and fluorescence quenching study.

PubMed

Secundo, Francesco; Russo, Consiglia; Giordano, Antonietta; Carrea, Giacomo; Rossi, Mosè; Raia, Carlo A

2005-08-23

A combination of hydrogen/deuterium exchange, fluorescence quenching, and kinetic studies was used to acquire experimental evidence for the crystallographically hypothesized increase in local flexibility which occurs in thermophilic NAD(+)-dependent Sulfolobus solfataricus alcohol dehydrogenase (SsADH) upon substitution Asn249Tyr. The substitution, located at the adenine-binding site, proved to decrease the affinity for both coenzyme and substrate, rendering the mutant enzyme 6-fold more active when compared to the wild-type enzyme [Esposito et al. (2003) FEBS Lett. 539, 14-18]. The amide H/D exchange data show that the wild-type and mutant enzymes have similar global flexibility at 22 and 60 degrees C. However, the temperature dependence of the Stern-Volmer constant determined by acrylamide quenching shows that the increase in temperature affects the local flexibility differently, since the K(SV) increment is significantly higher for the wild-type than for the mutant enzyme over the range 18-45 degrees C. Interestingly, the corresponding van't Hoff plot (log K(SV) vs 1/T) proves nonlinear for the apo and holo wild-type and apo mutant enzymes, with a break at approximately 45 degrees C in all three cases due to a conformational change affecting the tryptophan microenvironment experienced by the quencher molecules. The Arrhenius and van't Hoff plots derived from the k(cat) and K(M) thermodependence measured with cyclohexanol and NAD(+) at different temperatures display an abrupt change of slope at 45-50 degrees C. This proves more pronounced in the case of the mutant enzyme compared to the wild-type enzyme due to a conformational change in the structure rather than to an overlapping of two or more rate-limiting reaction steps with different temperature dependencies of their rate constants. Three-dimensional analysis indicates that the observed conformational change induced by temperature is associated with the flexible loops directly involved in the substrate and coenzyme binding.

Fractionating the Binding Process: Neuropsychological Evidence from Reversed Search Efficiencies

ERIC Educational Resources Information Center

Humphreys, Glyn W.; Hodsoll, John; Riddoch, M. Jane

2009-01-01

The authors present neuropsychological evidence distinguishing binding between form, color, and size (cross-domain binding) and binding between form elements. They contrasted conjunctive search with difficult feature search using control participants and patients with unilateral parietal or fronto/temporal lesions. To rule out effects of task…

Non-B-Form DNA Is Enriched at Centromeres

PubMed Central

Henikoff, Steven

2018-01-01

Abstract Animal and plant centromeres are embedded in repetitive “satellite” DNA, but are thought to be epigenetically specified. To define genetic characteristics of centromeres, we surveyed satellite DNA from diverse eukaryotes and identified variation in <10-bp dyad symmetries predicted to adopt non-B-form conformations. Organisms lacking centromeric dyad symmetries had binding sites for sequence-specific DNA-binding proteins with DNA-bending activity. For example, human and mouse centromeres are depleted for dyad symmetries, but are enriched for non-B-form DNA and are associated with binding sites for the conserved DNA-binding protein CENP-B, which is required for artificial centromere function but is paradoxically nonessential. We also detected dyad symmetries and predicted non-B-form DNA structures at neocentromeres, which form at ectopic loci. We propose that centromeres form at non-B-form DNA because of dyad symmetries or are strengthened by sequence-specific DNA binding proteins. This may resolve the CENP-B paradox and provide a general basis for centromere specification. PMID:29365169

Observation of Shear-Induced Turbulence Using HARLIE

NASA Technical Reports Server (NTRS)

Miller, David O.; Schwemmer, Geary K.; Wilkerson, Thomas D.; Sanders, Jason; Guerra, David; Moody, Steven

2000-01-01

Ground-based measurements of atmospheric aerosol structure were made using the Holographic Airborne Rotating Lidar Instrument Experiment (HARLIE) during the HOLO-1 field campaign. The scanning ability of HARLIE affords a unique opportunity to view various atmospheric phenomena. Shear-induced turbulence plays an important role in the transport of kinetic energy in the atmosphere and on March 10, 1999, several instances of shear-induced turbulence were observed via HARLIE. Using the data collected and upper-air wind profiles the nature of the instabilities is discussed.

Remote Sensing and Characterization of Oil on Water Using Coherent Fringe Projection and Holographic in-Line Interferometry

DTIC Science & Technology

2013-03-01

holo- graphic recording on photo-thermo-plastic structure ,” J. Modern Opt. 57(10), 854–858 (2010). 6. N. Kukhtarev and T. Kukhtareva, “ Dynamic ...RESPONSIBLE PERSON 19b. TELEPHONE NUMBER (Include area code) 21-10-2013 Journal Article Remote Sensing and Characterization of Oil on Water Using...green-blue region can also degrade oil. This finding indicates that properly structured laser clean-up can be an alternative method of decontamination

Replacing the Axial Ligand Tyrosine 75 or Its Hydrogen Bond Partner Histidine 83 Minimally Affects Hemin Acquisition by the Hemophore HasAp from Pseudomonas aeruginosa

PubMed Central

2015-01-01

Hemophores from Pseudomonas aeruginosa (HasAp), Serratia marcescens (HasAsm), and Yersinia pestis (HasAyp) bind hemin between two loops. One of the loops harbors conserved axial ligand Tyr75 (Y75 loop) in all three structures, whereas the second loop (H32 loop) contains axial ligand His32 in HasAp and HasAsm, but a noncoordinating Gln32 in HasAyp. Binding of hemin to the Y75 loop of HasAp or HasAsm causes a large rearrangement of the H32 loop that allows His32 coordination. The Q32 loop in apo-HasAyp is already in the closed conformation, such that binding of hemin to the conserved Y75 loop occurs with minimal structural rearrangement and without coordinative interaction with the Q32 loop. In this study, structural and spectroscopic investigations of the hemophore HasAp were conducted to probe (i) the role of the conserved Tyr75 loop in hemin binding and (ii) the proposed requirement of the His83–Tyr75 hydrogen bond to allow the coordination of hemin by Tyr75. High-resolution crystal structures of H83A holo-HasAp obtained at pH 6.5 (0.89 Å) and pH 5.4 (1.25 Å) show that Tyr75 remains coordinated to the heme iron, and that a water molecule can substitute for Nδ of His83 to interact with the Oη atom of Tyr75, likely stabilizing the Tyr75–Fe interaction. Nuclear magnetic resonance spectroscopy revealed that in apo-Y75A and apo-H83A HasAp, the Y75 loop is disordered, and that disorder propagates to nearby elements of secondary structure, suggesting that His83 Nδ–Tyr75 Oη interaction is important to the organization of the Y75 loop in apo-HasA. Kinetic analysis of hemin loading conducted via stopped-flow UV–vis and rapid-freeze-quench resonance Raman shows that both mutants load hemin with biphasic kinetic parameters that are not significantly dissimilar from those previously observed for wild-type HasAp. When the structural and kinetic data are taken together, a tentative model emerges, which suggests that HasA hemophores utilize hydrophobic, π–π stacking, and van der Waals interactions to load hemin efficiently, while axial ligation likely functions to slow hemin release, thus allowing the hemophore to meet the challenge of capturing hemin under inhospitable conditions and delivering it selectively to its cognate receptor. PMID:24625274

Mechanism of Copper Uptake from Blood Plasma Ceruloplasmin by Mammalian Cells

PubMed Central

Ramos, Danny; Vargas, Rebecca; Gaite, Michaella; Montgomery, Aaron; Linder, Maria C.

2016-01-01

Ceruloplasmin, the main copper binding protein in blood plasma, has been of particular interest for its role in efflux of iron from cells, but has additional functions. Here we tested the hypothesis that it releases its copper for cell uptake by interacting with a cell surface reductase and transporters, producing apoceruloplasmin. Uptake and transepithelial transport of copper from ceruloplasmin was demonstrated with mammary epithelial cell monolayers (PMC42) with tight junctions grown in bicameral chambers, and purified human 64Cu-labeled ceruloplasmin secreted by HepG2 cells. Monolayers took up virtually all the 64Cu over 16h and secreted half into the apical (milk) fluid. This was partly inhibited by Ag(I). The 64Cu in ceruloplasmin purified from plasma of 64Cu-injected mice accumulated linearly in mouse embryonic fibroblasts (MEFs) over 3-6h. Rates were somewhat higher in Ctr1+/+ versus Ctr1-/- cells, and 3-fold lower at 2°C. The ceruloplasmin-derived 64Cu could not be removed by extensive washing or trypsin treatment, and most was recovered in the cytosol. Actual cell copper (determined by furnace atomic absorption) increased markedly upon 24h exposure to holoceruloplasmin. This was accompanied by a conversion of holo to apoceruloplasmin in the culture medium and did not occur during incubation in the absence of cells. Four different endocytosis inhibitors failed to prevent 64Cu uptake from ceruloplasmin. High concentrations of non-radioactive Cu(II)- or Fe(III)-NTA (substrates for cell surface reductases), or Cu(I)-NTA (to compete for transporter uptake) almost eliminated uptake of 64Cu from ceruloplasmin. MEFs had cell surface reductase activity and expressed Steap 2 (but not Steaps 3 and 4 or dCytB). However, six-day siRNA treatment was insufficient to reduce activity or uptake. We conclude that ceruloplasmin is a circulating copper transport protein that may interact with Steap2 on the cell surface, forming apoceruloplasmin, and Cu(I) that enters cells through CTR1 and an unknown copper uptake transporter. PMID:26934375

The future of holographic technologies and their use by artists

NASA Astrophysics Data System (ADS)

Oliveira, S.; Richardson, M.

2013-02-01

The use of holographic technologies in the past has faced resistance in the artistic field. The most conservative artists and critiques saw the term "holographic" more as a technical subject than an artistic one. Nevertheless, to explore new forms to create art has been a constant challenge for any artist, whatever their field. At the end of the 20-century the concept that art can explore any field or subject, create a vision that is somehow technological, is part of the evolving artistic world. In the last two decades, in the search for new terminologies, scientists and artists have used the expression "Holographic" as a synonym of evolution, but with different meanings. Artists are using it as a new form of art call "Holo Art"; scientists see it as a "new" science technique where light takes an important part in the process, and can be used in various aspects of daily life, such as, security and medicine. This paper will explore artists who take the challenge of combining their art with new technologies and how they are viewed in a world where the question of 'what is and what isn't art' is very debatable. Other questions that will be explored are 'How can these techniques be useful to artists?' and 'How do artists challenge themselves to analyse the pros and cons of the results that can be obtained?'

The effects of distant healing performed by a spiritual healer on chronic pain: a randomized controlled trial.

PubMed

Tsubono, Kenjiro; Thomlinson, Paul; Shealy, C Norman

2009-01-01

Many individuals suffer from various kinds of chronic pain. Some controlled studies on distant healing for chronic pain exist, but no definitive conclusion has been established. To study the effects of distant healing performed by a professional Japanese healer on chronic pain. A double-blind randomized controlled study. Holos University, Fair Grove, Missouri. People suffering from chronic pain (not caused by clear organic diseases or that persists long after a reasonable period of healing following injuries or surgery) were recruited through local radio and newspaper advertising. Subjects were randomly assigned to a treatment group or control group using a double-blind procedure. All subjects met the healer at the initial session at Holos University. At the session, a 20-minute group meditation was performed. The healer went back to Japan after the session and started distant healing only to the treatment group for a 2-month period. All participants were asked to meditate for 20 minutes every day during this 2-month period. The visual analog scale and McGill Pain Questionnaire. A total of 17 subjects were recruited, and 16 subjects completed the study. Comparison of pretreatment and posttreatment visual analog scale indicated a slightly significant effect of distant healing (P=.056). The Present Pain Intensity Scale showed significant improvement in the treatment group compared to the control group (P=.0016). The Pain Rating Index showed improvement in the treatment group, but the difference between both groups was not statistically significant (P=.12).

The Hologram in My Hand: How Effective is Interactive Exploration of 3D Visualizations in Immersive Tangible Augmented Reality?

PubMed

Bach, Benjamin; Sicat, Ronell; Beyer, Johanna; Cordeil, Maxime; Pfister, Hanspeter

2018-01-01

We report on a controlled user study comparing three visualization environments for common 3D exploration. Our environments differ in how they exploit natural human perception and interaction capabilities. We compare an augmented-reality head-mounted display (Microsoft HoloLens), a handheld tablet, and a desktop setup. The novel head-mounted HoloLens display projects stereoscopic images of virtual content into a user's real world and allows for interaction in-situ at the spatial position of the 3D hologram. The tablet is able to interact with 3D content through touch, spatial positioning, and tangible markers, however, 3D content is still presented on a 2D surface. Our hypothesis is that visualization environments that match human perceptual and interaction capabilities better to the task at hand improve understanding of 3D visualizations. To better understand the space of display and interaction modalities in visualization environments, we first propose a classification based on three dimensions: perception, interaction, and the spatial and cognitive proximity of the two. Each technique in our study is located at a different position along these three dimensions. We asked 15 participants to perform four tasks, each task having different levels of difficulty for both spatial perception and degrees of freedom for interaction. Our results show that each of the tested environments is more effective for certain tasks, but that generally the desktop environment is still fastest and most precise in almost all cases.

Protein composition of catalytically active U7-dependent processing complexes assembled on histone pre-mRNA containing biotin and a photo-cleavable linker

PubMed Central

Skrajna, Aleksandra; Yang, Xiao-cui; Dadlez, Michał; Marzluff, William F; Dominski, Zbigniew

2018-01-01

Abstract 3′ end cleavage of metazoan replication-dependent histone pre-mRNAs requires the multi-subunit holo-U7 snRNP and the stem–loop binding protein (SLBP). The exact composition of the U7 snRNP and details of SLBP function in processing remain unclear. To identify components of the U7 snRNP in an unbiased manner, we developed a novel approach for purifying processing complexes from Drosophila and mouse nuclear extracts. In this method, catalytically active processing complexes are assembled in vitro on a cleavage-resistant histone pre-mRNA containing biotin and a photo-sensitive linker, and eluted from streptavidin beads by UV irradiation for direct analysis by mass spectrometry. In the purified processing complexes, Drosophila and mouse U7 snRNP have a remarkably similar composition, always being associated with CPSF73, CPSF100, symplekin and CstF64. Many other proteins previously implicated in the U7-dependent processing are not present. Drosophila U7 snRNP bound to histone pre-mRNA in the absence of SLBP contains the same subset of polyadenylation factors but is catalytically inactive and addition of recombinant SLBP is sufficient to trigger cleavage. This result suggests that Drosophila SLBP promotes a structural rearrangement of the processing complex, resulting in juxtaposition of the CPSF73 endonuclease with the cleavage site in the pre-mRNA substrate. PMID:29529248

Metallochaperone for Cu,Zn-superoxide dismutase (CCS) protein but not mRNA is higher in organs from copper-deficient mice and rats.

PubMed

Prohaska, Joseph R; Broderius, Margaret; Brokate, Bruce

2003-09-15

Cu,Zn-superoxide dismutase (SOD1) is an abundant metalloenzyme important in scavenging superoxide ions. Cu-deficient rats and mice have lower SOD1 activity and protein, possibly because apo-SOD1 is degraded faster than holo-SOD1. SOD1 interacts with and requires its metallochaperone CCS for donating copper. We produced dietary Cu deficiency in rodents to determine if the reduction in SOD1 was related to the level of its specific metallochaperone CCS. CCS levels determined by immunoblot were 2- to 3-fold higher in liver, heart, kidney, and brain from male Cu-deficient rats and mice under a variety of conditions. CCS was also higher in livers of Cu-deficient dams. Interestingly, CCS levels in brain of Cu-deficient mice were also higher even though SOD1 activity and protein were not altered, suggesting that the rise in CCS is correlated with altered Cu status rather than a direct result of lower SOD1. A DNA probe specific for rat CCS detected a single transcript by Northern blot hybridization with liver RNA. CCS mRNA levels in mouse and rat liver were not altered by dietary treatment. These results suggest a posttranscriptional mechanism for higher CCS protein when Cu is limiting in the cell, perhaps due to slower protein turnover. Elevation in CCS level is one of the most dramatic alterations in Cu binding proteins accompanying Cu deficiency and may be useful to assess Cu status.

The transformed glucocorticoid receptor has a lower steroid-binding affinity than the nontransformed receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nemoto, Takayuki; Ohara-Nemoto, Yuko; Denis, M.

1990-02-20

High-salt treatment of cytosolic glucocorticoid receptor (GR) preparations reduces the steroid-binding ability of the receptor and induces the conversion of the receptor from a nontransformed (non-DNA-binding) 9S form to a transformed (DNA-binding) 4S entity. Therefore, the authors decided to investigate the possible relationship between these two phenomena. The binding of ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) to the 9S form was almost saturated at a concentration of 20 nM, whereas ({sup 3}H)TA was hardly bound to the 4S form at this concentration. The 4S form was efficiently labeled at 200 nM. Scatchard analysis of the GR showed the presence of twomore » types of binding sites. In the absence of molybdate, the ratio of the lower affinity site was increased, but the total number of binding sites was not modified. The GR with the low ({sup 3}H)TA-binding affinity bound to DNA-cellulose even in its unliganded state, whereas the form with the high affinity did not. These results indicate that the transformed GR has a reduced ({sup 3}H)TA-binding affinity as compared to the nontransformed GR. The steroid-binding domain (amino acids 477-777) and the DNA- and steroid-binding domains (amino acids 415-777) of the human GR were expressed in Escherichia coli as protein A fused proteins. Taken together, these results suggest that the component(s) associating with the nontransformed GR, possibly the heat shock protein hsp 90, play(s) an important role in stabilizing the GR in a high-affinity state for steroids.« less

Different binding mechanisms of neutral and anionic poly-/perfluorinated chemicals to human transthyretin revealed by In silico models.

PubMed

Yang, Xianhai; Lyakurwa, Felichesmi; Xie, Hongbin; Chen, Jingwen; Li, Xuehua; Qiao, Xianliang; Cai, Xiyun

2017-09-01

Chemical forms-dependent binding interactions between phenolic compounds and human transthyretin (hTTR) have been elaborated previously. However, it is not known whether the binding interactions between ionizable halogenated alphatic compounds and hTTR also have the same manner. In this study, poly-/perfluorinated chemicals (PFCs) were selected as model compounds and molecular dynamic simulation was performed to investigate the binding mechanisms between PFCs and hTTR. Results show the binding interactions between the halogenated aliphatic compounds and hTTR are related to the chemical forms. The ionized groups of PFCs can form electrostatic interactions with the -NH + 3 groups of Lys 15 residues in hTTR and form hydrogen bonds with the residues of hTTR. By analyzing the molecular orbital energies of PFCs, we also found that the anionic groups (nucleophile) in PFCs could form electron donor - acceptor interactions with the -NH + 3 groups (electrophile) in Lys 15. The aforementioned orientational interactions make the ionized groups of the PFCs point toward the entry port of the binding site. The roles of fluorine atoms in the binding interactions were also explored. The fluorine atoms can influence the binding interactions via inductive effects. Appropriate molecular descriptors were selected to characterize these interactions, and two quantitative structure-activity relationship models were developed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Location of Bromide Ions in Tetragonal Lysozyme Crystals

NASA Technical Reports Server (NTRS)

Lim, Kap; Nadarajah, Arunan; Forsythe, Elizabeth L.; Pusey, Marc L.

1998-01-01

Anions have been shown to play a dominant role in the crystallization of chicken egg white lysozyme from salt solutions. Previous studies employing X-ray crystallography had found one chloride ion binding site in the tetragonal crystal form of the protein and four nitrate ion binding sites in the monoclinic form. In this study the anion positions in the tetragonal form were determined from the difference Fourier map obtained from lysozyme crystal grown in bromide and chloride solutions. Five possible anion binding sites were found in this manner. Some of these sites were in pockets containing basic residues while others were near neutral, but polar, residues. The sole chloride ion binding site found in previous studies was confirmed, while four of these sites corresponded to four binding sites found for nitrate ions in monoclinic crystals. The study suggests that most of the anion binding sites in lysozyme remain unchanged, even when different anions and different crystal forms of lysozyme are employed.

Locations of Bromide Ions in Tetragonal Lysozyme Crystals

NASA Technical Reports Server (NTRS)

Lim, Kap; Nadarajah, Arunan; Forsythe, Elizabeth L.; Pusey, Marc L.

1998-01-01

Anions have been shown to play a dominant role in the crystallization of chicken egg-white lysozyme from salt solutions. Previous studies employing X-ray crystallography have found one chloride ion binding site in the tetragonal crystal form of the protein and four nitrate ion binding sites in the monoclinic form. In this study the anion positions in the tetragonal form were determined from the difference Fourier map obtained from lysozyme crystals grown in bromide and chloride solutions. Five possible anion-binding sites were found in this manner. Some of these sites were in pockets containing basic residues while others were near neutral, but polar, residues. The sole chloride ion binding site found in previous studies was confirmed, while four further sites were found which corresponded to the four binding sites found for nitrate ions in monoclinic crystals. The study suggests that most of the anion-binding sites in lysozyme remain unchanged even when different anions and different crystal forms of lysozyme are employed.

Conformational change of adenosine deaminase during ligand-exchange in a crystal.

PubMed

Kinoshita, Takayoshi; Tada, Toshiji; Nakanishi, Isao

2008-08-15

Adenosine deaminase (ADA) perpetuates chronic inflammation by degrading extracellular adenosine which is toxic for lymphocytes. ADA has two distinct conformations: open form and closed form. From the crystal structures with various ligands, the non-nucleoside type inhibitors bind to the active site occupying the critical water-binding-position and sustain the open form of apo-ADA. In contrast, substrate mimics do not occupy the critical position, and induce the large conformational change to the closed form. However, it is difficult to predict the binding of (+)-erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), as it possesses characteristic parts of both the substrate and the non-nucleoside inhibitors. The crystal structure shows that EHNA binds to the open form through a novel recognition of the adenine base accompanying conformational change from the closed form of the PR-ADA complex in crystalline state.

Native American Languages Act. Hearing before the Committee on Indian Affairs, United States Senate. One Hundred Eighth Congress, First Session on S. 575 To Amend the Native American Languages Act To Provide for the Support of Native American Language Survival Schools (May 15, 2003).

ERIC Educational Resources Information Center

Congress of the U.S., Washington, DC. Senate Committee on Indian Affairs.

This document includes statements given at this hearing by the following: William Y. Brown; John Cheek, Jennifer Chock; Rita Coosewon; David Dinwoodie; William Demmert, Jr.; Joycelyn DesRosier; Mary Hermes; Carla Herrera; Leanne Hinton; Holo Ho'opai; Hon. Daniel K. Inouye; Lawrence D. Kaplan; Keiki Kawaiaea; Rosalyn, LaPier; Lisa LaRonge; Vina…

Cooperative Allosteric Ligand Binding in Calmodulin

NASA Astrophysics Data System (ADS)

Nandigrami, Prithviraj

Conformational dynamics is often essential for a protein's function. For example, proteins are able to communicate the effect of binding at one site to a distal region of the molecule through changes in its conformational dynamics. This so called allosteric coupling fine tunes the sensitivity of ligand binding to changes in concentration. A conformational change between a "closed" (apo) and an "open" (holo) conformation upon ligation often produces this coupling between binding sites. Enhanced sensitivity between the unbound and bound ensembles leads to a sharper binding curve. There are two basic conceptual frameworks that guide our visualization about ligand binding mechanisms. First, a ligand can stabilize the unstable "open" state from a dynamic ensemble of conformations within the unbound basin. This binding mechanism is called conformational selection. Second, a ligand can weakly bind to the low-affinity "closed" state followed by a conformational transition to the "open" state. In this dissertation, I focus on molecular dynamics simulations to understand microscopic origins of ligand binding cooperativity. A minimal model of allosteric binding transitions must include ligand binding/unbinding events, while capturing the transition mechanism between two distinct meta-stable free energy basins. Due in part to computational timescales limitations, work in this dissertation describes large-scale conformational transitions through a simplified, coarse-grained model based on the energy basins defined by the open and closed conformations of the protein Calmodulin (CaM). CaM is a ubiquitous calcium-binding protein consisting of two structurally similar globular domains connected by a flexible linker. The two domains of CaM, N-terminal domain (nCaM) and C-terminal domain (cCaM) consists of two helix-loop-helix motifs (the EF-hands) connected by a flexible linker. Each domain of CaM consists of two binding loops and binds 2 calcium ions each. The intact domain binds up to 4 calcium ions. The simulations use a coupled molecular dynamics/monte carlo scheme where the protein dynamics is simulated explicitly, while ligand binding/unbinding are treated implicitly. In the model, ligand binding/unbinding events coupled with a conformational change of the protein within the grand canonical ensemble. Here, ligand concentration is controlled through the chemical potential (micro). This allows us to use a simple thermodynamic model to analyze the simulated data and quantify binding cooperativity. Simulated binding titration curves are calculated through equilibrium simulations at different values of micro. First, I study domain opening transitions of isolated nCaM and cCaM in the absence of calcium. This work is motivated by results from a recent analytic variational model that predicts distinct domain opening transition mechanism for the domains of CaM. This is a surprising result because the domains have the same folded state topology. In the simulations, I find the two domains of CaM have distinct transition mechanism over a broad range of temperature, in harmony with the analytic predictions. In particular, the simulated transition mechanism of nCaM follows a two-state behavior, while domain opening in cCaM involves global unfolding and refolding of the tertiary structure. The unfolded intermediate also appears in the landscape of nCaM, but at a higher temperature than it appears in cCaM's energy landscape. This is consistent with nCaM's higher thermal stability. Under approximate physiological conditions, majority of the sampled transitions in cCaM involves unfolding and refolding during conformational change. Kinetically, the transient unfolding and refolding in cCaM significantly slows the domain opening and closing rates in cCaM. Second, I investigate the structural origins of binding affinity and allosteric cooperativity of binding 2 calcium-ions to each domain of CaM. In my work, I predict the order of binding strength of CaM's loops. I analyze simulated binding curves within the framework of the classic Monod-Wyman-Changeux (MWC) model of allostery to extract the binding free energies to the closed and open ensembles. The simulations predict that cCaM binds calcium with higher affinity and greater cooperativity than nCaM. Where it is possible to compare, these predictions are in good agreement with experimental results. The analysis of the simulations offers a rationale for why the two domains differ in cooperativity: the higher cooperativity of cCaM is due to larger difference in affinity of its binding loops. Third, I extend the work to investigate structural origins of binding cooperativity of 4 calcium-ions to intact CaM. I characterize the microscopic cooperativities of each ligation state and provide a kinetic description of the binding mechanism. Due to the heterogeneous nature of CaM's loops, as predicted in our simulations of isolated domains, I focus on investigating the influence of this heterogeneity on the kinetic flux of binding pathways as a function of concentration. The formalism developed for Network Models of protein folding kinetics, is used to evaluate the directed flux of all possible pathways between unligated and fully loaded CaM. (Abstract shortened by ProQuest.).

Construction of in vitro transcription system for Corynebacterium glutamicum and its use in the recognition of promoters of different classes.

PubMed

Holátko, Jiří; Silar, Radoslav; Rabatinová, Alžbeta; Sanderová, Hana; Halada, Petr; Nešvera, Jan; Krásný, Libor; Pátek, Miroslav

2012-10-01

To facilitate transcription studies in Corynebacterium glutamicum, we have developed an in vitro transcription system for this bacterium used as an industrial producer of amino acids and a model organism for actinobacteria. This system consists of C. glutamicum RNA polymerase (RNAP) core (α2, β, β'), a sigma factor and a promoter-carrying DNA template, that is specifically recognized by the RNAP holoenzyme formed. The RNAP core was purified from the C. glutamicum strain with the modified rpoC gene, which produced His-tagged β' subunit. The C. glutamicum sigA and sigH genes were cloned and overexpressed using the Escherichia coli plasmid vector, and the sigma subunits σ(A) and σ(H) were purified by affinity chromatography. Using the reconstituted C. glutamicum holo-RNAPs, recognition of the σ(A)- and σ(H)-dependent promoters and synthesis of the specific transcripts was demonstrated. The developed in vitro transcription system is a novel tool that can be used to directly prove the specific recognition of a promoter by the particular σ factor(s) and to analyze transcriptional control by various regulatory proteins in C. glutamicum.

Investigation of Inhibition Mechanism of Chemokine Receptor CCR5 by Micro-second Molecular Dynamics Simulations.

PubMed

Salmas, Ramin Ekhteiari; Yurtsever, Mine; Durdagi, Serdar

2015-08-24

Chemokine receptor 5 (CCR5) belongs to G protein coupled receptors (GPCRs) and plays an important role in treatment of human immunodeficiency virus (HIV) infection since HIV uses CCR5 protein as a co-receptor. Recently, the crystal structure of CCR5-bound complex with an approved anti-retroviral drug (maroviroc) was resolved. During the crystallization procedure, amino acid residues (i.e., Cys224, Arg225, Asn226 and Glu227) at the third intra-cellular loop were replaced by the rubredoxin for stability reasons. In the current study, we aimed to understand the impact of the incorporated rubredoxin on the conformations of TM domains of the target protein. For this reason, rubredoxin was deleted from the crystal structure and the missing amino acids were engineered. The resultant structure was subjected to long (μs) molecular dynamics (MD) simulations to shed light into the inhibitory mechanism. The derived model structure displayed a significant deviation in the cytoplasmic domain of TM5 and IC3 in the absence of rubredoxin. The principal component analyses (PCA) and MD trajectory analyses revealed important structural and dynamical differences at apo and holo forms of the CCR5.

Identification of Nucleic Acid Binding Sites on Translin-Associated Factor X (TRAX) Protein

PubMed Central

Gupta, Gagan Deep; Kumar, Vinay

2012-01-01

Translin and TRAX proteins play roles in very important cellular processes such as DNA recombination, spatial and temporal expression of mRNA, and in siRNA processing. Translin forms a homomeric nucleic acid binding complex and binds to ssDNA and RNA. However, a mutant translin construct that forms homomeric complex lacking nucleic acid binding activity is able to form fully active heteromeric translin-TRAX complex when co-expressed with TRAX. A substantial progress has been made in identifying translin sites that mediate its binding activity, while TRAX was thought not to bind DNA or RNA on its own. We here for the first time demonstrate nucleic acid binding to TRAX by crosslinking radiolabeled ssDNA to heteromeric translin-TRAX complex using UV-laser. The TRAX and translin, photochemically crosslinked with ssDNA, were individually detected on SDS-PAGE. We mutated two motifs in TRAX and translin, designated B2 and B3, to help define the nucleic acid binding sites in the TRAX sequence. The most pronounced effect was observed in the mutants of B3 motif that impaired nucleic acid binding activity of the heteromeric complexes. We suggest that both translin and TRAX are binding competent and contribute to the nucleic acid binding activity. PMID:22427937

Pulse-radiolysis studies on the interaction of one-electron reduced species with blue oxidases. Reduction of native and type-2-copper-depleted Vietnamese-lacquer-tree and Japanese-lacquer-tree laccases.

PubMed

O'Neill, P; Fielden, E M; Morpurgo, L; Agostinelli, E

1984-08-15

The interactions of one-electron reduced metronidazole (ArNO2.-) and O2.- with native and Type-2-copper-depleted Vietnamese- and Japanese-lacquer-tree laccases were studied in aqueous solution at pH 6.0 and 7.4 by using the technique of pulse radiolysis. On reaction with ArNO2.-, in the absence of O2, the holo- and the Type-2-copper-depleted proteins accept, with reduction of Type 1 copper, 2 and 1 reducing equivalents respectively. On reaction with O2.- of both holo- and Type-2-copper-depleted Vietnamese-lacquer-tree laccase, almost complete reduction of Type 1 copper was observed and, after completion of the reaction, some (less than 20%) reoxidation of Type 1 copper occurs. Reduction of Type 1 copper of the laccases by these one-electron donors occurs via a bimolecular step; however, the rate of reduction of Vietnamese-lacquer-tree laccase is over 10 times that of Japanese-lacquer-tree laccase. It is inferred that electrons enter the protein via Type 1 copper with, in the case of the holoprotein, subsequent rapid intramolecular transfer of 1 reducing equivalent within the protein. Furthermore it is suggested that intra-molecular electron transfer to Type 3 copper atoms is slow and, in the case of Type-2-copper-depleted protein, may not occur. This slow process may partially account for the variation of the catalytic activities of 'blue' oxidases.

A rapid fluorometric assay for the S-malonyltransacylase FabD and other sulfhydryl utilizing enzymes.

PubMed

Marcella, Aaron M; Barb, Adam W

The development of biorenewable chemicals will support green chemistry initiatives and supplement the catalog of starting materials available to the chemical industry. Bacterial fatty acid biosynthesis is being pursued as a source of protein catalysts to synthesize novel reduced carbon molecules in fermentation systems. The availability of methods to measure enzyme catalysis for native and engineered enzymes from this pathway remains a bottleneck because a simple quantitative enzyme assay for numerous enzymes does not exist. Here we present two variations of a fluorescence assay that is readily extendable to high-throughput screening and is appropriate for thiol consuming and generating enzymes including the Escherichia coli enzymes malonyl-coenzyme A transacylase (FabD) and keto-acylsynthase III (FabH). We measured K M values of 60 ± 20 µM (acetyl-CoA) and 20 ± 10 µM (malonyl-ACP) and a k cat of 7.4-9.0 s -1 with FabH. Assays of FabD included a precipitation step to remove the thiol-containing substrate holo-ACP from the reaction product coenzyme-A to estimate reaction rates. Analysis of initial velocity measurements revealed K M values of 60 ± 20 µM (malonyl-CoA) and 40 ± 10 µM (holo-ACP) and a k cat of 2100-2600 s -1 for the FabD enzyme. Our data show similar results when compared to existing radioactive and continuous coupled assays in terms of sensitivity while providing the benefit of simplicity, scalability and repeatability. Fluorescence detection also eliminates the need for radioactive substrates traditionally used to assay these enzymes.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Venkadesh, S.; Mandal, P.K.; Gautham, N., E-mail: n_gautham@hotmail.com

Highlights: {yields} This is the first crystal structure of a four-way junction with sticky ends. {yields} Four junction structures bind to each other and form a rhombic cavity. {yields} Each rhombus binds to others to form 'infinite' 2D tiles. {yields} This is an example of bottom-up fabrication of a DNA nano-lattice. -- Abstract: We report here the crystal structure of the partially self-complementary decameric sequence d(CGGCGGCCGC), which self assembles to form a four-way junction with sticky ends. Each junction binds to four others through Watson-Crick base pairing at the sticky ends to form a rhombic structure. The rhombuses bind tomore » each other and form two dimensional tiles. The tiles stack to form the crystal. The crystal diffracted in the space group P1 to a resolution of 2.5 A. The junction has the anti-parallel stacked-X conformation like other junction structures, though the formation of the rhombic net noticeably alters the details of the junction geometry.« less

Hydrolysis at One of the Two Nucleotide-binding Sites Drives the Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zoghbi, M. E.; Altenberg, G. A.

The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides “sandwiched” at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we usedmore » luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation.« less

Polymorphisms A387P in thrombospondin-4 and N700S in thrombospondin-1 perturb calcium binding sites.

PubMed

Stenina, Olga I; Ustinov, Valentin; Krukovets, Irene; Marinic, Tina; Topol, Eric J; Plow, Edward F

2005-11-01

Recent genetic studies have associated members of the thrombospondin (TSP) gene family with premature cardiovascular disease. The disease-associated polymorphisms lead to single amino acid changes in TSP-4 (A387P) and TSP-1 (N700S). These substitutions reside in adjacent domains of these highly homologous proteins. Secondary structural predictive programs and the homology of the domains harboring these amino acid substitutions to those in other proteins pointed to potential alterations of putative Ca2+ binding sites that reside in close proximity to the polymorphic amino acids. Since Ca2+ binding is critical for the structure and function of TSP family members, direct evidence for differences in Ca2+ binding by the polymorphic forms was sought. Using synthetic peptides and purified recombinant variant fragments bearing the amino acid substitutions, we measured differences in Tb3+ luminescence as an index of Ca2+ binding. The Tb3+ binding constants placed the TSP-1 region affected by N700S polymorphism among other high-affinity Ca2+ binding sites. The affinity of Ca2+ binding was lower for peptides (3.5-fold) and recombinant fragments (10-fold) containing the S700 vs. the N700 form. In TSP-4, the P387 form acquired an additional Ca2+ binding site absent in the A387 form. The results of our study suggest that both substitutions (A387P in TSP-4 and N700S in TSP-1) alter Ca2+ binding properties. Since these substitutions exert the opposite effects on Ca2+ binding, a decrease in TSP-1 and an increase in TSP-4, the two TSP variants are likely to influence cardiovascular functions in distinct but yet pathogenic ways.

Differential recognition of syk-binding sites by each of the two phosphotyrosine-binding pockets of the Vav SH2 domain.

PubMed

Chen, Chih-Hong; Piraner, Dan; Gorenstein, Nina M; Geahlen, Robert L; Beth Post, Carol

2013-11-01

The association of spleen tyrosine kinase (Syk), a central tyrosine kinase in B cell signaling, with Vav SH2 domain is controlled by phosphorylation of two closely spaced tyrosines in Syk linker B: Y342 and Y346. Previous studies established both singly phosphorylated and doubly phosphorylated forms play a role in signaling. The structure of the doubly phosphorylated form identified a new recognition of phosphotyrosine whereby two phosphotyrosines bind simultaneously to the Vav SH2 domain, one in the canonical pTyr pocket and one in the specificity pocket on the opposite side of the central β-sheet. It is unknown if the specificity pocket can bind phosphotyrosine independent of phosphotyrosine binding the pTyr pocket. To address this gap in knowledge, we determined the structure of the complex between Vav1 SH2 and a peptide (SykLB-YpY) modeling the singly phosphorylated-Y346 form of Syk with unphosphorylated Y342. The nuclear magnetic resonance (NMR) data conclusively establish that recognition of phosphotyrosine is swapped between the two pockets; phosphorylated pY346 binds the specificity pocket of Vav1 SH2, and unphosphorylated Y342 occupies what is normally the pTyr binding pocket. Nearly identical changes in chemical shifts occurred upon binding all three forms of singly and doubly phosphorylated peptides; however, somewhat smaller shift perturbations for SykLB-YpY from residues in regions of high internal mobility suggest that internal motions are coupled to binding affinity. The differential recognition that includes this swapped binding of phosphotyrosine to the specificity pocket of Vav SH2 increases the repertoire of possible phosphotyrosine binding by SH2 domains in regulating protein-protein interactions in cellular signaling. Copyright © 2013 Wiley Periodicals, Inc.

Structures of human thymidylate synthase R163K with dUMP, FdUMP and glutathione show asymmetric ligand binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gibson, Lydia M.; Celeste, Lesa R.; Lovelace, Leslie L.

Thymidylate synthase (TS) is a well validated target in cancer chemotherapy. Here, a new crystal form of the R163K variant of human TS (hTS) with five subunits per asymmetric part of the unit cell, all with loop 181-197 in the active conformation, is reported. This form allows binding studies by soaking crystals in artificial mother liquors containing ligands that bind in the active site. Using this approach, crystal structures of hTS complexes with FdUMP and dUMP were obtained, indicating that this form should facilitate high-throughput analysis of hTS complexes with drug candidates. Crystal soaking experiments using oxidized glutathione revealed thatmore » hTS binds this ligand. Interestingly, the two types of binding observed are both asymmetric. In one subunit of the physiological dimer covalent modification of the catalytic nucleophile Cys195 takes place, while in another dimer a noncovalent adduct with reduced glutathione is formed in one of the active sites.« less

Domain-Swapped Dimers of Intracellular Lipid-Binding Proteins: Evidence for Ordered Folding Intermediates.

PubMed

Assar, Zahra; Nossoni, Zahra; Wang, Wenjing; Santos, Elizabeth M; Kramer, Kevin; McCornack, Colin; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James H

2016-09-06

Human Cellular Retinol Binding Protein II (hCRBPII), a member of the intracellular lipid-binding protein family, is a monomeric protein responsible for the intracellular transport of retinol and retinal. Herein we report that hCRBPII forms an extensive domain-swapped dimer during bacterial expression. The domain-swapped region encompasses almost half of the protein. The dimer represents a novel structural architecture with the mouths of the two binding cavities facing each other, producing a new binding cavity that spans the length of the protein complex. Although wild-type hCRBPII forms the dimer, the propensity for dimerization can be substantially increased via mutation at Tyr60. The monomeric form of the wild-type protein represents the thermodynamically more stable species, making the domain-swapped dimer a kinetically trapped entity. Hypothetically, the wild-type protein has evolved to minimize dimerization of the folding intermediate through a critical hydrogen bond (Tyr60-Glu72) that disfavors the dimeric form. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of glycation on meloxicam binding to human serum albumin

NASA Astrophysics Data System (ADS)

Trynda-Lemiesz, Lilianna; Wiglusz, Katarzyna

2011-05-01

The current study reports a binding of meloxicam a pharmacologically important new generation, non-steroidal anti-inflammatory drug to glycated form of the human serum albumin (HSA). The interaction of the meloxicam with nonglycated and glycated albumin has been studied at pH 7.4 in 0.05 M sodium phosphate buffer with 0.1 M NaCl, using fluorescence quenching technique and circular dichroism spectroscopy. Results of the present study have shown that the meloxicam could bind both forms of albumin glycated and nonglycated at a site, which was close to the tryptophan residues. Similarly, how for native albumin glycated form has had one high affinity site for the drug with association constants of the order of 10 5 M -1. The glycation process of the HSA significantly has affected the impact of the meloxicam on the binding of other ligands such as warfarin and bilirubin. The affinity of the glycated albumin for bilirubin as for native albumin has been reduced by meloxicam but observed effect was weaker by half (about 20%) compared with nonglycated albumin. In contrast to the native albumin meloxicam binding to glycated form of the protein only slightly affected the binding of warfarin. It seemed possible that the effects on warfarin binding might be entirely attributable to the Lys 199 modification which was in site I.

Role of the Iron Axial Ligands of Heme Carrier HasA in Heme Uptake and Release*

PubMed Central

Caillet-Saguy, Célia; Piccioli, Mario; Turano, Paola; Lukat-Rodgers, Gudrun; Wolff, Nicolas; Rodgers, Kenton R.; Izadi-Pruneyre, Nadia; Delepierre, Muriel; Lecroisey, Anne

2012-01-01

The hemophore protein HasA from Serratia marcescens cycles between two states as follows: the heme-bound holoprotein, which functions as a carrier of the metal cofactor toward the membrane receptor HasR, and the heme-free apoprotein fishing for new porphyrin to be taken up after the heme has been delivered to HasR. Holo- and apo-forms differ for the conformation of the two loops L1 and L2, which provide the axial ligands of the iron through His32 and Tyr75, respectively. In the apo-form, loop L1 protrudes toward the solvent far away from loop L2; in the holoprotein, closing of the loops on the heme occurs upon establishment of the two axial coordination bonds. We have established that the two variants obtained via single point mutations of either axial ligand (namely H32A and Y75A) are both in the closed conformation. The presence of the heme and one out of two axial ligands is sufficient to establish a link between L1 and L2, thanks to the presence of coordinating solvent molecules. The latter are stabilized in the iron coordination environment by H-bond interactions with surrounding protein residues. The presence of such a water molecule in both variants is revealed here through a set of different spectroscopic techniques. Previous studies had shown that heme release and uptake processes occur via intermediate states characterized by a Tyr75-iron-bound form with open conformation of loop L1. Here, we demonstrate that these states do not naturally occur in the free protein but can only be driven by the interaction with the partner proteins. PMID:22700962

New Insights into Vitamin D Sterol-VDR Proteolysis, Allostery, Structure-Function from the Perspective of a Conformational Ensemble Model

PubMed Central

Mizwicki, Mathew T.; Bula, Craig M.; Bishop, June E.; Norman, Anthony W.

2007-01-01

Recently, we have developed a vitamin D sterol (VDS)-VDR conformational ensemble model. This model can be broken down into three individual, yet interlinked parts: a) the conformationally flexible VDS, b) the apo/holo-VDR helix-12 (H12) conformational ensemble, and c) the presence of two VDR ligand binding pockets (LBPs); one thermodynamically favored (the genomic pocket, G-pocket) and the other kinetically favored by VDSs (the alternative pocket, A-pocket). One focus of this study is to use directed VDR mutagenesis to 1) demonstrate H12 is stabilized in the transcriptionally active closed conformation (hVDR-c1) by three salt-bridges that span the length of H12 (cationic residues R154, K264 and R402), 2) to elucidate the VDR trypsin sites [R173 (hVDR-c1), K413 (hVDR-c2) and R402 (hVDR-c3)] and 3) demonstrate the apo-VDR H12 equilibrium can be shifted. The other focus of this study is to apply the model to generate a mechanistic understanding to discrepancies observed in structure-function data obtained with a variety of 1α,25(OH)2-vitamin D3 (1,25D) A-ring and side-chain analogs, and side-chain metabolites. We will demonstrate that these structure-function conundrums can be rationalized, for the most part by focusing on alterations in the VDS conformational flexibility and the elementary interaction between the VDS and the VDR A- and G-pockets, relative to the control, 1,25D. PMID:17368177

Binding patterns of vanadium ions with different valence states to human serum transferrin studied by HPLC/high-resolution ICP-MS.

PubMed

Nagaoka, Megumi Hamano; Yamazaki, Takeshi; Maitani, Tamio

2002-09-06

Vanadium (V) is an essential metal for mammals and has different valence states. In blood, V is bound to serum transferrin (Tf), a glycoprotein which has two metal-binding sites, and carbonate is generally required for the binding. In this study, the binding patterns of V(III), V(IV), and V(V) to human serum Tf (hTf) were analyzed using an HPLC system equipped with an anion-exchange column and directly connected to a high-resolution inductively coupled plasma-mass spectrometer for metal detection (51V). In affinity to hTf, the three ions were ranked V(III)>V(IV)>V(V) in the presence of bicarbonate and V(III) reverse congruent V(IV)>V(V) in the absence. Intermediates in the "open forms" binding to the respective sites were detected at the initial stage. V(IV) and V(V) were bound to the N-lobe site in the "closed form" and "open form," respectively. In the absence of bicarbonate, V ions with respective valence states were bound to hTf in the "open form." In terms of binding to hTf, tri-valent V was most favorable in the presence of bicarbonate.

HoloHands: games console interface for controlling holographic optical manipulation

NASA Astrophysics Data System (ADS)

McDonald, C.; McPherson, M.; McDougall, C.; McGloin, D.

2013-03-01

The increasing number of applications for holographic manipulation techniques has sparked the development of more accessible control interfaces. Here, we describe a holographic optical tweezers experiment which is controlled by gestures that are detected by a Microsoft Kinect. We demonstrate that this technique can be used to calibrate the tweezers using the Stokes drag method and compare this to automated calibrations. We also show that multiple particle manipulation can be handled. This is a promising new line of research for gesture-based control which could find applications in a wide variety of experimental situations.

Essential motions and energetic contributions of individual residues in a peptide bound to an SH3 domain.

PubMed Central

Kolafa, J; Perram, J W; Bywater, R P

2000-01-01

We have studied protein-ligand interactions by molecular dynamics simulations using software designed to exploit parallel computing architectures. The trajectories were analyzed to extract the essential motions and to estimate the individual contributions of fragments of the ligand to overall binding enthalpy. Two forms of the bound ligand are compared, one with the termini blocked by covalent derivatization, and one in the underivatized, zwitterionic form. The ends of the peptide tend to bind more loosely in the capped form. We can observe significant motions in the bound ligand and distinguish between motions of the peptide backbone and of the side chains. This could be useful in designing ligands, which fit optimally to the binding protein. We show that it is possible to determine the different contributions of each residue in a peptide to the enthalpy of binding. Proline is a major net contributor to binding enthalpy, in keeping with the known propensity for this family of proteins to bind proline-rich peptides. PMID:10919999

Point mutation increases a form of the NK1 receptor with high affinity for neurokinin A and B and septide

PubMed Central

Ciucci, Alessandra; Palma, Carla; Manzini, Stefano; Werge, Thomas M

1998-01-01

The binding modalities of substance P and neurokinin A on the wild type and Gly166 to-Cys mutant NK1 receptors expressed on CHO cells were investigated in homologous and heterologous binding experiments using both radiolabelled substance P and neurokinin A.On the wild type NK1 receptor NKA displaces radiolabelled substance P with very low apparent affinity, despite its high-affinity binding constant (determined in homologous binding experiments). The Gly166 to-Cys substitution in the NK1 tachykinin receptor greatly enhances the apparent affinity of neurokinin A in competition for radiolabelled substance P, but it does not change the binding constant of neurokinin A. The mutation, thereby, eliminates the discrepancy between the low apparent affinity and the high binding constant of neurokinin A.On the wild type receptor the binding capacity of neurokinin A is significantly smaller than that of substance P. In contrast, the two tachykinins bind to approximately the same number of sites on the mutant receptor.Simultaneous mass action law analysis of binding data in which multiple radioligands were employed in parallel demonstrated that a one-site model was unable to accommodate all the experimental data, whereas a two-site model provided a dramatically better description.These two receptor-sites display equally high affinity for substance P, while neurokinin A strongly discriminates between a high and a low affinity component. The binding affinities of neurokinin A are not affected by the mutation, which instead specifically alters the distribution between receptor sites in favour of a high affinity neurokinin A binding form.The low apparent affinity and binding capacity of neurokinin A on the wild type receptor results from neurokinin A binding with high affinity only to a fraction of the sites labelled by substance P. The mutation increases the proportion of this site, and consequently enhances the apparent affinity and binding capacity of neurokinin A.The binding modalities of septide-like ligands (i.e. neurokinin B, SP(6-11), SP-methyl ester) are affected similarly to neurokinin A and are better resolved into two sites. The mutation leaves the affinity of these ligands for the two receptor forms unchanged, but increases the fraction of high-affinity sites. On the other hand, the binding of non-peptide and peptide antagonists (SR140.333 and FK888) behaved similarly to substance P with a single high affinity site that is unaffected by the mutation.These findings may suggest that the NK1 receptor exists in two different forms with similar affinity for substance P and NK1 antagonists, but with a high and a low affinity for neurokinin A and septide-like ligands. Hence, the Gly166 in the NK1 receptor would seem to control the distribution between a pan-reactive form and a substance P-selective form of the receptor. PMID:9786514

Crystal structure of the solute-binding protein BxlE from Streptomyces thermoviolaceus OPC-520 complexed with xylobiose.

PubMed

Tomoo, Koji; Miki, Yasuhiro; Morioka, Hideaki; Seike, Kiho; Ishida, Toshimasa; Ikenishi, Sadao; Miyamoto, Katsushiro; Hasegawa, Tomokazu; Yamano, Akihito; Hamada, Kensaku; Tsujibo, Hiroshi

2017-06-01

BxlE from Streptomyces thermoviolaceus OPC-520 is a xylo-oligosaccharide (mainly xylobiose)-binding protein that serves as the initial receptor for the bacterial ABC-type xylo-oligosaccharide transport system. To determine the ligand-binding mechanism of BxlE, X-ray structures of ligand-free (open form) and ligand (xylobiose)-bound (closed form) BxlE were determined at 1.85 Å resolution. BxlE consists of two globular domains that are linked by two β-strands, with the cleft at the interface of the two domains creating the ligand-binding pocket. In the ligand-free open form, this pocket consists of a U-shaped and negatively charged groove located between the two domains. In the xylobiose-bound closed form of BxlE, both the N and C domains move to fold the ligand without conformational changes in either domain. Xylobiose is buried in the groove and wrapped by the N-domain mainly via hydrogen bond interactions and by the C-domain primarily via non-polar interactions with Trp side chains. In addition to the concave shape matching the binding of xylobiose, an inter-domain salt bridge between Asp-47 and Lys-294 limits the space in the ligand-binding site. This domain-stabilized mechanism of ligand binding to BxlE is a unique feature that is not observed with other solute-binding proteins. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Free energy changes and components implicit in the MWC allosteric model for the cooperative oxygen binding of hemoglobin.#

PubMed Central

Bucci, Enrico

2013-01-01

Hill’s plots of oxygen binding isotherms reveal the presence of a transition between two different oxygen affinities at the beginning and end of the isotherm. They correspond to the two conformations anticipated by the MWC model, namely the T and R conformations at the beginning and end of oxygen binding, when the lower affinity of the T form develops into the higher affinity of the R form. The difference between the binding Gibbs free energies changes of the two affinities (ΔGL) is the free energy of binding cooperativity. Notably ΔGL is positive in favor of the T form, that moves to a higher energy level upon oxygen release. Osmotic stress reveals a higher volume/surface ratio of deoxyHb, with a positive ΔGW also in favor of the T form . Increasing protein concentration shifts the isotherms to the right indicating the formation of intermediate polymeric forms. Enthalpy of the intermediates show a strong absorption of heat at the third oxygenation step due to polymers formation with quinary, and above, structures. The disassembly of intermediate polymers releases energy with a negative ΔG that compensates and allow the positivity of ΔGL. High energy polymers are the barrier preventing the relaxation of the T and R conformations into one another. The MWC allosteric model is the best justification of oxygen binding cooperativity . PMID:23710673

Mismatch repair factor MSH2-MSH3 binds and alters the conformation of branched DNA structures predicted to form during genetic recombination.

PubMed

Surtees, Jennifer A; Alani, Eric

2006-07-14

Genetic studies in Saccharomyces cerevisiae predict that the mismatch repair (MMR) factor MSH2-MSH3 binds and stabilizes branched recombination intermediates that form during single strand annealing and gene conversion. To test this model, we constructed a series of DNA substrates that are predicted to form during these recombination events. We show in an electrophoretic mobility shift assay that S. cerevisiae MSH2-MSH3 specifically binds branched DNA substrates containing 3' single-stranded DNA and that ATP stimulates its release from these substrates. Chemical footprinting analyses indicate that MSH2-MSH3 specifically binds at the double-strand/single-strand junction of branched substrates, alters its conformation and opens up the junction. Therefore, MSH2-MSH3 binding to its substrates creates a unique nucleoprotein structure that may signal downstream steps in repair that include interactions with MMR and nucleotide excision repair factors.

DISTINCT ANTIBODY SPECIES: STRUCTURAL DIFFERENCES CREATING THERAPEUTIC OPPORTUNITIES

PubMed Central

Muyldermans, Serge; Smider, Vaughn V.

2016-01-01

Antibodies have been a remarkably successful class of molecules for binding a large number of antigens in therapeutic, diagnostic, and research applications. Typical antibodies derived from mouse or human sources use the surface formed by complementarity determining regions (CDRs) on the variable regions of the heavy chain/light chain heterodimer, which typically forms a relatively flat binding surface. Alternative species, particularly camelids and bovines, provide a unique paradigm for antigen recognition through novel domains which form the antigen binding paratope. For camelids, heavy chain antibodies bind antigen with only a single heavy chain variable region, in the absence of light chains. In bovines, ultralong CDR-H3 regions form an independently folding minidomain, which protrudes from the surface of the antibody and is diverse in both its sequence and disulfide patterns. The atypical paratopes of camelids and bovines potentially provide the ability to interact with different epitopes, particularly recessed or concave surfaces, compared to traditional antibodies. PMID:26922135

Expanded RNA-binding activities of mammalian Argonaute 2

PubMed Central

Tan, Grace S.; Garchow, Barry G.; Liu, Xuhang; Yeung, Jennifer; Morris, John P.; Cuellar, Trinna L.; McManus, Michael T.; Kiriakidou, Marianthi

2009-01-01

Mammalian Argonaute 2 (Ago2) protein associates with microRNAs (miRNAs) or small interfering RNAs (siRNAs) forming RNA-induced silencing complexes (RISCs/miRNPs). In the present work, we characterize the RNA-binding and nucleolytic activity of recombinant mouse Ago2. Our studies show that recombinant mouse Ago2 binds efficiently to miRNAs forming active RISC. Surprisingly, we find that recombinant mouse Ago2 forms active RISC using pre-miRNAs or long unstructured single stranded RNAs as guides. Furthermore, we demonstrate that, in vivo, endogenous human Ago2 binds directly to pre-miRNAs independently of Dicer, and that Ago2:pre-miRNA complexes are found both in the cytoplasm and in the nucleus of human cells. PMID:19808937

Membrane binding of human immunodeficiency virus type 1 matrix protein in vivo supports a conformational myristyl switch mechanism.

PubMed Central

Spearman, P; Horton, R; Ratner, L; Kuli-Zade, I

1997-01-01

The interaction of the human immunodeficiency virus (HIV) Gag protein with the plasma membrane of a cell is a critical event in the assembly of HIV particles. The matrix protein region (MA) of HIV type 1 (HIV-1) Pr55Gag has previously been demonstrated to confer membrane-binding properties on the precursor polyprotein. Both the myristic acid moiety and additional determinants within MA are essential for plasma membrane binding and subsequent particle formation. In this study, we demonstrated the myristylation-dependent membrane interaction of MA in an in vivo membrane-binding assay. When expressed within mammalian cells, MA was found both in association with cellular membranes and in a membrane-free form. In contrast, the intact precursor Pr55Gag molecule analyzed in an identical manner was found almost exclusively bound to membranes. Both membrane-bound and membrane-free forms of MA were myristylated and phosphorylated. Differential membrane binding was not due to the formation of multimers, as dimeric and trimeric forms of MA were also found in both membrane-bound and membrane-free fractions. To define the requirements for membrane binding of MA, we analyzed the membrane binding of a series of MA deletion mutants. Surprisingly, deletions within alpha-helical regions forming the globular head of MA led to a dramatic increase in overall membrane binding. The stability of the MA-membrane interaction was not affected by these deletions, and no deletion eliminated membrane binding of the molecule. These results establish that myristic acid is a primary determinant of the stability of the Gag protein-membrane interaction and provide support for the hypothesis that a significant proportion of HIV-1 MA molecules may adopt a conformation in which myristic acid is hidden and unavailable for membrane interaction. PMID:9261380

Binding of Phenazinium Dye Safranin T to Polyriboadenylic Acid: Spectroscopic and Thermodynamic Study

PubMed Central

Roy, Snigdha; Das, Suman

2014-01-01

Here, we report results from experiments designed to explore the association of the phenazinium dye safranin T (ST, 3,7-diamino-2,8-dimethyl-5-phenylphenazinium chloride) with single and double stranded form of polyriboadenylic acid (hereafter poly-A) using several spectroscopic techniques. We demonstrate that the dye binds to single stranded polyriboadenylic acid (hereafter ss poly-A) with high affinity while it does not interact at all with the double stranded (ds) form of the polynucleotide. Fluorescence and absorption spectral studies reveal the molecular aspects of binding of ST to single stranded form of the polynucleotide. This observation is also supported by the circular dichroism study. Thermodynamic data obtained from temperature dependence of binding constant reveals that association is driven by negative enthalpy change and opposed by negative entropy change. Ferrocyanide quenching studies have shown intercalative binding of ST to ss poly-A. Experiments on viscosity measurements confirm the binding mode of the dye to be intercalative. The effect of [Na+] ion concentration on the binding process suggests the role of electrostatic forces in the complexation. Present studies reveal the utility of the dye in probing nucleic acid structure. PMID:24498422

Binding of phenazinium dye safranin T to polyriboadenylic acid: spectroscopic and thermodynamic study.

PubMed

Pradhan, Ankur Bikash; Haque, Lucy; Roy, Snigdha; Das, Suman

2014-01-01

Here, we report results from experiments designed to explore the association of the phenazinium dye safranin T (ST, 3,7-diamino-2,8-dimethyl-5-phenylphenazinium chloride) with single and double stranded form of polyriboadenylic acid (hereafter poly-A) using several spectroscopic techniques. We demonstrate that the dye binds to single stranded polyriboadenylic acid (hereafter ss poly-A) with high affinity while it does not interact at all with the double stranded (ds) form of the polynucleotide. Fluorescence and absorption spectral studies reveal the molecular aspects of binding of ST to single stranded form of the polynucleotide. This observation is also supported by the circular dichroism study. Thermodynamic data obtained from temperature dependence of binding constant reveals that association is driven by negative enthalpy change and opposed by negative entropy change. Ferrocyanide quenching studies have shown intercalative binding of ST to ss poly-A. Experiments on viscosity measurements confirm the binding mode of the dye to be intercalative. The effect of [Na⁺] ion concentration on the binding process suggests the role of electrostatic forces in the complexation. Present studies reveal the utility of the dye in probing nucleic acid structure.

Unorthodox Acetylcholine Binding Sites Formed by α5 and β3 Accessory Subunits in α4β2* Nicotinic Acetylcholine Receptors.

PubMed

Jain, Akansha; Kuryatov, Alexander; Wang, Jingyi; Kamenecka, Theodore M; Lindstrom, Jon

2016-11-04

All nicotinic acetylcholine receptors (nAChRs) evolved from homomeric nAChRs in which all five subunits are involved in forming acetylcholine (ACh) binding sites at their interfaces. Heteromeric α4β2* nAChRs typically have two ACh binding sites at α4/β2 interfaces and a fifth accessory subunit surrounding the central cation channel. β2 accessory subunits do not form ACh binding sites, but α4 accessory subunits do at the α4/α4 interface in (α4β2) 2 α4 nAChRs. α5 and β3 are closely related subunits that had been thought to act only as accessory subunits and not take part in forming ACh binding sites. The effect of agonists at various subunit interfaces was determined by blocking homologous sites at these interfaces using the thioreactive agent 2-((trimethylammonium)ethyl) methanethiosulfonate (MTSET). We found that α5/α4 and β3/α4 interfaces formed ACh binding sites in (α4β2) 2 α5 and (α4β2) 2 β3 nAChRs. The α4/α5 interface in (β2α4) 2 α5 nAChRs also formed an ACh binding site. Blocking of these sites with MTSET reduced the maximal ACh evoked responses of these nAChRs by 30-50%. However, site-selective agonists NS9283 (for the α4/α4 site) and sazetidine-A (for the α4/β2 site) did not act on the ACh sites formed by the α5/α4 or β3/α4 interfaces. This suggests that unorthodox sites formed by α5 and β3 subunits have unique ligand selectivity. Agonists or antagonists for these unorthodox sites might be selective and effective drugs for modulating nAChR function to treat nicotine addiction and other disorders. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Growth, inter- and intraspecific variation, palaeobiogeography, taphonomy and systematics of the Cenozoic ghost shrimp Glypturus

PubMed Central

Klompmaker, Adiël A.; Hyžný, Matúš; Portell, Roger W.; Kowalewski, Michał

2015-01-01

Studies in systematic palaeontology are greatly aided when numerous, well-preserved specimens are available so that quantitative methods can be used to substantiate qualitative observations. This is often not the case for fossil decapod crustaceans due to their relatively low preservation potential. Here, we examined primarily two large collections of the well-preserved ghost shrimp Glypturus from the Holo-Pleistocene of Panama and the late Miocene of Florida. Using descriptive, bivariate, multivariate and geometric morphometric methods, two new species are described based on appendage material: Glypturus panamacanalensis sp. nov. and G. sikesi sp. nov. New characters are identified, and size-related and intraspecific variation are assessed for these taxa and modern G. acanthochirus. Taxonomic placement of single specimens from other localities was confirmed by multivariate methods. Furthermore, Glypturus is revised, especially with regard to Western Atlantic species that inhabited both carbonate and siliciclastic environments. Callianassa anguillensis, C. latidigata, and Neocallichirus? quisquellanus are referred to as Glypturus sp. until more material is available to determine the validity of these species. Diversity within Glypturus may thus be underestimated, thereby also impacting the assessment of phylogenetic relationships. Minor propodi appear under-represented relative to major propodi, suggesting a taphonomic bias. Single specimens of interest include a specimen of G. panamacanalensis sp. nov. exhibiting a peculiar swelling in the fixed finger and another showing damage on the propodal upper margin, suggesting failed predation or antagonistic behaviour. Glypturus is first found in the Oligocene in the Western Atlantic and may have expanded its palaeobiogeographical range since the Miocene. The genus was still present on the Pacific side of the Isthmus of Panama in the Holo-Pleistocene, but is only known from the Western Atlantic today, suggesting a relatively recent extinction on the Pacific side. PMID:26819570

Pregnant women of South Asian ethnicity in Canada have substantially lower vitamin B12 status compared with pregnant women of European ethnicity.

PubMed

Schroder, Theresa H; Sinclair, Graham; Mattman, Andre; Jung, Benjamin; Barr, Susan I; Vallance, Hilary D; Lamers, Yvonne

2017-09-01

Maternal vitamin B12 (B12) status has been inversely associated with adverse pregnancy outcomes and positively with fetal growth and infant development. South Asians, Canada's largest ethnic minority, are prone to B12 deficiency. Yet, data are lacking on B12 status in South Asian pregnant women in North America. We sought to determine B12 status, using multiple biomarkers, in 1st and 2nd trimester pregnant women of South Asian and, for comparison, European ethnicity living in Vancouver, Canada. In this retrospective cohort study, total B12, holotranscobalamin (holoTC), methylmalonic acid (MMA), and total homocysteine concentrations were quantified in two routinely collected (mean gestational week: 11·5 (range 8·3-13·9) and 16·5 (range 14·9-20·9)), banked serum samples of 748 healthy pregnant South Asian (n 371) and European (n 377) women. South Asian pregnant women had significantly lower B12 status than European pregnant women at both time points, as indicated by lower serum total B12 and holoTC concentrations, and higher MMA concentrations (all P≤0·001). The largest difference, which was substantial (Cohen's d≥0·5), was observed in mean serum total B12 concentrations (1st trimester: 189 (95 % CI 180, 199) v. 246 (95 % CI 236, 257) pmol/l; 2nd trimester: 176 (95 % CI 168, 185) v. 226 (95 % CI 216, 236) pmol/l). Further, South Asian ethnicity was a significant negative predictor of B12 status during pregnancy. South Asian women living in Vancouver have substantially lower B12 status during early pregnancy. Future research identifying predictors and health consequences of this observed difference is needed to allow for targeted interventions.

Characteristics of structure, composition, mass spectra, and iron release from the ferritin of shark liver (Sphyrna zygaena).

PubMed

Huang, He-Qing; Xiao, Zhi-Qun; Chen, Xu; Lin, Qing-Mei; Cai, Zong-Wei; Chen, Ping

2004-11-01

The ferritin consists of a protein shell constructed of 24 subunits and an iron core. The liver ferritin of Sphyrna zygaena (SZLF) purified by column chromatography is a protein composed of eight ferritins containing varying iron numbers ranging from 400+/-20 Fe3+/SZLF to 1890+/-20 Fe3+/SZLF within the protein shell. Nature SZLF (SZLFN) consisting of holoSZLF and SZLF with unsaturated iron (SZLFUI) to have been purified with polyacrylamide gel electrophoresis (PAGE) exhibited five ferritin bands with different pI values ranging from 4.0 to 7.0 in the gel slab of isoelectric focusing (IEF). HoloSZLF purified by PAGE (SZLFE) not only had 1890+/-20 Fe3+/SZLFE but also showed an identical size of iron core observed by transmission electron microscopy (TEM). Molecular weight of approximately 21 kDa for SZLFE subunit was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Four peaks of molecular ions at mass/charge (m/z) ratios of 10611.07, 21066.52, 41993.16, and 63555.64 that come from the SZLFE were determined by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS), which were identified as molecular ions of the ferritin subunit (M+) and its polymers, namely, [M]2+, [M]+, [2M]+, and [3M]+, respectively. Both SZLFE and a crude extract from shark liver of S. zygaena showed similar kinetic characteristics of complete iron release with biphasic behavior. In addition, a combined technique of visible spectrometry and column chromatography was used for studying ratio of phosphate to Fe3+ within the SZLFE core. Interestingly, this ratio maintained invariable even after the iron release, which differed from that of other mammal ferritins.

Minute Virus of Mice Initiator Protein NS1 and a Host KDWK Family Transcription Factor Must Form a Precise Ternary Complex with Origin DNA for Nicking To Occur

PubMed Central

Christensen, Jesper; Cotmore, Susan F.; Tattersall, Peter

2001-01-01

Parvoviral rolling hairpin replication generates palindromic genomic concatemers whose junctions are resolved to give unit-length genomes by a process involving DNA replication initiated at origins derived from each viral telomere. The left-end origin of minute virus of mice (MVM), oriL, contains binding sites for the viral initiator nickase, NS1, and parvovirus initiation factor (PIF), a member of the emerging KDWK family of transcription factors. oriL is generated as an active form, oriLTC, and as an inactive form, oriLGAA, which contains a single additional nucleotide inserted between the NS1 and PIF sites. Here we examined the interactions on oriLTC which lead to activation of NS1 by PIF. The two subunits of PIF, p79 and p96, cooperatively bind two ACGT half-sites, which can be flexibly spaced. When coexpressed from recombinant baculoviruses, the PIF subunits preferentially form heterodimers which, in the presence of ATP, show cooperative binding with NS1 on oriL, but this interaction is preferentially enhanced on oriLTC compared to oriLGAA. Without ATP, NS1 is unable to bind stably to its cognate site, but PIF facilitates this interaction, rendering the NS1 binding site, but not the nick site, resistant to DNase I. Varying the spacing of the PIF half-sites shows that the distance between the NS1 binding site and the NS1-proximal half-site is critical for nickase activation, whereas the position of the distal half-site is unimportant. When expressed separately, both PIF subunits form homodimers that bind site specifically to oriL, but only complexes containing p79 activate the NS1 nickase function. PMID:11435581

Structural basis of recognition of farnesylated and methylated KRAS4b by PDEδ.

PubMed

Dharmaiah, Srisathiyanarayanan; Bindu, Lakshman; Tran, Timothy H; Gillette, William K; Frank, Peter H; Ghirlando, Rodolfo; Nissley, Dwight V; Esposito, Dominic; McCormick, Frank; Stephen, Andrew G; Simanshu, Dhirendra K

2016-11-01

Farnesylation and carboxymethylation of KRAS4b (Kirsten rat sarcoma isoform 4b) are essential for its interaction with the plasma membrane where KRAS-mediated signaling events occur. Phosphodiesterase-δ (PDEδ) binds to KRAS4b and plays an important role in targeting it to cellular membranes. We solved structures of human farnesylated-methylated KRAS4b in complex with PDEδ in two different crystal forms. In these structures, the interaction is driven by the C-terminal amino acids together with the farnesylated and methylated C185 of KRAS4b that binds tightly in the central hydrophobic pocket present in PDEδ. In crystal form II, we see the full-length structure of farnesylated-methylated KRAS4b, including the hypervariable region. Crystal form I reveals structural details of farnesylated-methylated KRAS4b binding to PDEδ, and crystal form II suggests the potential binding mode of geranylgeranylated-methylated KRAS4b to PDEδ. We identified a 5-aa-long sequence motif (Lys-Ser-Lys-Thr-Lys) in KRAS4b that may enable PDEδ to bind both forms of prenylated KRAS4b. Structure and sequence analysis of various prenylated proteins that have been previously tested for binding to PDEδ provides a rationale for why some prenylated proteins, such as KRAS4a, RalA, RalB, and Rac1, do not bind to PDEδ. Comparison of all four available structures of PDEδ complexed with various prenylated proteins/peptides shows the presence of additional interactions due to a larger protein-protein interaction interface in KRAS4b-PDEδ complex. This interface might be exploited for designing an inhibitor with minimal off-target effects.

The Diabetes Drug Target MitoNEET Governs a Novel Trafficking Pathway to Rebuild an Fe-S Cluster into Cytosolic Aconitase/Iron Regulatory Protein 1*

PubMed Central

Ferecatu, Ioana; Gonçalves, Sergio; Golinelli-Cohen, Marie-Pierre; Clémancey, Martin; Martelli, Alain; Riquier, Sylvie; Guittet, Eric; Latour, Jean-Marc; Puccio, Hélène; Drapier, Jean-Claude; Lescop, Ewen; Bouton, Cécile

2014-01-01

In eukaryotes, mitochondrial iron-sulfur cluster (ISC), export and cytosolic iron-sulfur cluster assembly (CIA) machineries carry out biogenesis of iron-sulfur (Fe-S) clusters, which are critical for multiple essential cellular pathways. However, little is known about their export out of mitochondria. Here we show that Fe-S assembly of mitoNEET, the first identified Fe-S protein anchored in the mitochondrial outer membrane, strictly depends on ISC machineries and not on the CIA or CIAPIN1. We identify a dedicated ISC/export pathway in which augmenter of liver regeneration, a mitochondrial Mia40-dependent protein, is specific to mitoNEET maturation. When inserted, the Fe-S cluster confers mitoNEET folding and stability in vitro and in vivo. The holo-form of mitoNEET is resistant to NO and H2O2 and is capable of repairing oxidatively damaged Fe-S of iron regulatory protein 1 (IRP1), a master regulator of cellular iron that has recently been involved in the mitochondrial iron supply. Therefore, our findings point to IRP1 as the missing link to explain the function of mitoNEET in the control of mitochondrial iron homeostasis. PMID:25012650

Three-dimensional structure of homodimeric cholesterol esterase-ligand complex at 1.4 Å resolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pletnev, V.; Addlagatta, A.; Wawrzak, Z.

2010-03-08

The three-dimensional structure of a Candida cylindracea cholesterol esterase (ChE) homodimer (534 x 2 amino acids) in complex with a ligand of proposed formula C{sub 23}H{sub 48}O{sub 2} has been determined at 1.4 {angstrom} resolution in space group P1 using synchrotron low-temperature data. The structure refined to R = 0.136 and R{sub free} = 0.169 and has revealed new stereochemical details in addition to those detected for the apo- and holo-forms at 1.9 and 2.0 {angstrom} resolution, respectively [Ghosh et al. (1995), Structure, 3, 279-288]. The cholesterol esterase structure is a dimer with four spatially separated interfacial contact areas andmore » two symmetry-related pairs of openings to an internal intradimer cavity. Hydrophobic active-site gorges in each subunit face each other across a central interfacial cavity. The ChE subunits have carbohydrate chains attached to their Asn314 and Asn351 residues, with two ordered N-acetyl-D-glucosoamine moieties visible at each site. The side chains of 14 residues have two alternative conformations with occupancy values of 0.5 {+-} 0.2. For each subunit the electron density in the enzyme active-site gorge is well modeled by a C{sub 23}-chain fatty acid.« less

The diabetes drug target MitoNEET governs a novel trafficking pathway to rebuild an Fe-S cluster into cytosolic aconitase/iron regulatory protein 1.

PubMed

Ferecatu, Ioana; Gonçalves, Sergio; Golinelli-Cohen, Marie-Pierre; Clémancey, Martin; Martelli, Alain; Riquier, Sylvie; Guittet, Eric; Latour, Jean-Marc; Puccio, Hélène; Drapier, Jean-Claude; Lescop, Ewen; Bouton, Cécile

2014-10-10

In eukaryotes, mitochondrial iron-sulfur cluster (ISC), export and cytosolic iron-sulfur cluster assembly (CIA) machineries carry out biogenesis of iron-sulfur (Fe-S) clusters, which are critical for multiple essential cellular pathways. However, little is known about their export out of mitochondria. Here we show that Fe-S assembly of mitoNEET, the first identified Fe-S protein anchored in the mitochondrial outer membrane, strictly depends on ISC machineries and not on the CIA or CIAPIN1. We identify a dedicated ISC/export pathway in which augmenter of liver regeneration, a mitochondrial Mia40-dependent protein, is specific to mitoNEET maturation. When inserted, the Fe-S cluster confers mitoNEET folding and stability in vitro and in vivo. The holo-form of mitoNEET is resistant to NO and H2O2 and is capable of repairing oxidatively damaged Fe-S of iron regulatory protein 1 (IRP1), a master regulator of cellular iron that has recently been involved in the mitochondrial iron supply. Therefore, our findings point to IRP1 as the missing link to explain the function of mitoNEET in the control of mitochondrial iron homeostasis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Pore-forming activity of clostridial binary toxins.

PubMed

Knapp, O; Benz, R; Popoff, M R

2016-03-01

Clostridial binary toxins (Clostridium perfringens Iota toxin, Clostridium difficile transferase, Clostridium spiroforme toxin, Clostridium botulinum C2 toxin) as Bacillus binary toxins, including Bacillus anthracis toxins consist of two independent proteins, one being the binding component which mediates the internalization into cell of the intracellularly active component. Clostridial binary toxins induce actin cytoskeleton disorganization through mono-ADP-ribosylation of globular actin and are responsible for enteric diseases. Clostridial and Bacillus binary toxins share structurally and functionally related binding components which recognize specific cell receptors, oligomerize, form pores in endocytic vesicle membrane, and mediate the transport of the enzymatic component into the cytosol. Binding components retain the global structure of pore-forming toxins (PFTs) from the cholesterol-dependent cytotoxin family such as perfringolysin. However, their pore-forming activity notably that of clostridial binding components is more related to that of heptameric PFT family including aerolysin and C. perfringens epsilon toxin. This review focuses upon pore-forming activity of clostridial binary toxins compared to other related PFTs. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale. Copyright © 2015 Elsevier B.V. All rights reserved.

Chloroquine Analog Interaction with C2- and Iota-Toxin in Vitro and in Living Cells.

PubMed

Kronhardt, Angelika; Beitzinger, Christoph; Barth, Holger; Benz, Roland

2016-08-10

C2-toxin from Clostridium botulinum and Iota-toxin from Clostridium perfringens belong both to the binary A-B-type of toxins consisting of two separately secreted components, an enzymatic subunit A and a binding component B that facilitates the entry of the corresponding enzymatic subunit into the target cells. The enzymatic subunits are in both cases actin ADP-ribosyltransferases that modify R177 of globular actin finally leading to cell death. Following their binding to host cells' receptors and internalization, the two binding components form heptameric channels in endosomal membranes which mediate the translocation of the enzymatic components Iota a and C2I from endosomes into the cytosol of the target cells. The binding components form ion-permeable channels in artificial and biological membranes. Chloroquine and related 4-aminoquinolines were able to block channel formation in vitro and intoxication of living cells. In this study, we extended our previous work to the use of different chloroquine analogs and demonstrate that positively charged aminoquinolinium salts are able to block channels formed in lipid bilayer membranes by the binding components of C2- and Iota-toxin. Similarly, these molecules protect cultured mammalian cells from intoxication with C2- and Iota-toxin. The aminoquinolinium salts did presumably not interfere with actin ADP-ribosylation or receptor binding but blocked the pores formed by C2IIa and Iota b in living cells and in vitro. The blocking efficiency of pores formed by Iota b and C2IIa by the chloroquine analogs showed interesting differences indicating structural variations between the types of protein-conducting nanochannels formed by Iota b and C2IIa.

Chloroquine Analog Interaction with C2- and Iota-Toxin in Vitro and in Living Cells

PubMed Central

Kronhardt, Angelika; Beitzinger, Christoph; Barth, Holger; Benz, Roland

2016-01-01

C2-toxin from Clostridium botulinum and Iota-toxin from Clostridium perfringens belong both to the binary A-B-type of toxins consisting of two separately secreted components, an enzymatic subunit A and a binding component B that facilitates the entry of the corresponding enzymatic subunit into the target cells. The enzymatic subunits are in both cases actin ADP-ribosyltransferases that modify R177 of globular actin finally leading to cell death. Following their binding to host cells’ receptors and internalization, the two binding components form heptameric channels in endosomal membranes which mediate the translocation of the enzymatic components Iota a and C2I from endosomes into the cytosol of the target cells. The binding components form ion-permeable channels in artificial and biological membranes. Chloroquine and related 4-aminoquinolines were able to block channel formation in vitro and intoxication of living cells. In this study, we extended our previous work to the use of different chloroquine analogs and demonstrate that positively charged aminoquinolinium salts are able to block channels formed in lipid bilayer membranes by the binding components of C2- and Iota-toxin. Similarly, these molecules protect cultured mammalian cells from intoxication with C2- and Iota-toxin. The aminoquinolinium salts did presumably not interfere with actin ADP-ribosylation or receptor binding but blocked the pores formed by C2IIa and Iota b in living cells and in vitro. The blocking efficiency of pores formed by Iota b and C2IIa by the chloroquine analogs showed interesting differences indicating structural variations between the types of protein-conducting nanochannels formed by Iota b and C2IIa. PMID:27517960

Mammalian molybdo-flavoenzymes, an expanding family of proteins: structure, genetics, regulation, function and pathophysiology.

PubMed Central

Garattini, Enrico; Mendel, Ralf; Romão, Maria João; Wright, Richard; Terao, Mineko

2003-01-01

The molybdo-flavoenzymes are structurally related proteins that require a molybdopterin cofactor and FAD for their catalytic activity. In mammals, four enzymes are known: xanthine oxidoreductase, aldehyde oxidase and two recently described mouse proteins known as aldehyde oxidase homologue 1 and aldehyde oxidase homologue 2. The present review article summarizes current knowledge on the structure, enzymology, genetics, regulation and pathophysiology of mammalian molybdo-flavoenzymes. Molybdo-flavoenzymes are structurally complex oxidoreductases with an equally complex mechanism of catalysis. Our knowledge has greatly increased due to the recent crystallization of two xanthine oxidoreductases and the determination of the amino acid sequences of many members of the family. The evolution of molybdo-flavoenzymes can now be traced, given the availability of the structures of the corresponding genes in many organisms. The genes coding for molybdo-flavoenzymes are expressed in a cell-specific fashion and are controlled by endogenous and exogenous stimuli. The recent cloning of the genes involved in the biosynthesis of the molybdenum cofactor has increased our knowledge on the assembly of the apo-forms of molybdo-flavoproteins into the corresponding holo-forms. Xanthine oxidoreductase is the key enzyme in the catabolism of purines, although recent data suggest that the physiological function of this enzyme is more complex than previously assumed. The enzyme has been implicated in such diverse pathological situations as organ ischaemia, inflammation and infection. At present, very little is known about the pathophysiological relevance of aldehyde oxidase, aldehyde oxidase homologue 1 and aldehyde oxidase homologue 2, which do not as yet have an accepted endogenous substrate. PMID:12578558

Metal binding stoichiometry and isotherm choice in biosorption

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schiewer, S.; Wong, M.H.

1999-11-01

Seaweeds that possess a high metal binding capacity may be used as biosorbents for the removal of toxic heavy metals from wastewater. The binding of Cu and Ni by three brown algae (Sargassum, Colpomenia, Petalonia) and one green alga (Ulva) was investigated at pH 4.0 and pH 3.0. The greater binding strength of Cu is reflected in a binding constant that is about 10 times as high as that of Ni. The extent of metal binding followed the order Petalonia {approximately} Sargassum > Colpomenia > Ulva. This was caused by a decreasing number of binding sites and by much lowermore » metal binding constants for Ulva as compared to the brown algae. Three different stoichiometric assumptions are compared for describing the metal binding, which assume either that each metal ion M binds to one binding site B forming a BM complex or that a divalent metal ion M binds to two monovalent sites B forming BM{sub 0.5} or B{sub 2}M complexes, respectively. Stoichiometry plots are proposed as tools to discern the relevant binding stoichiometry. The pH effect in metal binding and the change in proton binding were well predicted for the B{sub 2}M or BM{sub 0.5} stoichiometries with the former being better for Cu and the latter preferable for Ni. Overall, the BM{sub 0.5} model is recommended because it avoids iterations.« less

Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import

PubMed Central

Buchanan, Susan K; Lukacik, Petra; Grizot, Sylvestre; Ghirlando, Rodolfo; Ali, Maruf M U; Barnard, Travis J; Jakes, Karen S; Kienker, Paul K; Esser, Lothar

2007-01-01

Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full-length colicin Ia to show that the channel forming domain is initially positioned 150 Å above the cell surface. Functional data using full-length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane. PMID:17464289

The Activation Domain of the Bovine Papillomavirus E2 Protein Mediates Association of DNA-Bound Dimers to form DNA Loops

NASA Astrophysics Data System (ADS)

Knight, Jonathan D.; Li, Rong; Botchan, Michael

1991-04-01

The E2 transactivator protein of bovine papillomavirus binds its specific DNA target sequence as a dimer. We have found that E2 dimers, performed in solution independent of DNA, exhibit substantial cooperativity of DNA binding as detected by both nitrocellulose filter retention and footprint analysis techniques. If the binding sites are widely spaced, E2 forms stable DNA loops visible by electron microscopy. When three widely separated binding sites reside on te DNA, E2 condenses the molecule into a bow-tie structure. This implies that each E2 dimer has at least two independent surfaces for multimerization. Two naturally occurring shorter forms of the protein, E2C and D8/E2, which function in vivo as repressors of transcription, do not form such loops. Thus, the looping function of E2 maps to the 161-amino acid activation domain. These results support the looping model of transcription activation by enhancers.

Hardware and software improvements to a low-cost horizontal parallax holographic video monitor.

PubMed

Henrie, Andrew; Codling, Jesse R; Gneiting, Scott; Christensen, Justin B; Awerkamp, Parker; Burdette, Mark J; Smalley, Daniel E

2018-01-01

Displays capable of true holographic video have been prohibitively expensive and difficult to build. With this paper, we present a suite of modularized hardware components and software tools needed to build a HoloMonitor with basic "hacker-space" equipment, highlighting improvements that have enabled the total materials cost to fall to $820, well below that of other holographic displays. It is our hope that the current level of simplicity, development, design flexibility, and documentation will enable the lay engineer, programmer, and scientist to relatively easily replicate, modify, and build upon our designs, bringing true holographic video to the masses.

The Intrinsically Disordered Regions of the Drosophila melanogaster Hox Protein Ultrabithorax Select Interacting Proteins Based on Partner Topology

PubMed Central

Hsiao, Hao-Ching; Gonzalez, Kim L.; Catanese, Daniel J.; Jordy, Kristopher E.; Matthews, Kathleen S.; Bondos, Sarah E.

2014-01-01

Interactions between structured proteins require a complementary topology and surface chemistry to form sufficient contacts for stable binding. However, approximately one third of protein interactions are estimated to involve intrinsically disordered regions of proteins. The dynamic nature of disordered regions before and, in some cases, after binding calls into question the role of partner topology in forming protein interactions. To understand how intrinsically disordered proteins identify the correct interacting partner proteins, we evaluated interactions formed by the Drosophila melanogaster Hox transcription factor Ultrabithorax (Ubx), which contains both structured and disordered regions. Ubx binding proteins are enriched in specific folds: 23 of its 39 partners include one of 7 folds, out of the 1195 folds recognized by SCOP. For the proteins harboring the two most populated folds, DNA-RNA binding 3-helical bundles and α-α superhelices, the regions of the partner proteins that exhibit these preferred folds are sufficient for Ubx binding. Three disorder-containing regions in Ubx are required to bind these partners. These regions are either alternatively spliced or multiply phosphorylated, providing a mechanism for cellular processes to regulate Ubx-partner interactions. Indeed, partner topology correlates with the ability of individual partner proteins to bind Ubx spliceoforms. Partners bind different disordered regions within Ubx to varying extents, creating the potential for competition between partners and cooperative binding by partners. The ability of partners to bind regions of Ubx that activate transcription and regulate DNA binding provides a mechanism for partners to modulate transcription regulation by Ubx, and suggests that one role of disorder in Ubx is to coordinate multiple molecular functions in response to tissue-specific cues. PMID:25286318

Structural basis of redox-dependent substrate binding of protein disulfide isomerase

PubMed Central

Yagi-Utsumi, Maho; Satoh, Tadashi; Kato, Koichi

2015-01-01

Protein disulfide isomerase (PDI) is a multidomain enzyme, operating as an essential folding catalyst, in which the b′ and a′ domains provide substrate binding sites and undergo an open–closed domain rearrangement depending on the redox states of the a′ domain. Despite the long research history of this enzyme, three-dimensional structural data remain unavailable for its ligand-binding mode. Here we characterize PDI substrate recognition using α-synuclein (αSN) as the model ligand. Our nuclear magnetic resonance (NMR) data revealed that the substrate-binding domains of PDI captured the αSN segment Val37–Val40 only in the oxidized form. Furthermore, we determined the crystal structure of an oxidized form of the b′–a′ domains in complex with an undecapeptide corresponding to this segment. The peptide-binding mode observed in the crystal structure with NMR validation, was characterized by hydrophobic interactions on the b′ domain in an open conformation. Comparison with the previously reported crystal structure indicates that the a′ domain partially masks the binding surface of the b′ domain, causing steric hindrance against the peptide in the reduced form of the b′–a′ domains that exhibits a closed conformation. These findings provide a structural basis for the mechanism underlying the redox-dependent substrate binding of PDI. PMID:26350503

The Gly-54-->Asp allelic form of human mannose-binding protein (MBP) fails to bind MBP-associated serine protease.

PubMed Central

Matsushita, M; Ezekowitz, R A; Fujita, T

1995-01-01

The human mannose-binding protein (MBP) is a pattern recognition molecule that appears to play a role in initial host defence. MBP activates the complement cascade and it may act as an opsonin both in the absence and in the presence of complement. A number of distinct MBP allelic forms exist in different population groups. An allele that occurs in 5-7% of Caucasians was identified by an inability to activate the complement system. A homozygous mutation at base pair 230 of the MBP gene results in a Gly-to-Asp substitution at the fifth collagen repeat. It appears that the resultant protein, MBPD, is able to form high-order multimers that bind bacteria but do not support complement activation. Recently a novel serine protease, the MBP-associated serine protease (MASP), has been described. MBP-MASP complexes circulate in serum and result in the direct activation of a novel complement pathway (lectin pathway) in the absence of the first complement components. In this study we demonstrate that MASP and its proenzyme proMASP are unable to bind to recombinant (r)MBPD. This lack of a MASP-rMBPD association corresponds to a failure of the Gly-54-->Asp form of MBP to activate complement. Our results provide a biochemical basis for the functional deficit in the Gly-54-->Asp allelic form of MBP and suggest that the proMASP/MASP binding site maps to the fifth collagen repeat of MBP. Images Figure 1 PMID:7487919

Binding of Intrinsic and Extrinsic Features in Working Memory

ERIC Educational Resources Information Center

Ecker, Ullrich K. H.; Maybery, Murray; Zimmer, Hubert D.

2013-01-01

There is ongoing debate concerning the mechanisms of feature binding in working memory. In particular, there is controversy regarding the extent to which these binding processes are automatic. The present article demonstrates that binding mechanisms differ depending on whether the to-be-integrated features are perceived as forming a coherent…

Two classes of ouabain binding sites in ferret heart and two forms of Na+-K+-ATPase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ng, Y.C.; Akera, T.

1987-05-01

In partially purified Na+-K+-adenosinetriphosphatase (ATPase) obtained from ferret heart, ouabain produced a monophasic inhibition curve; however, the curve spanned over 5 logarithmic units, indicating the presence of more than one classes of enzyme. (/sup 3/H)ouabain binding studies revealed high-and low-affinity binding sites in approximately equal abundance, with apparent dissociation constants of 10 and 230 nM, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of phosphoenzyme formed from (gamma-/sup 32/P)ATP showed two distinct K+-sensitive bands of approximately 100,000 molecular weight. Phosphoenzyme formation from the high-molecular-weight alpha(+) form was selectively inhibited by N-ethylmaleimide. Ouabain caused a 50% inhibition of phosphorylation of the alpha(+) formmore » at 40 nM and the lower-molecular-weight alpha form at 300 nM. In papillary muscle preparations, 1-30 nM ouabain produced a modest positive inotropic effect that reached an apparent plateau at 30 nM. Further increases in ouabain concentrations, however, produced additional and prominent inotropic effects at 0.1-10 microM. These results indicate for the first time in cardiac muscle that the high- and low-affinity ouabain binding sites are associated with the alpha(+) and alpha forms of the Na+-K+-ATPase, respectively, and that binding of ouabain to either of these sites causes enzyme inhibition and the positive inotropic effect.« less

Solution structure of an ATP-binding RNA aptamer reveals a novel fold.

PubMed Central

Dieckmann, T; Suzuki, E; Nakamura, G K; Feigon, J

1996-01-01

In vitro selection has been used to isolate several RNA aptamers that bind specifically to biological cofactors. A well-characterized example in the ATP-binding RNA aptamer family, which contains a conserved 11-base loop opposite a bulged G and flanked by regions of double-stranded RNA. The nucleotides in the consensus sequence provide a binding pocket for ATP (or AMP), which binds with a Kd in the micromolar range. Here we present the three-dimensional solution structure of a 36-nucleotide ATP-binding RNA aptamer complexed with AMP, determined from NMR-derived distance and dihedral angle restraints. The conserved loop and bulged G form a novel compact, folded structure around the AMP. The backbone tracing of the loop nucleotides can be described by a Greek zeta (zeta). Consecutive loop nucleotides G, A, A form a U-turn at the bottom of the zeta, and interact with the AMP to form a structure similar to a GNRA tetraloop, with AMP standing in for the final A. Two asymmetric G. G base pairs close the stems flanking the internal loop. Mutated aptamers support the existence of the tertiary interactions within the consensus nucleotides and with the AMP found in the calculated structures. PMID:8756406

Unmatter Plasma revisited

NASA Astrophysics Data System (ADS)

Smarandache, Florentin

2017-10-01

Unmmatter Plasma is a novel form of plasma, exclusively made of matter and its antimatter counterpart. The electron-positron beam plasma was generated in the laboratory in the beginning of 2015. This experimental fact shows that unmatter, a new form of matter that is formed by matter and antimatter bind together (mathematically predicted since 2004) really exists. That is the electron-positron plasma experiment of 2015 is the experimentum crucis verifying the mathematically predicted unmatter. Unmatter is formed by combinations of matter and antimatter that bind together, or by long-range mixture of matter and antimatter forming a weakly-coupled phase. Binding and bound state means that the interaction is sufficiently strong to tie together the particles of a system, therefore hindering them from becoming free. For example, a usual liquid is a bound state of molecules, while a gas is an un-bounded where the molecules can move freely in successive collisions.

Evaluation of the bioactivity of recombinant human lactoferrins toward murine osteoblast-like cells for bone tissue engineering.

PubMed

Amini, Ashley A; Nair, Lakshmi S

2013-05-01

Lactoferrin (LF), which belongs to the iron-binding transferrin family, is an important regulator of the levels of free iron in the body fluids. LF has raised significant interest as a bioactive protein due to its wide array of physiological effects on many different cell types, including osteoblasts and osteoclasts. The glycoprotein's degree of iron saturation has a pivotal influence on its physical structure. The objective of this study is to investigate the biological effects of apo (low iron saturation), pis (partially iron saturated), and holo (high iron saturation) recombinant human LF (rhLF) on MC3T3-E1 cells to identify the suitable candidate for bone tissue engineering application. Our studies demonstrated a dose-dependent mitogenic response of MC3T3 to rhLF treatment irrespective of the iron concentration. Furthermore, rhLF induced the cells to produce transcription factors, chemokines, and cytokines as determined by β-catenin activation, phosphorylation of Akt, vascular endothelial growth factor, and interleukin (IL-6) expression. The iron saturation of rhLF did not have any significant effect on these biological activities of MC3T3 cells. In addition, the overall pattern of gene regulation in MC3T3-E1 cells upon rhLF treatment was followed by a global microarray analysis. Among the 45,200 genes tested, only 251 genes were found to be regulated by rhLFs of different iron concentrations. Of these, the transferrin receptor (Tfrc) was the only gene differentially regulated by the iron saturated and iron depleted (apo) rhLFs. In conclusion, the study demonstrated that rhLF is a bioactive protein and that the iron saturation of rhLF may not play a significant role in modulating osteoblast functions.

Cancer-Related NEET Proteins Transfer 2Fe-2S Clusters to Anamorsin, a Protein Required for Cytosolic Iron-Sulfur Cluster Biogenesis.

PubMed

Lipper, Colin H; Paddock, Mark L; Onuchic, José N; Mittler, Ron; Nechushtai, Rachel; Jennings, Patricia A

2015-01-01

Iron-sulfur cluster biogenesis is executed by distinct protein assembly systems. Mammals have two systems, the mitochondrial Fe-S cluster assembly system (ISC) and the cytosolic assembly system (CIA), that are connected by an unknown mechanism. The human members of the NEET family of 2Fe-2S proteins, nutrient-deprivation autophagy factor-1 (NAF-1) and mitoNEET (mNT), are located at the interface between the mitochondria and the cytosol. These proteins have been implicated in cancer cell proliferation, and they can transfer their 2Fe-2S clusters to a standard apo-acceptor protein. Here we report the first physiological 2Fe-2S cluster acceptor for both NEET proteins as human Anamorsin (also known as cytokine induced apoptosis inhibitor-1; CIAPIN-1). Anamorsin is an electron transfer protein containing two iron-sulfur cluster-binding sites that is required for cytosolic Fe-S cluster assembly. We show, using UV-Vis spectroscopy, that both NAF-1 and mNT can transfer their 2Fe-2S clusters to apo-Anamorsin with second order rate constants similar to those of other known human 2Fe-2S transfer proteins. A direct protein-protein interaction of the NEET proteins with apo-Anamorsin was detected using biolayer interferometry. Furthermore, electrospray mass spectrometry of holo-Anamorsin prepared by cluster transfer shows that it receives both of its 2Fe-2S clusters from the NEETs. We propose that mNT and NAF-1 can provide parallel routes connecting the mitochondrial ISC system and the CIA. 2Fe-2S clusters assembled in the mitochondria are received by NEET proteins and when needed transferred to Anamorsin, activating the CIA.

Responses of Mn2+ speciation in Deinococcus radiodurans and Escherichia coli to γ-radiation by advanced paramagnetic resonance methods

PubMed Central

Sharma, Ajay; Gaidamakova, Elena K.; Matrosova, Vera Y.; Bennett, Brian; Daly, Michael J.; Hoffman, Brian M.

2013-01-01

The remarkable ability of bacterium Deinococcus radiodurans to survive extreme doses of γ-rays (12,000 Gy), 20 times greater than Escherichia coli, is undiminished by loss of Mn-dependent superoxide dismutase (SodA). D. radiodurans radiation resistance is attributed to the accumulation of low-molecular-weight (LMW) “antioxidant” Mn2+–metabolite complexes that protect essential enzymes from oxidative damage. However, in vivo information about such complexes within D. radiodurans cells is lacking, and the idea that they can supplant reactive-oxygen-species (ROS)–scavenging enzymes remains controversial. In this report, measurements by advanced paramagnetic resonance techniques [electron-spin-echo (ESE)-EPR/electron nuclear double resonance/ESE envelope modulation (ESEEM)] reveal differential details of the in vivo Mn2+ speciation in D. radiodurans and E. coli cells and their responses to 10 kGy γ-irradiation. The Mn2+ of D. radiodurans exists predominantly as LMW complexes with nitrogenous metabolites and orthophosphate, with negligible EPR signal from Mn2+ of SodA. Thus, the extreme radiation resistance of D. radiodurans cells cannot be attributed to SodA. Correspondingly, 10 kGy irradiation causes no change in D. radiodurans Mn2+ speciation, despite the paucity of holo-SodA. In contrast, the EPR signal of E. coli is dominated by signals from low-symmetry enzyme sites such as that of SodA, with a minority pool of LMW Mn2+ complexes that show negligible coordination by nitrogenous metabolites. Nonetheless, irradiation of E. coli majorly changes LMW Mn2+ speciation, with extensive binding of nitrogenous ligands created by irradiation. We infer that E. coli is highly susceptible to radiation-induced ROS because it lacks an adequate supply of LMW Mn antioxidants. PMID:23536297

Malaria diagnosis and treatment under the strategy of the integrated management of childhood illness (IMCI): relevance of laboratory support from the rapid immunochromatographic tests of ICT Malaria P.f/P.v and OptiMal.

PubMed

Tarimo, D S; Minjas, J N; Bygbjerg, I C

2001-07-01

The algorithm developed for the integrated management of childhood illness (IMCI) provides guidelines for the treatment of paediatric malaria. In areas where malaria is endemic, for example, the IMCI strategy may indicate that children who present with fever, a recent history of fever and/or pallor should receive antimalarial chemotherapy. In many holo-endemic areas, it is unclear whether laboratory tests to confirm that such signs are the result of malaria would be very relevant or useful. Children from a holo-endemic region of Tanzania were therefore checked for malarial parasites by microscopy and by using two rapid immunochromatographic tests (RIT) for the diagnosis of malaria (ICT Malaria P.f/P.v and OptiMal. At the time they were tested, each of these children had been targeted for antimalarial treatment (following the IMCI strategy) because of fever and/or pallor. Only 70% of the 395 children classified to receive antimalarial drugs by the IMCI algorithm had malarial parasitaemias (68.4% had Plasmodium falciparum trophozoites, 1.3% only P. falciparum gametocytes, 0.3% P. ovale and 0.3% P. malariae). As indicators of P. falciparum trophozoites in the peripheral blood, fever had a sensitivity of 93.0% and a specificity of 15.5% whereas pallor had a sensitivity of 72.2% and a specificity of 50.8%. The RIT both had very high corresponding sensitivities (of 100.0% for the ICT and 94.0% for OptiMal) but the specificity of the ICT (74.0%) was significantly lower than that for OptiMal (100.0%). Fever and pallor were significantly associated with the P. falciparum asexual parasitaemias that equalled or exceeded the threshold intensity (2000/microl) that has the optimum sensitivity and specificity for the definition of a malarial episode. Diagnostic likelihood ratios (DLR) showed that a positive result in the OptiMal test (DLR = infinity) was a better indication of malaria than a positive result in the ICT (DLR = 3.85). In fact, OptiMal had diagnostic reliability (0.93) which approached that of an ideal test and, since it only detects live parasites, OptiMal is superior to the ICT in monitoring therapeutic responses. Although the RIT may seem attractive for use in primary health facilities because relatively inexperienced staff can perform them, the high cost of these tests is prohibitive. In holo-endemic areas, use of RIT or microscopical examination of bloodsmears may only be relevant when malaria needs to be excluded as a cause of illness (e.g. prior to treatment with toxic or expensive drugs, or during malaria epidemics). Wherever the effective drugs for the first-line treatment of malaria are cheap (e.g. chloroquine and Fansidar), treatment based on clinical diagnosis alone should prove cost-saving in health facilities without microscopy.

Calorimetric studies of the interactions of metalloenzyme active site mimetics with zinc-binding inhibitors.

PubMed

Robinson, Sophia G; Burns, Philip T; Miceli, Amanda M; Grice, Kyle A; Karver, Caitlin E; Jin, Lihua

2016-07-19

The binding of drugs to metalloenzymes is an intricate process that involves several interactions, including binding of the drug to the enzyme active site metal, as well as multiple interactions between the drug and the enzyme residues. In order to determine the free energy contribution of Zn(2+) binding by known metalloenzyme inhibitors without the other interactions, valid active site zinc structural mimetics must be formed and binding studies need to be performed in biologically relevant conditions. The potential of each of five ligands to form a structural mimetic with Zn(2+) was investigated in buffer using Isothermal Titration Calorimetry (ITC). All five ligands formed strong 1 : 1 (ligand : Zn(2+)) binary complexes. The complexes were used in further ITC experiments to study their interaction with 8-hydroxyquinoline (8-HQ) and/or acetohydroxamic acid (AHA), two bidentate anionic zinc-chelating enzyme inhibitors. It was found that tetradentate ligands were not suitable for creating zinc structural mimetics for inhibitor binding in solution due to insufficient coordination sites remaining on Zn(2+). A stable binary complex, [Zn(BPA)](2+), which was formed by a tridentate ligand, bis(2-pyridylmethyl)amine (BPA), was found to bind one AHA in buffer or a methanol : buffer mixture (60 : 40 by volume) at pH 7.25 or one 8-HQ in the methanol : buffer mixture at pH 6.80, making it an effective structural mimetic for the active site of zinc metalloenzymes. These results are consistent with the observation that metalloenzyme active site zinc ions have three residues coordinated to them, leaving one or two sites open for inhibitors to bind. Our findings indicate that Zn(BPA)X2 can be used as an active site structural mimetic for zinc metalloenzymes for estimating the free energy contribution of zinc binding to the overall inhibitor active site interactions. Such use will help aid in the rational design of inhibitors to a variety of zinc metalloenzymes.

Self-Organization of Metal Nanoparticles in Light: Electrodynamics-Molecular Dynamics Simulations and Optical Binding Experiments.

PubMed

McCormack, Patrick; Han, Fei; Yan, Zijie

2018-02-01

Light-driven self-organization of metal nanoparticles (NPs) can lead to unique optical matter systems, yet simulation of such self-organization (i.e., optical binding) is a complex computational problem that increases nonlinearly with system size. Here we show that a combined electrodynamics-molecular dynamics simulation technique can simulate the trajectories and predict stable configurations of silver NPs in optical fields. The simulated dynamic equilibrium of a two-NP system matches the probability density of oscillations for two optically bound NPs obtained experimentally. The predicted stable configurations for up to eight NPs are further compared to experimental observations of silver NP clusters formed by optical binding in a Bessel beam. All configurations are confirmed to form in real systems, including pentagonal clusters with five-fold symmetry. Our combined simulations and experiments have revealed a diverse optical matter system formed by anisotropic optical binding interactions, providing a new strategy to discover artificial materials.

The influence of Cu+ binding to hypoxanthine on stabilization of mismatches involving hypoxanthine and DNA bases: A DFT study.

PubMed

Masoodi, Hamid Reza; Bagheri, Sotoodeh; Ghaderi, Zahra

2018-05-14

In the present work, the influence of Cu + binding to N3- and N7-positions of hypoxanthine on energetic, geometrical and topological properties of hypoxanthine-guanine, hypoxanthine-adenine, hypoxanthine-cytosine, hypoxanthine-thymine and hypoxanthine-hypoxanthine mismatches is theoretically investigated. The calculations, in gas phase, are performed at B3LYP/6-311++G(3df,3pd) level of theory. Unlike the other mispairs, Cu + binding to N3-position of hypoxanthine causes the proton transfer process from enol form of hypoxanthine to imino forms of adenine and cytosine. This process also occurs in all mismatches having enol form of hypoxanthine when Cu + binds to N7-position of hypoxanthine. The mismatches are stabilized by hydrogen bonds. The influence of Cu + on hydrogen bonds is also examined by atoms in molecules (AIM) and natural bond orbital (NBO) analyses.

Native Hydrophobic Binding Interactions at the Transition State for Association between the TAZ1 Domain of CBP and the Disordered TAD-STAT2 Are Not a Requirement.

PubMed

Lindström, Ida; Dogan, Jakob

2017-08-15

A significant fraction of the eukaryotic proteome consists of proteins that are either partially or completely disordered under native-like conditions. Intrinsically disordered proteins (IDPs) are common in protein-protein interactions and are involved in numerous cellular processes. Although many proteins have been identified as disordered, much less is known about the binding mechanisms of the coupled binding and folding reactions involving IDPs. Here we have analyzed the rate-limiting transition state for binding between the TAZ1 domain of CREB binding protein and the intrinsically disordered transactivation domain of STAT2 (TAD-STAT2) by site-directed mutagenesis and kinetic experiments (Φ-value analysis) and found that the native protein-protein binding interface is not formed at the transition state for binding. Instead, native hydrophobic binding interactions form late, after the rate-limiting barrier has been crossed. The association rate constant in the absence of electrostatic enhancement was determined to be rather high. This is consistent with the Φ-value analysis, which showed that there are few or no obligatory native contacts. Also, linear free energy relationships clearly demonstrate that native interactions are cooperatively formed, a scenario that has usually been observed for proteins that fold according to the so-called nucleation-condensation mechanism. Thus, native hydrophobic binding interactions at the rate-limiting transition state for association between TAD-STAT2 and TAZ1 are not a requirement, which is generally in agreement with previous findings on other IDP systems and might be a common mechanism for IDPs.

Manipulation of P-TEFb control machinery by HIV: recruitment of P-TEFb from the large form by Tat and binding of HEXIM1 to TAR

PubMed Central

Sedore, Stanley C.; Byers, Sarah A.; Biglione, Sebastian; Price, Jason P.; Maury, Wendy J.; Price, David H.

2007-01-01

Basal transcription of the HIV LTR is highly repressed and requires Tat to recruit the positive transcription elongation factor, P-TEFb, which functions to promote the transition of RNA polymerase II from abortive to productive elongation. P-TEFb is found in two forms in cells, a free, active form and a large, inactive complex that also contains 7SK RNA and HEXIM1 or HEXIM2. Here we show that HIV infection of cells led to the release of P-TEFb from the large form. Consistent with Tat being the cause of this effect, transfection of a FLAG-tagged Tat in 293T cells caused a dramatic shift of P-TEFb out of the large form to a smaller form containing Tat. In vitro, Tat competed with HEXIM1 for binding to 7SK, blocked the formation of the P-TEFb–HEXIM1–7SK complex, and caused the release P-TEFb from a pre-formed P-TEFb–HEXIM1–7SK complex. These findings indicate that Tat can acquire P-TEFb from the large form. In addition, we found that HEXIM1 binds tightly to the HIV 5′ UTR containing TAR and recruits and inhibits P-TEFb activity. This suggests that in the absence of Tat, HEXIM1 may bind to TAR and repress transcription elongation of the HIV LTR. PMID:17576689

The monomeric form of Neisseria DNA mimic protein DMP19 prevents DNA from binding to the histone-like HU protein

PubMed Central

Ko, Tzu-Ping; Liao, Yi-Ting; Hsu, Kai-Cheng

2017-01-01

DNA mimicry is a direct and effective strategy by which the mimic competes with DNA for the DNA binding sites on other proteins. Until now, only about a dozen proteins have been shown to function via this strategy, including the DNA mimic protein DMP19 from Neisseria meningitides. We have shown previously that DMP19 dimer prevents the operator DNA from binding to the transcription factor NHTF. Here, we provide new evidence that DMP19 monomer can also interact with the Neisseria nucleoid-associated protein HU. Using BS3 crosslinking, gel filtration and isothermal titration calorimetry assays, we found that DMP19 uses its monomeric form to interact with the Neisseria HU dimer. Crosslinking conjugated mass spectrometry was used to investigate the binding mode of DMP19 monomer and HU dimer. Finally, an electrophoretic mobility shift assay (EMSA) confirmed that the DNA binding affinity of HU is affected by DMP19. These results showed that DMP19 is bifunctional in the gene regulation of Neisseria through its variable oligomeric forms. PMID:29220372

The binding of glucose to yeast hexokinase monomers is independent of ionic strength.

PubMed

Mayes, E L; Hoggett, J G; Kellett, G L

1982-05-01

Hoggett & Kellett [Eur. J. Biochem. 66, 65-77 (1976)] have reported that the binding of glucose to the monomer of hexokinase PII isoenzyme is independent of ionic strength, in contrast to the subsequent claim of Feldman & Kramp [Biochemistry 17, 1541-1547 (1978)] that the binding is strongly dependent on ionic strength. Since measurements with native hexokinase P forms are complicated by the fact that the enzyme exists in a monomer-dimer association-dissociation equilibrium, we have now studied the binding of glucose to the proteolytically-modified S forms which are monomeric. At pH 8.5, the affinity of glucose for both SI and SII monomers is independent of salt concentration over the range of KCl concentrations 0-1.0 mol . dm-3 and is in good agreement with that of the corresponding P forms in both low and high salt. These observations confirm that the binding of glucose to hexokinase P monomers is independent of ionic strength and that the affinity of glucose for the hexokinase PII monomer is about an order of magnitude greater than that for the dimer.

The binding of glucose to yeast hexokinase monomers is independent of ionic strength.

PubMed Central

Mayes, E L; Hoggett, J G; Kellett, G L

1982-01-01

Hoggett & Kellett [Eur. J. Biochem. 66, 65-77 (1976)] have reported that the binding of glucose to the monomer of hexokinase PII isoenzyme is independent of ionic strength, in contrast to the subsequent claim of Feldman & Kramp [Biochemistry 17, 1541-1547 (1978)] that the binding is strongly dependent on ionic strength. Since measurements with native hexokinase P forms are complicated by the fact that the enzyme exists in a monomer-dimer association-dissociation equilibrium, we have now studied the binding of glucose to the proteolytically-modified S forms which are monomeric. At pH 8.5, the affinity of glucose for both SI and SII monomers is independent of salt concentration over the range of KCl concentrations 0-1.0 mol . dm-3 and is in good agreement with that of the corresponding P forms in both low and high salt. These observations confirm that the binding of glucose to hexokinase P monomers is independent of ionic strength and that the affinity of glucose for the hexokinase PII monomer is about an order of magnitude greater than that for the dimer. PMID:7052060

The oligomerization state determines regulatory properties and inhibitor sensitivity of type 4 cAMP-specific phosphodiesterases.

PubMed

Richter, Wito; Conti, Marco

2004-07-16

PDE4 splice variants are classified into long and short forms depending on the presence or absence of two unique N-terminal domains termed upstream conserved regions 1 and 2 (UCR1 and -2). We have shown previously that the UCR module mediates dimerization of PDE4 long forms, whereas short forms, which lack UCR1, behave as monomers. In the present study, we demonstrate that dimerization is an essential structural element that determines the regulatory properties and inhibitor sensitivities of PDE4 enzymes. Comparing the properties of the dimeric wild type PDE4D3 with several monomeric mutant PDE4D3 constructs revealed that disruption of dimerization ablates the activation of PDE4 long forms by either protein kinase A phosphorylation or phosphatidic acid binding. Moreover, the analysis of heterodimers consisting of a catalytically active and a catalytically inactive PDE4D3 subunit indicates that protein kinase A phosphorylation of both subunits is essential to fully activate PDE4 enzymes. In addition to affecting enzyme regulation, disruption of dimerization reduces the sensitivity of the enzymes toward the prototypical PDE4 inhibitor rolipram. Parallel binding assays indicated that this shift in rolipram sensitivity is likely mediated by a decrease in the number of inhibitor binding sites in the high affinity rolipram binding state. Thus, although dimerization is not a requirement for high affinity rolipram binding, it functions to stabilize PDE4 long forms in their high affinity rolipram binding conformation. Taken together, our data indicate that dimerization defines the properties of PDE4 enzymes and suggest a common structural and functional organization for all PDEs.

Substituent Effects on the Coordination Chemistry of Metal-Binding Pharmacophores

DOE Office of Scientific and Technical Information (OSTI.GOV)

Craig, Whitney R.; Baker, Tessa W.; Marts, Amy R.

2017-09-12

A combination of XAS, UV–vis, NMR, and EPR was used to examine the binding of a series of α-hydroxythiones to CoCA. All three appear to bind preferentially in their neutral, protonated forms. Two of the three clearly bind in a monodentate fashion, through the thione sulfur alone. Thiomaltol (TM) appears to show some orientational preference, on the basis of the NMR, while it appears that thiopyromeconic acid (TPMA) retains rotational freedom. In contrast, allothiomaltol (ATM), after initially binding in its neutral form, presumably through the thione sulfur, forms a final complex that is five-coordinate via bidentate coordination of ATM. Onmore » the basis of optical titrations, we speculate that this may be due to the lower initial pKa of ATM (8.3) relative to those of TM (9.0) and TPMA (9.5). Binding through the thione is shown to reduce the hydroxyl pKa by ~0.7 pH unit on metal binding, bringing only ATM’s pKa close to the pH of the experiment, facilitating deprotonation and subsequent coordination of the hydroxyl. The data predict the presence of a solvent-exchangeable proton on TM and TPMA, and Q-band 2-pulse ESEEM experiments on CoCA + TM suggest that the proton is present. ESE-detected EPR also showed a surprising frequency dependence, giving only a subset of the expected resonances at X-band.« less

DOTAP cationic liposomes prefer relaxed over supercoiled plasmids.

PubMed

Even-Chen, S; Barenholz, Y

2000-12-20

Cationic liposomes and DNA interact electrostatically to form complexes called lipoplexes. The amounts of unbound (free) DNA in a mixture of cationic liposomes and DNA at different cationic lipid:DNA molar ratios can be used to describe DNA binding isotherms; these provide a measure of the binding efficiency of DNA to different cationic lipid formulations at various medium conditions. In order to quantify the ratio between the various forms of naked DNA and supercoiled, relaxed and single-stranded DNA, and the ratio between cationic lipid bound and unbound DNA of various forms we developed a simple, sensitive quantitative assay using agarose gel electrophoresis, followed by staining with the fluorescent cyanine DNA dyes SYBR Green I or SYBR Gold. This assay was compared with that based on the use of ethidium bromide (the most commonly used nucleic acid stain). Unlike ethidium bromide, SYBR Green I DNA sensitivity and concentration-dependent fluorescence intensity were identical for supercoiled and nicked-relaxed forms. DNA detection by SYBR Green I in solution is approximately 40-fold more sensitive than by ethidium bromide for double-stranded DNA and approximately 10-fold for single-stranded DNA, and in agarose gel it is 16-fold more sensitive for double-stranded DNA compared with ethidium bromide. SYBR Gold performs similarly to SYBR Green I. This study shows that: (a) there is no significant difference in DNA binding isotherms to the monocationic DOTAP (DOTAP/DOPE) liposomes and to the polycationic DOSPA (DOSPA/DOPE) liposomes, even when four DOSPA positive charges are involved in the electrostatic interaction with DNA; (b) the helper lipids affect DNA binding, as DOTAP/DOPE liposomes bind more DNA than DOTAP/cholesterol; (c) in the process of lipoplex formation, when the DNA is a mixture of two forms, supercoiled and nicked-relaxed (open circular), there is a preference for the binding to the cationic liposomes of plasmid DNA in the nicked-relaxed over the supercoiled form. This preference is much more pronounced when the cationic liposome formulation is based on the monocationic lipid DOTAP than on the polycationic lipid DOSPA. The preference of DOTAP formulations to bind to the relaxed DNA plasmid suggests that the binding of supercoiled DNA is weaker and easier to dissociate from the complex.

A Study of the Interaction of Bovine Hemoglobin with Synthetic Dyes Using Spectroscopic Techniques and Molecular Docking.

PubMed

Kamaljeet; Bansal, Saurabh; SenGupta, Uttara

2016-01-01

Synthetic dyes are a very efficient class of dyes that are ingested or come into contact with the skin from numerous sources (cosmetics, textiles, leather, paper, and drugs). An important component of their safety profile is the interactions that they form after they enter the body. Hemoglobin is a functionally important protein that can form multiple interactions with soluble compounds present in the blood, and hence forms an important aspect of the toxicological or safety profile of the dyes. Here we study the interaction between bovine hemoglobin and organic dyes using UV-Vis absorbance and fluorescence spectroscopy. Molecular modeling was used to visualize the binding site and partners of the dye molecules, within the hemoglobin molecule. We find that all four dyes studied form sufficiently strong interactions with hemoglobin to allow for the formation of potentially toxic interactions. Molecular modeling showed that all four dyes bind within the central cavity of the hemoglobin molecule. However, binding partners could not be identified as multiple binding conformations with very similar energies were possible for each dye.

Recognition and Binding of Human Telomeric G-Quadruplex DNA by Unfolding Protein 1

PubMed Central

2015-01-01

The specific recognition by proteins of G-quadruplex structures provides evidence of a functional role for in vivo G-quadruplex structures. As previously reported, the ribonucleoprotein, hnRNP Al, and it is proteolytic derivative, unwinding protein 1 (UP1), bind to and destabilize G-quadruplex structures formed by the human telomeric repeat d(TTAGGG)n. UP1 has been proposed to be involved in the recruitment of telomerase to telomeres for chain extension. In this study, a detailed thermodynamic characterization of the binding of UP1 to a human telomeric repeat sequence, the d[AGGG(TTAGGG)3] G-quadruplex, is presented and reveals key insights into the UP1-induced unfolding of the G-quadruplex structure. The UP1–G-quadruplex interactions are shown to be enthalpically driven, exhibiting large negative enthalpy changes for the formation of both the Na+ and K+ G-quadruplex–UP1 complexes (ΔH values of −43 and −19 kcal/mol, respectively). These data reveal three distinct enthalpic contributions from the interactions of UP1 with the Na+ form of G-quadruplex DNA. The initial interaction is characterized by a binding affinity of 8.5 × 108 M–1 (strand), 200 times stronger than the binding of UP1 to a single-stranded DNA with a comparable but non-quadruplex-forming sequence [4.1 × 106 M–1 (strand)]. Circular dichroism spectroscopy reveals the Na+ form of the G-quadruplex to be completely unfolded by UP1 at a binding ratio of 2:1 (UP1:G-quadruplex DNA). The data presented here demonstrate that the favorable energetics of the initial binding event are closely coupled with and drive the unfolding of the G-quadruplex structure. PMID:24831962

Competition between Ski and CREB-binding protein for binding to Smad proteins in transforming growth factor-beta signaling.

PubMed

Chen, Weijun; Lam, Suvana S; Srinath, Hema; Schiffer, Celia A; Royer, William E; Lin, Kai

2007-04-13

The family of Smad proteins mediates transforming growth factor-beta (TGF-beta) signaling in cell growth and differentiation. Smads repress or activate TGF-beta signaling by interacting with corepressors (e.g. Ski) or coactivators (e.g. CREB-binding protein (CBP)), respectively. Specifically, Ski has been shown to interfere with the interaction between Smad3 and CBP. However, it is unclear whether Ski competes with CBP for binding to Smads and whether they can interact with Smad3 at the same binding surface on Smad3. We investigated the interactions among purified constructs of Smad, Ski, and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Here, we show that Ski-(16-192) interacted directly with a homotrimer of receptor-regulated Smad protein (R-Smad), e.g. Smad2 or Smad3, to form a hexamer; Ski-(16-192) interacted with an R-Smad.Smad4 heterotrimer to form a pentamer. CBP-(1941-1992) was also found to interact directly with an R-Smad homotrimer to form a hexamer and with an R-Smad.Smad4 heterotrimer to form a pentamer. Moreover, these domains of Ski and CBP competed with each other for binding to Smad3. Our mutational studies revealed that domains of Ski and CBP interacted with Smad3 at a portion of the binding surface of the Smad anchor for receptor activation. Our results suggest that Ski negatively regulates TGF-beta signaling by replacing CBP in R-Smad complexes. Our working model suggests that Smad protein activity is delicately balanced by Ski and CBP in the TGF-beta pathway.

Assessing past and present P Retention in Sediments in Lake Ontario (Bay of Quinte) by Reaction-Transport Diagenetic Modeling

NASA Astrophysics Data System (ADS)

Doan, Phuong; Berry, Sandra; Markovic, Stefan; Watson, Sue; Mugalingam, Shan; Dittrich, Maria

2016-04-01

Phosphorus (P) is an important macronutrient that can limit aquatic primary production and the risk of harmful algal blooms. Although there is considerable evidence that P release from sediments can represent a significant source of P and burial in sediments returns P to the geological sink; these processes have been poorly characterised. In this study, we applied a non-steady state reactive transport diagenetic model to gain insights into the dynamics of phosphorus binding forms in sediments and the phosphorus cycling of the Bay of Quinte, an embayment of Lake Ontario, Canada. The three basins of the bay (Belleville, Hay Bay and Napanee) that we investigated had differences in their phosphorus binding forms and phosphorus release, reflecting the distinct spatial temporal patterns of land use and urbanization levels in the watershed. Sediment cores from the three stations were collected during summer and under ice cover in 2013-14. Oxygen, pH and redox potential were monitored by microsensors; porewater and sediment solid matter were analyzed for P content, and a sequential extraction was used to analyze P binding forms. In the reaction-transport model, total phosphorus was divided into adsorbed phosphorus, phosphorus bound with aluminium, organic phosphorus, redox sensitive and apatite phosphorus. Using the fluxes of organic and inorganic matter as dynamic boundary conditions, we simulated the depth profiles of solute and solid components. The model closely reproduced the fractionation data of phosphorus binding forms and soluble reactive phosphorus. The past and present P fluxes were calculated and estimated; they related to geochemical conditions, and P binding forms in sediments. Our results show that P release from sediments is dominated by the redox-sentive P fraction accounting for higher percentage at Napanee station. The main P binding form that can be immobilized through diagenesis is apatite P contributing highest P retention at HayBay station. The mass balance of P was closed by our model.

The mongoose acetylcholine receptor alpha-subunit: analysis of glycosylation and alpha-bungarotoxin binding.

PubMed

Asher, O; Jensen, B S; Lupu-Meiri, M; Oron, Y; Fuchs, S

1998-04-17

The mongoose AChR alpha-subunit has been cloned and shown to be highly homologous to other AChR alpha-subunits, with only six differences in amino acid residues at positions that are conserved in animal species that bind alpha-bungarotoxin (alpha-BTX). Four of these six substitutions cluster in the ligand binding site, and one of them, Asn-187, forms a consensus N-glycosylation site. The mongoose glycosylated alpha-subunit has a higher apparent molecular mass than that of the rat glycosylated alpha-subunit, probably resulting from the additional glycosylation at Asn-187 of the mongoose subunit. The in vitro translated mongoose alpha-subunit, in a glycosylated or non-glycosylated form, does not bind alpha-BTX, indicating that lack of alpha-BTX binding can be achieved also in the absence of glycosylation.

Convergent evolution of adenosine aptamers spanning bacterial, human, and random sequences revealed by structure-based bioinformatics and genomic SELEX

PubMed Central

Vu, Michael M. K.; Jameson, Nora E.; Masuda, Stuart J.; Lin, Dana; Larralde-Ridaura, Rosa; Lupták, Andrej

2012-01-01

SUMMARY Aptamers are structured macromolecules in vitro evolved to bind molecular targets, whereas in nature they form the ligand-binding domains of riboswitches. Adenosine aptamers of a single structural family were isolated several times from random pools but they have not been identified in genomic sequences. We used two unbiased methods, structure-based bioinformatics and human genome-based in vitro selection, to identify aptamers that form the same adenosine-binding structure in a bacterium, and several vertebrates, including humans. Two of the human aptamers map to introns of RAB3C and FGD3 genes. The RAB3C aptamer binds ATP with dissociation constants about ten times lower than physiological ATP concentration, while the minimal FGD3 aptamer binds ATP only co-transcriptionally. PMID:23102219

Selectivity of substrate binding and ionization of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase.

PubMed

Luanloet, Thikumporn; Sucharitakul, Jeerus; Chaiyen, Pimchai

2015-08-01

2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (EC 1.14.12.4) from Pseudomonas sp. MA-1 is a flavin-dependent monooxygenase that catalyzes a hydroxylation and aromatic ring cleavage reaction. The functional roles of two residues, Tyr223 and Tyr82, located ~ 5 Å away from MHPC, were characterized using site-directed mutagenesis, along with ligand binding, product analysis and transient kinetic experiments. Mutation of Tyr223 resulted in enzyme variants that were impaired in their hydroxylation activity and had Kd values for substrate binding 5-10-fold greater than the wild-type enzyme. Because this residue is adjacent to the water molecule that is located next to the 3-hydroxy group of MHPC, the results indicate that the interaction between Tyr223, H2 O and the 3-hydroxyl group of MHPC are important for substrate binding and hydroxylation. By contrast, the Kd for substrate binding of Tyr82His and Tyr82Phe variants were similar to that of the wild-type enzyme. However, only ~ 40-50% of the substrate was hydroxylated in the reactions of both variants, whereas most of the substrate was hydroxylated in the wild-type enzyme reaction. In free solution, MHPC or 5-hydroxynicotinic acid exists in a mixture of monoanionic and tripolar ionic forms, whereas only the tripolar ionic form binds to the wild-type enzyme. The binding of tripolar ionic MHPC would allow efficient hydroxylation through an electrophilic aromatic substitution mechanism. For the Tyr82His and Tyr82Phe variants, both forms of substrates can bind to the enzymes, indicating that the mutation at Tyr82 abolished the selectivity of the enzyme towards the tripolar ionic form. Transient kinetic studies indicated that the hydroxylation rate constants of both Tyr82 variants are approximately two- to 2.5-fold higher than that of the wild-type enzyme. Altogether, our findings suggest that Tyr82 is important for the binding selectivity of MHPC oxygenase towards the tripolar ionic species, whereas the interaction between Tyr223 and the substrate is important for ensuring hydroxylation. These results highlight how the active site of a flavoenzyme is able to deal with the presence of multiple forms of a substrate in solution and ensure efficient hydroxylation. © 2015 FEBS.

Molecular Control of Polyene Macrolide Biosynthesis

PubMed Central

Santos-Aberturas, Javier; Vicente, Cláudia M.; Guerra, Susana M.; Payero, Tamara D.; Martín, Juan F.; Aparicio, Jesús F.

2011-01-01

Control of polyene macrolide production in Streptomyces natalensis is mediated by the transcriptional activator PimM. This regulator, which combines an N-terminal PAS domain with a C-terminal helix-turn-helix motif, is highly conserved among polyene biosynthetic gene clusters. PimM, truncated forms of the protein without the PAS domain (PimMΔPAS), and forms containing just the DNA-binding domain (DBD) (PimMDBD) were overexpressed in Escherichia coli as GST-fused proteins. GST-PimM binds directly to eight promoters of the pimaricin cluster, as demonstrated by electrophoretic mobility shift assays. Assays with truncated forms of the protein revealed that the PAS domain does not mediate specificity or the distinct recognition of target genes, which rely on the DBD domain, but significantly reduces binding affinity up to 500-fold. Transcription start points were identified by 5′-rapid amplification of cDNA ends, and the binding regions of PimMDBD were investigated by DNase I protection studies. In all cases, binding took place covering the −35 hexamer box of each promoter, suggesting an interaction of PimM and RNA polymerase to cause transcription activation. Information content analysis of the 16 sequences protected in target promoters was used to deduce the structure of the PimM-binding site. This site displays dyad symmetry, spans 14 nucleotides, and adjusts to the consensus TVGGGAWWTCCCBA. Experimental validation of this binding site was performed by using synthetic DNA duplexes. Binding of PimM to the promoter region of one of the polyketide synthase genes from the Streptomyces nodosus amphotericin cluster containing the consensus binding site was also observed, thus proving the applicability of the findings reported here to other antifungal polyketides. PMID:21187288

The trehalose/maltose-binding protein as the sensitive element of a glucose biosensor

NASA Astrophysics Data System (ADS)

Fonin, A. V.; Povarova, O. I.; Staiano, M.; D'Auria, S.; Turoverov, K. K.; Kuznetsova, I. M.

2014-08-01

The promising direction of the development of a modern glucometer is the construction of sensing element on the basis of stained (dyed) protein which changes its fluorescence upon glucose binding. One of the proteins that can be used for this purpose is the D-trehalose/D-maltose-binding protein (TMBP) from the thermophilic bacteria Thermococcus litoralis. We investigated the physical-chemical properties of the protein and evaluated its stability to the denaturing action of GdnHCl and heating. It was confirmed that TMBP is an extremely stable protein. In vivo, the intrinsic ligands of TMBP are trehalose and maltose, but TMBP can also bind glucose. The dissociation constant of the TMBP-glucose complex is in the range of 3-8 mM. The binding of glucose does not noticeably change the intrinsic fluorescence of the TMBP. To register protein-glucose binding, we used the fluorescence of the thiol-reactive dye BADAN attached to TMBP. Because the fluorescence of BADAN attached to the cysteine Cys182 of TMBP does not change upon glucose binding, the mutant forms ТМВР/C182S/X_Cys were created. In these mutant proteins, Cys182 is replaced by Ser, removing intrinsic binding site of BADAN and a new dye binding sites were introduced. The largest increase (by 1.4 times) in the intensity of the dye fluorescence was observed upon TMBP/C182S/A14C-BADAN-Glc complex formation. The dissociation constant of this complex is 3.4 ± 0.1 mM. We consider TMBP/C182S/A14C mutant form with attached fluorescent dye BADAN as a good basis for further research aimed to develop of series of TMBP mutant forms with different affinities to glucose labeled with fluorescent dyes.

The mechanism of formation, structure and physiological relevance of covalent hemoglobin attachment to the erythrocyte membrane.

PubMed

Welbourn, Elizabeth M; Wilson, Michael T; Yusof, Ashril; Metodiev, Metodi V; Cooper, Chris E

2017-02-01

Covalent hemoglobin binding to membranes leads to band 3 (AE1) clustering and the removal of erythrocytes from the circulation; it is also implicated in blood storage lesions. Damaged hemoglobin, with the heme being in a redox and oxygen-binding inactive hemichrome form, has been implicated as the binding species. However, previous studies used strong non-physiological oxidants. In vivo hemoglobin is constantly being oxidised to methemoglobin (ferric), with around 1% of hemoglobin being in this form at any one time. In this study we tested the ability of the natural oxidised form of hemoglobin (methemoglobin) in the presence or absence of the physiological oxidant hydrogen peroxide to initiate membrane binding. The higher the oxidation state of hemoglobin (from Fe(III) to Fe(V)) the more binding was observed, with approximately 50% of this binding requiring reactive sulphydryl groups. The hemoglobin bound was in a high molecular weight complex containing spectrin, ankyrin and band 4.2, which are common to one of the cytoskeletal nodes. Unusually, we showed that hemoglobin bound in this way was redox active and capable of ligand binding. It can initiate lipid peroxidation showing the potential to cause cell damage. In vivo oxidative stress studies using extreme endurance exercise challenges showed an increase in hemoglobin membrane binding, especially in older cells with lower levels of antioxidant enzymes. These are then targeted for destruction. We propose a model where mild oxidative stress initiates the binding of redox active hemoglobin to the membrane. The maximum lifetime of the erythrocyte is thus governed by the redox activity of the cell; from the moment of its release into the circulation the timer is set. Copyright © 2016. Published by Elsevier Inc.

Destination Mars Grand Opening

NASA Image and Video Library

2016-09-18

A ceremonial ribbon is cut for the opening of new Destination: Mars experience at the Kennedy Space Center Visitor Complex. From the left are Therrin Protze, chief operating officer of the visitor complex, center director Bob Cabana, Apollo 11 astronaut Buzz Aldrin, Kudo Tsunoda of Microsoft, and Jeff Norris of NASA’s Jet Propulsion Laboratory (JPL) in Pasadena, California. Destination: Mars gives guests an opportunity to “visit” several sites on Mars using real imagery from NASA’s Curiosity Mars Rover. Based on OnSight, a tool created by JPL, the experience brings guests together with a holographic version of Aldrin as they are guided to Mars using Microsoft HoloLens mixed reality headset. Photo credit: NASA/Charles Babir

A divergent Pumilio repeat protein family for pre-rRNA processing and mRNA localization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, Chen; McCann, Kathleen L.; Wine, Robert N.

Pumilio/feminization of XX and XO animals (fem)-3 mRNA-binding factor (PUF) proteins bind sequence specifically to mRNA targets using a single-stranded RNA-binding domain comprising eight Pumilio (PUM) repeats. PUM repeats have now been identified in proteins that function in pre-rRNA processing, including human Puf-A and yeast Puf6. This is a role not previously ascribed to PUF proteins. In this paper we present crystal structures of human Puf-A that reveal a class of nucleic acid-binding proteins with 11 PUM repeats arranged in an “L”-like shape. In contrast to classical PUF proteins, Puf-A forms sequence-independent interactions with DNA or RNA, mediated by conservedmore » basic residues. We demonstrate that equivalent basic residues in yeast Puf6 are important for RNA binding, pre-rRNA processing, and mRNA localization. Finally, PUM repeats can be assembled into alternative folds that bind to structured nucleic acids in addition to forming canonical eight-repeat crescent-shaped RNA-binding domains found in classical PUF proteins.« less

Nanomechanical mapping of first binding steps of a virus to animal cells

NASA Astrophysics Data System (ADS)

Alsteens, David; Newton, Richard; Schubert, Rajib; Martinez-Martin, David; Delguste, Martin; Roska, Botond; Müller, Daniel J.

2017-02-01

Viral infection is initiated when a virus binds to cell surface receptors. Because the cell membrane is dynamic and heterogeneous, imaging living cells and simultaneously quantifying the first viral binding events is difficult. Here, we show an atomic force and confocal microscopy set-up that allows the surface receptor landscape of cells to be imaged and the virus binding events within the first millisecond of contact with the cell to be mapped at high resolution (<50 nm). We present theoretical approaches to contour the free-energy landscape of early binding events between an engineered virus and cell surface receptors. We find that the first bond formed between the viral glycoprotein and its cognate cell surface receptor has relatively low lifetime and free energy, but this increases as additional bonds form rapidly (≤1 ms). The formation of additional bonds occurs with positive allosteric modulation and the three binding sites of the viral glycoprotein are quickly occupied. Our quantitative approach can be readily applied to study the binding of other viruses to animal cells.

Structure of Epstein-Barr Virus Glycoprotein 42 Suggests a Mechanism for Triggering Receptor-Activated Virus Entry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kirschner, Austin N.; Sorem, Jessica; Longnecker, Richard

Epstein-Barr virus requires glycoproteins gH/gL, gB, and gp42 to fuse its lipid envelope with B cells. Gp42 is a type II membrane protein consisting of a flexible N-terminal region, which binds gH/gL, and a C-terminal lectin-like domain that binds to the B-cell entry receptor human leukocyte antigen (HLA) class II. Gp42 triggers membrane fusion after HLA binding, a process that requires simultaneous binding to gH/gL and a functional hydrophobic pocket in the lectin domain adjacent to the HLA binding site. Here we present the structure of gp42 in its unbound form. Comparisons to the previously determined structure of a gp42:HLAmore » complex reveals additional N-terminal residues forming part of the gH/gL binding site and structural changes in the receptor binding domain. Although the core of the lectin domain remains similar, significant shifts in two loops and an {alpha} helix bordering the essential hydrophobic pocket suggest a structural mechanism for triggering fusion.« less

A divergent Pumilio repeat protein family for pre-rRNA processing and mRNA localization

DOE PAGES

Qiu, Chen; McCann, Kathleen L.; Wine, Robert N.; ...

2014-12-15

Pumilio/feminization of XX and XO animals (fem)-3 mRNA-binding factor (PUF) proteins bind sequence specifically to mRNA targets using a single-stranded RNA-binding domain comprising eight Pumilio (PUM) repeats. PUM repeats have now been identified in proteins that function in pre-rRNA processing, including human Puf-A and yeast Puf6. This is a role not previously ascribed to PUF proteins. In this paper we present crystal structures of human Puf-A that reveal a class of nucleic acid-binding proteins with 11 PUM repeats arranged in an “L”-like shape. In contrast to classical PUF proteins, Puf-A forms sequence-independent interactions with DNA or RNA, mediated by conservedmore » basic residues. We demonstrate that equivalent basic residues in yeast Puf6 are important for RNA binding, pre-rRNA processing, and mRNA localization. Finally, PUM repeats can be assembled into alternative folds that bind to structured nucleic acids in addition to forming canonical eight-repeat crescent-shaped RNA-binding domains found in classical PUF proteins.« less

Identification of Collagen-Binding Proteins in Lactobacillus spp. with Surface-Enhanced Laser Desorption/Ionization–Time of Flight ProteinChip Technology

PubMed Central

Howard, Jeffrey C.; Heinemann, Christine; Thatcher, Bradley J.; Martin, Brian; Gan, Bing Siang; Reid, Gregor

2000-01-01

Biosurfactants produced by Lactobacillus fermentum RC-14, L. rhamnosus GR-1 and 36, and L. casei Shirota were found to contain proteins that bind to both collagen types III and VI, as determined by surface-enhanced laser desorption/ionization (SELDI)–time of flight mass spectrometry. Both collagen types III and VI immobilized on SELDI preactivated ProteinChip arrays detected several different sizes (2 to 48 kDa) of collagen-binding proteins. Overall, the RC-14-produced biosurfactant contained the greatest number of collagen-binding proteins (RC-14 > GR-1 > 36 > Shirota), including the mature form of a previously cloned 29-kDa collagen-binding protein (referred to in its mature 26-kDa form). Although biosurfactants isolated from L. casei Shirota and L. rhamnosus 36 and GR-1 also contain several collagen-binding proteins, they do not contain the 26-kDa collagen-binding protein. Together, these results demonstrate the utility of the SELDI system as a means of rapidly characterizing clinically important but complex biosurfactant solutions. PMID:11010889

MCM ring hexamerization is a prerequisite for DNA-binding

DOE PAGES

Froelich, Clifford A.; Nourse, Amanda; Enemark, Eric J.

2015-09-13

The hexameric Minichromosome Maintenance (MCM) protein complex forms a ring that unwinds DNA at the replication fork in eukaryotes and archaea. Our recent crystal structure of an archaeal MCM N-terminal domain bound to single-stranded DNA (ssDNA) revealed ssDNA associating across tight subunit interfaces but not at the loose interfaces, indicating that DNA-binding is governed not only by the DNA-binding residues of the subunits (MCM ssDNA-binding motif, MSSB) but also by the relative orientation of the subunits. We now extend these findings to show that DNA-binding by the MCM N-terminal domain of the archaeal organism Pyrococcus furiosus occurs specifically in themore » hexameric oligomeric form. We show that mutants defective for hexamerization are defective in binding ssDNA despite retaining all the residues observed to interact with ssDNA in the crystal structure. One mutation that exhibits severely defective hexamerization and ssDNA-binding is at a conserved phenylalanine that aligns with the mouse Mcm4(Chaos3) mutation associated with chromosomal instability, cancer, and decreased intersubunit association.« less

Oligomycin frames a common drug-binding site in the ATP synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Symersky, Jindrich; Osowski, Daniel; Walters, D. Eric

We report the high-resolution (1.9 {angstrom}) crystal structure of oligomycin bound to the subunit c10 ring of the yeast mitochondrial ATP synthase. Oligomycin binds to the surface of the c10 ring making contact with two neighboring molecules at a position that explains the inhibitory effect on ATP synthesis. The carboxyl side chain of Glu59, which is essential for proton translocation, forms an H-bond with oligomycin via a bridging water molecule but is otherwise shielded from the aqueous environment. The remaining contacts between oligomycin and subunit c are primarily hydrophobic. The amino acid residues that form the oligomycin-binding site are 100%more » conserved between human and yeast but are widely different from those in bacterial homologs, thus explaining the differential sensitivity to oligomycin. Prior genetics studies suggest that the oligomycin-binding site overlaps with the binding site of other antibiotics, including those effective against Mycobacterium tuberculosis, and thereby frames a common 'drug-binding site.' We anticipate that this drug-binding site will serve as an effective target for new antibiotics developed by rational design.« less

Lectins discriminate between pathogenic and nonpathogenic South American trypanosomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

de Miranda Santos, I.K.; Pereira, M.E.

1984-09-01

Cell surface carbohydrates of Trypanosoma cruzi, Trypanosoma rangeli, and Trypanosoma conorhini were analyzed by a micro-agglutination assay employing 27 highly purified lectins and by binding assays using various /sup 125/I-labeled lectins. The following seven lectins discriminated between the trypanosomes: 1) tomato lectin (an N-acetyl-D-glucosamine-binding protein), both in purified form and as crude tomato juice; 2) Bauhinea purpurea and Sophora japonica lectins (both N-acetyl-D-galactosamine-binding proteins), which selectively agglutinated T. cruzi; 3) Vicia villosa (an N-acetyl-D-galactosamine-binding protein) which was specific for T. rangeli; 4) peanut lectin (a D-galactose-binding protein) both in purified form and as crude saline extract; and 5) Ulex europaeusmore » and Lotus tetragonolobus (both L-fucose-binding proteins) lectins which reacted only with T. conorhini. Binding studies with 125I-labeled lectins were performed to find whether unagglutinated cells of the three different species of trypanosomes might have receptors for these lectins, in which case absence of agglutination could be due to a peculiar arrangement of the receptors. These assays essentially confirmed the agglutination experiments.« less

Effect of glycosylation on hydration behavior at the ice-binding surface of the Ocean Pout type III antifreeze protein: a molecular dynamics simulation.

PubMed

Halder, Swagata; Mukhopadhyay, Chaitali

2017-12-01

Antifreeze proteins (AFPs), found in certain vertebrates, plants, fungi and bacteria have the ability to permit their survival in subzero environments by thermal hysteresis mechanism. However, the exact mechanism of ice growth inhibition is still not clearly understood. Here, four long explicit molecular dynamics (MD) simulations have been carried out at two different temperatures (277 and 298 K) with and without glycan to study the conformational rigidity of the Ocean pout type III antifreeze protein in aqueous medium and the structural arrangements of water molecules hydrating its ice-binding surface. It is found that irrespective of the temperature the ice-binding surface (IBS) of the protein is relatively more rigid than its non ice-binding surface (NonIBS) in its native and glycosylated form. Hydrophilic residues N14, T18 and Q44 are essential to antifreeze activity. Radial distribution, density distribution function and nearest neighbor orientation plots with respect to individual two surfaces confirm that density of water molecule near these binding surface in native and glycosylated form are relatively more than the nonbinding surface. The glycosylated form shows a strong peak than the native one. From rotational auto correlation function of water molecules around ice-binding sites, it is prominent that with increase in temperature, strong interaction between the water oxygen and the hydrogen bond acceptor group on the protein-binding surface decreases. This provides a possible molecular reason behind the ice-binding activity of ocean pout at the prism plane of ice.

Red Light-enhanced Phytochrome Pelletability

PubMed Central

Pratt, Lee H.; Marmé, Dieter

1976-01-01

Red light-enhanced pelletability of phytochrome was observed in extracts of all 11 plants tested: Avena sativa L., Secale cereale L., Zea mays L., Cucurbita pepo L., Sinapis alba L., Pisum sativum L., Helianthus anuus L., Raphanus sativus L., Glycine max (L.) Merr., Phaseolus vulgaris L., and Lupinus albus L. This enhanced pelletability was observed in all 11 plants following in situ irradiation (in vivo binding) but only in Sinapis and Cucurbita after irradiation of crude extracts (in vitro binding). In vivo binding was not strongly dependent upon pH and, with few exceptions, was not markedly sensitive to high salt concentration, whereas in vitro binding was completely reversed by both high pH and high salt concentration. However, both binding phenomena were observed only with a divalent cation in the extract buffer. In vivo binding was further characterized using Avena which showed an increase in pelletability from less than 10% in dark control extracts to more than 60% in extracts of red light-irradiated shoots. The half-life for binding was 40 seconds at 0.5 C and was strongly temperature-dependent, binding being complete within 5 to 10 sec at 22 C. If pelletable phytochrome in the far red-absorbing form was photoconverted back to the red-absorbing form in situ, phytochrome was released from the pelletable condition with a half-life of 25 minutes at 25 C and 100 minutes at both 13 C and 3 C. No cooperativity in red light-enhanced pelletability with respect to phytochrome-far red-absorbing form was observed. PMID:16659745

Synthesis, characterization, DNA-binding and cleavage studies of polypyridyl copper(II) complexes

NASA Astrophysics Data System (ADS)

Gubendran, Ammavasi; Rajesh, Jegathalaprathaban; Anitha, Kandasamy; Athappan, Periyakaruppan

2014-10-01

Six new mixed-ligand copper(II) complexes were synthesized namely [Cu(phen)2OAc]ClO4ṡH2O(1), [Cu(bpy)2OAc]ClO4ṡH2O(2), [Cu(o-ampacac)(phen)]ClO4(3), [Cu(o-ampbzac)(phen)]ClO4(4), [Cu(o-ampacac)(bpy)]ClO4(5), and [Cu(o-ampbzac)(bpy)]ClO4(6) (phen = 1,10-phenanthroline, bpy = 2, 2‧-bipyridine, o-ampacac = (Z)-4-(2-hydroxylamino)pent-3-ene-2-one,o-ampbzac = (Z)-4-(2-hydroxylamino)-4-phenylbut-3-ene-2-one)and characterized by UV-Vis, IR, EPR and cyclic voltammetry. Ligands were characterized by NMR spectra. Single crystal X-ray studies of the complex 1 shows Cu(II) ions are located in a highly distorted octahedral environment. Absorption spectral studies reveal that the complexes 1-6 exhibit hypochromicity during the interaction with DNA and binding constant values derived from spectral and electrochemical studies indicate that complexes 1, 2 and 3 bind strongly with DNA possibly by an intercalative mode. Electrochemical studies reveal that the complexes 1-4 prefer to bind with DNA in Cu(I) rather than Cu(II) form. The shift in the formal potentials E1/2 and CD spectral studies suggest groove or electrostatic binding mode for the complexes 4-6. Complex 1 can cleave supercoiled (SC) pUC18 DNA efficiently into nicked form II under photolytic conditions and into an open circular form (form II) and linear form (form III) in the presence of H2O2 at pH 8.0 and 37 °C, while the complex 2 does not cleave DNA under similar conditions.

Interactions of solute (3p, 4p, 5p and 6p) with solute, vacancy and divacancy in bcc Fe

NASA Astrophysics Data System (ADS)

You, Yu-Wei; Kong, Xiang-Shan; Wu, Xue-Bang; Liu, Wei; Liu, C. S.; Fang, Q. F.; Chen, J. L.; Luo, G.-N.; Wang, Zhiguang

2014-12-01

Solute-vacancy binding energy is a key quantity in understanding solute diffusion kinetics and phase segregation, and may help choice of alloy compositions for future material design. However, the binding energy of solute with vacancy is notoriously difficult to measure and largely unknown in bcc Fe. With first-principles method, we systemically calculate the binding energies of solute (3p, 4p, 5p and 6p alloying solutes are included) with vacancy, divacancy and solute in bcc Fe. The binding energy of Si with vacancy in the present work is in good consistent with experimental value available. All the solutes considered are able to form stable solute-vacancy, solute-divacancy complexes, and the binding strength of solute-divacancy is about two times larger than that of solute-vacancy. Most solutes could not form stable solute-solute complexes except S, Se, In and Tl. The factors controlling the binding energies are analyzed at last.

Structure determination of a sugar-binding protein from the phytopathogenic bacterium Xanthomonas citri

PubMed Central

Medrano, Francisco Javier; de Souza, Cristiane Santos; Romero, Antonio; Balan, Andrea

2014-01-01

The uptake of maltose and related sugars in Gram-negative bacteria is mediated by an ABC transporter encompassing a periplasmic component (the maltose-binding protein or MalE), a pore-forming membrane protein (MalF and MalG) and a membrane-associated ATPase (MalK). In the present study, the structure determination of the apo form of the putative maltose/trehalose-binding protein (Xac-MalE) from the citrus pathogen Xanthomonas citri in space group P6522 is described. The crystals contained two protein molecules in the asymmetric unit and diffracted to 2.8 Å resolution. Xac-MalE conserves the structural and functional features of sugar-binding proteins and a ligand-binding pocket with similar characteristics to eight different orthologues, including the residues for maltose and trehalose interaction. This is the first structure of a sugar-binding protein from a phytopathogenic bacterium, which is highly conserved in all species from the Xanthomonas genus. PMID:24817711

Mass spectrometry reveals that the antibiotic simocyclinone D8 binds to DNA gyrase in a "bent-over" conformation: evidence of positive cooperativity in binding.

PubMed

Edwards, Marcus J; Williams, Mark A; Maxwell, Anthony; McKay, Adam R

2011-05-03

DNA topoisomerases are enzymes that control DNA topology and are vital targets for antimicrobial and anticancer drugs. Here we present a mass spectrometry study of complexes formed between the A subunit of the topoisomerase DNA gyrase and the bifunctional inhibitor simocyclinone D8 (SD8), an antibiotic isolated from Streptomyces. These studies show that, in an alternative mode of interaction to that found by X-ray crystallography, each subunit binds a single bifunctional inhibitor with separate binding pockets for the two ends of SD8. The gyrase subunits form constitutive dimers, and fractional occupancies of inhibitor-bound states show that there is strong allosteric cooperativity in the binding of two bifunctional ligands to the dimer. We show that the mass spectrometry data can be fitted to a general model of cooperative binding via an extension of the "tight-binding" approach, providing a rigorous determination of the dissociation constants and degree of cooperativity. This general approach will be applicable to other systems with multiple binding sites and highlights mass spectrometry's role as a powerful emerging tool for unraveling the complexities of biomolecular interactions.

Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chakraborty, Kaushik; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in

2015-07-28

Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging themore » ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.« less

Resolution of the diadenosine 5',5"'-P1,P4-tetraphosphate binding subunit from a multiprotein form of HeLa cell DNA polymerase alpha.

PubMed Central

Baril, E; Bonin, P; Burstein, D; Mara, K; Zamecnik, P

1983-01-01

A diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding subunit has been resolved from a high molecular weight (640,000) multiprotein form of DNA polymerase alpha [deoxynucleoside triphosphate:DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] from HeLa cells [DNA polymerase alpha 2 of Lamothe, P., Baril, B., Chi, A., Lee, L. & Baril, E. (1981) Proc. Natl. Acad. Sci. USA 78, 4723-4727]. The Ap4A binding activity copurifies with the DNA polymerizing activity during the course of purification. Hydrophobic chromatography on butylagarose resolves the Ap4A binding activity from the DNA polymerase. The Ap4A binding activity is protein in nature since the binding of Ap4A is abolished by treatment of the isolated binding activity with proteinase K but is insensitive to treatment with DNase or RNase. The molecular weight of the Ap4A binding protein, as determined by polyacrylamide gel electrophoresis under nondenaturing conditions or by NaDodSO4/polyacrylamide gel electrophoresis after photoaffinity labeling of the protein with [32P]Ap4A is 92,000 or 47,000. The binding activity of this protein is highly specific for Ap4A. Images PMID:6576366

Metallomics for drug development: a further insight into intracellular activation chemistry of a ruthenium(III)-based anticancer drug gained using a multidimensional analytical approach.

PubMed

Matczuk, Magdalena; Prządka, Monika; Aleksenko, Svetlana S; Czarnocki, Zbigniew; Pawlak, Katarzyna; Timerbaev, Andrei R; Jarosz, Maciej

2014-01-01

The mechanism by which the most relevant ruthenium anticancer drugs are activated in tumors to commence their tumor-inhibiting action remains one of the challenging research tasks of present-day metallomics. This contribution aims to capture and identify eventually more reactive species of one of two bis-indazole tetrachloridoruthenate(III) compounds that are progressing in clinical trials. In view of the fact that the transport of ruthenium into cancer cells is governed by transferrin receptors, the susceptibility of the Ru drug adduct with holo-transferrin to exposure by glutathione and ascorbic acid (at their cancer cytosol concentrations) was studied by inductively coupled plasma mass spectrometry (ICP-MS), following isolation of the reaction products by ultrafiltration. Next, capillary electrophoresis coupled to ICP-MS was applied to monitor changes in the Ru speciation both under simulated cancer cytosol conditions and in real cytosol and to assign the charge state of novel metal species. The latter were identified by using tandem electrospray ionization MS in the respective ion mode. The formation of ruthenium(II) species was for the first time revealed, in which the central metal is coordinated by the reduced (GSH) or the oxidized (GSSG) form of glutathione, i.e. [Ru(II)HindCl4(GSH)](2-) and [Ru(II)HindCl4(GSSG)](2-), respectively (Hind = indazole). Ascorbic acid released the ruthenium functionality from the protein-bound form in a different way, the products of adduct cleavage containing aqua ligands. Distribution of low-molecular mass species of Ru in human cytosol was found to have very much in common with the ruthenium speciation assayed under simulated cytosol conditions.

Changes in the distribution of mechanically dependent plants along a gradient of past hurricane impact

PubMed Central

Batke, Sven P.; Kelly, Daniel L.

2015-01-01

The severity of the effects that large disturbance events such as hurricanes can have on the forest canopy and the associated mechanically dependent plant community (epiphytes, climbers, etc.) is dependent on the frequency and intensity of the disturbance events. Here we investigate the effects of different structural and environmental properties of the host trees and previously modelled past hurricanes on dependent plants in Cusuco National Park, Honduras. Tree-climbing methods were employed to sample different dependent life-forms in ten 150 × 150 m plots. We identified 7094 individuals of dependent plants from 214 different species. For holo- and hemi-epiphytes, we found that diversity was significantly negatively related to past hurricane impact. The abundance of dependent plants was greatly influenced by their position in tree canopy and hurricane disturbance regimes. The relationship between abundance and mean branch height shifts across a gradient of hurricane impact (from negative to positive), which might result from a combination of changes in abundance of individual species and composition of the dependent flora across sites. Mechanically dependent plants also responded to different structural and environmental conditions along individual branches. The variables that explained much of the community differences of life-forms and families among branches were branch surface area and bryophyte cover. The factors that explained most variation at a plot level were mean vapour pressure deficit and elevation. At the level of the individual tree, the most important factors were canopy openness and past hurricane impact. We believe that more emphasis needs to be placed on the effects that past disturbance events have on mechanically dependent plant communities, particularly in areas that are prone to catastrophic perturbations. PMID:26286220

Functionalization of Recombinant Amelogenin Nanospheres Allows Their Binding to Cellulose Materials.

PubMed

Butler, Samuel J; Bülow, Leif; Bonde, Johan

2016-10-01

Protein engineering to functionalize the self-assembling enamel matrix protein amelogenin with a cellulose binding domain (CBD) is used. The purpose is to examine the binding of the engineered protein, rh174CBD, to cellulose materials, and the possibility to immobilize self-assembled amelogenin nanospheres on cellulose. rh174CBD assembled to nanospheres ≈35 nm in hydrodynamic diameter, very similar in size to wild type amelogenin (rh174). Uniform particles are formed at pH 10 for both rh174 and rh174CBD, but only rh174CBD nanospheres showes significant binding to cellulose (Avicel). Cellulose binding of rh174CBD is promoted when the protein is self-assembled to nanospheres, compared to being in a monomeric form, suggesting a synergistic effect of the multiple CBDs on the nanospheres. The amount of bound rh174CBD nanospheres reached ≈15 mg/g Avicel, which corresponds to 4.2 to 6.3 × 10 -7 mole/m 2 . By mixing rh174 and rh174CBD, and then inducing self-assembly, composite nanospheres with a high degree of cellulose binding can be formed, despite a lower proportion of rh174CBD. This demonstrates that amelogenin variants like rh174 can be incorporated into the nanospheres, and still retain most of the binding to cellulose. Engineered amelogenin nanoparticles can thus be utilized to construct a range of new cellulose based hybrid materials, e.g. for wound treatment. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Constitutively active rhodopsin mutants causing night blindness are effectively phosphorylated by GRKs but differ in arrestin-1 binding

PubMed Central

Vishnivetskiy, Sergey. A.; Ostermaier, Martin K.; Singhal, Ankita; Panneels, Valerie; Homan, Kristoff T.; Glukhova, Alisa; Sligar, Stephen G.; Tesmer, John J. G.; Schertler, Gebhard F.X.; Standfuss, Joerg; Gurevich, Vsevolod V.

2013-01-01

The effects of activating mutations associated with night blindness on the stoichiometry of rhodopsin interactions with G protein-coupled receptor kinase 1 (GRK1) and arrestin-1 have not been reported. Here we show that the monomeric form of WT rhodopsin and its constitutively active mutants M257Y, G90D, and T94I, reconstituted into HDL particles are effectively phosphorylated by GRK1, as well as two more ubiquitously expressed subtypes, GRK2 and GRK5. All versions of arrestin-1 tested (WT, pre-activated, and constitutively monomeric mutants) bind to monomeric rhodopsin and show the same selectivity for different functional forms of rhodopsin as in native disc membranes. Rhodopsin phosphorylation by GRK1 and GRK2 promotes arrestin-1 binding to a comparable extent, whereas similar phosphorylation by GRK5 is less effective, suggesting that not all phosphorylation sites on rhodopsin are equivalent in promoting arrestin-1 binding. The binding of WT arrestin-1 to phospho-opsin is comparable to the binding to its preferred target, P-Rh*, suggesting that in photoreceptors arrestin-1 only dissociates after opsin regeneration with 11-cis-retinal, which converts phospho-opsin into inactive phospho-rhodopsin that has lower affinity for arrestin-1. Reduced binding of arrestin-1 to the phospho-opsin form of G90D mutant likely contributes to night blindness caused by this mutation in humans. PMID:23872075

Constitutively active rhodopsin mutants causing night blindness are effectively phosphorylated by GRKs but differ in arrestin-1 binding.

PubMed

Vishnivetskiy, Sergey A; Ostermaier, Martin K; Singhal, Ankita; Panneels, Valerie; Homan, Kristoff T; Glukhova, Alisa; Sligar, Stephen G; Tesmer, John J G; Schertler, Gebhard F X; Standfuss, Joerg; Gurevich, Vsevolod V

2013-11-01

The effects of activating mutations associated with night blindness on the stoichiometry of rhodopsin interactions with G protein-coupled receptor kinase 1 (GRK1) and arrestin-1 have not been reported. Here we show that the monomeric form of WT rhodopsin and its constitutively active mutants M257Y, G90D, and T94I, reconstituted into HDL particles are effectively phosphorylated by GRK1, as well as two more ubiquitously expressed subtypes, GRK2 and GRK5. All versions of arrestin-1 tested (WT, pre-activated, and constitutively monomeric mutants) bind to monomeric rhodopsin and show the same selectivity for different functional forms of rhodopsin as in native disc membranes. Rhodopsin phosphorylation by GRK1 and GRK2 promotes arrestin-1 binding to a comparable extent, whereas similar phosphorylation by GRK5 is less effective, suggesting that not all phosphorylation sites on rhodopsin are equivalent in promoting arrestin-1 binding. The binding of WT arrestin-1 to phospho-opsin is comparable to the binding to its preferred target, P-Rh*, suggesting that in photoreceptors arrestin-1 only dissociates after opsin regeneration with 11-cis-retinal, which converts phospho-opsin into inactive phospho-rhodopsin that has lower affinity for arrestin-1. Reduced binding of arrestin-1 to the phospho-opsin form of G90D mutant likely contributes to night blindness caused by this mutation in humans. © 2013.

LINC Complexes Form by Binding of Three KASH Peptides to Domain Interfaces of Trimeric SUN Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sosa, Brian A.; Rothballer, Andrea; Kutay, Ulrike

Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the nuclear envelope and are composed of KASH and SUN proteins residing in the outer and inner nuclear membrane, respectively. LINC formation relies on direct binding of KASH and SUN in the perinuclear space. Thereby, molecular tethers are formed that can transmit forces for chromosome movements, nuclear migration, and anchorage. We present crystal structures of the human SUN2-KASH1/2 complex, the core of the LINC complex. The SUN2 domain is rigidly attached to a trimeric coiled coil that prepositions it to bind three KASH peptides. The peptides bind in three deep and expansivemore » grooves formed between adjacent SUN domains, effectively acting as molecular glue. In addition, a disulfide between conserved cysteines on SUN and KASH covalently links both proteins. The structure provides the basis of LINC complex formation and suggests a model for how LINC complexes might arrange into higher-order clusters to enhance force-coupling.« less

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA

PubMed Central

Mori, Tetsuya; Saveliev, Sergei V.; Xu, Yao; Stafford, Walter F.; Cox, Michael M.; Inman, Ross B.; Johnson, Carl H.

2002-01-01

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecA/DnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecA/DnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns. PMID:12477935

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA.

PubMed

Mori, Tetsuya; Saveliev, Sergei V; Xu, Yao; Stafford, Walter F; Cox, Michael M; Inman, Ross B; Johnson, Carl H

2002-12-24

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecADnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecADnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns.

Dense Bicoid hubs accentuate binding along the morphogen gradient

PubMed Central

Mir, Mustafa; Reimer, Armando; Haines, Jenna E.; Li, Xiao-Yong; Stadler, Michael; Garcia, Hernan

2017-01-01

Morphogen gradients direct the spatial patterning of developing embryos; however, the mechanisms by which these gradients are interpreted remain elusive. Here we used lattice light-sheet microscopy to perform in vivo single-molecule imaging in early Drosophila melanogaster embryos of the transcription factor Bicoid that forms a gradient and initiates patterning along the anteroposterior axis. In contrast to canonical models, we observed that Bicoid binds to DNA with a rapid off rate throughout the embryo such that its average occupancy at target loci is on-rate-dependent. We further observed Bicoid forming transient “hubs” of locally high density that facilitate binding as factor levels drop, including in the posterior, where we observed Bicoid binding despite vanishingly low protein levels. We propose that localized modulation of transcription factor on rates via clustering provides a general mechanism to facilitate binding to low-affinity targets and that this may be a prevalent feature of other developmental transcription factors. PMID:28982761

Characterization and inhibitor discovery of one novel malonyl-CoA: acyl carrier protein transacylase (MCAT) from Helicobacter pylori.

PubMed

Liu, Weizhi; Han, Cong; Hu, Lihong; Chen, Kaixian; Shen, Xu; Jiang, Hualiang

2006-01-23

Type II fatty acid synthesis (FAS II) is an essential process for bacteria survival, and malonyl-CoA:acyl carrier protein transacylase (MCAT) is a key enzyme in FAS II pathway, which is responsible for transferring the malonyl group from malonyl-CoA to the holo-ACP by forming malonyl-ACP. In this work, we described the cloning, characterization and enzymatic inhibition of a new MCAT from Helicobacter pylori strain SS1 (HpMCAT), and the gene sequence of HpfabD was deposited in the GenBank database (Accession No. AY738332 ). Enzymatic characterization of HpMCAT showed that the K(m) value for malonyl-CoA was 21.01+/-2.3 microM, and the thermal- and guanidinium hydrochloride-induced unfolding processes for HpMCAT were quantitatively investigated by circular dichroism spectral analyses. Moreover, a natural product, corytuberine, was discovered to demonstrate inhibitory activity against HpMCAT with IC(50) value at 33.1+/-3.29 microM. Further enzymatic assay results indicated that corytuberine inhibits HpMCAT in an uncompetitive manner. To our knowledge, this is the firstly reported MCAT inhibitor to date. This current work is hoped to supply useful information for better understanding the MCAT features of H. pylori strain, and corytuberine might be used as a potential lead compound in the discovery of the antibacterial agents using HpMCAT as target.

Crystallization and X-ray diffraction analysis of a putative bacterial class I labdane-related diterpene synthase.

PubMed

Serrano-Posada, Hugo; Centeno-Leija, Sara; Rojas-Trejo, Sonia; Stojanoff, Vivian; Rodríguez-Sanoja, Romina; Rudiño-Piñera, Enrique; Sánchez, Sergio

2015-09-01

Labdane-related diterpenoids are natural products with potential pharmaceutical applications that are rarely found in bacteria. Here, a putative class I labdane-related diterpene synthase (LrdC) identified by genome mining in a streptomycete was successfully crystallized using the microbatch method. Crystals of the LrdC enzyme were obtained in a holo form with its natural cofactor Mg(2+) (LrdC-Mg(2+)) and in complex with inorganic pyrophosphate (PPi) (LrdC-Mg(2+)-PPi). Crystals of native LrdC-Mg(2+) diffracted to 2.50 Å resolution and belonged to the trigonal space group P3221, with unit-cell parameters a = b = 107.1, c = 89.2 Å. Crystals of the LrdC-Mg(2+)-PPi complex grown in the same conditions as the native enzyme with PEG 8000 diffracted to 2.36 Å resolution and also belonged to the trigonal space group P3221. Crystals of the LrdC-Mg(2+)-PPi complex grown in a second crystallization condition with PEG 3350 diffracted to 2.57 Å resolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 49.9, b = 104.1, c = 66.5 Å, β = 111.4°. The structure was determined by the single-wavelength anomalous dispersion (SAD) technique using the osmium signal from a potassium hexachloroosmate (IV) derivative.

The hepta-beta-glucoside elicitor-binding proteins from legumes represent a putative receptor family.

PubMed

Mithöfer, A; Fliegmann, J; Neuhaus-Url, G; Schwarz, H; Ebel, J

2000-08-01

The ability of legumes to recognize and respond to beta-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound beta-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to beta-glucans. The cDNAs for the beta-glucan-binding proteins of bean and soybean were cloned. The deduced 75-kDa proteins are predominantly hydrophilic and constitute a unique class of glucan-binding proteins with no currently recognizable functional domains. Heterologous expression of the soybean beta-glucan-binding protein in tomato cells resulted in the generation of a high-affinity binding site for the elicitor-active hepta-beta-glucoside conjugate (Kd = 4.5 nM). Ligand competition experiments with the recombinant binding sites demonstrated similar ligand specificities when compared with soybean. In both soybean and transgenic tomato, membrane-bound, active forms of the glucan-binding proteins coexist with immunologically detectable, soluble but inactive forms of the proteins. Reconstitution of a soluble protein fraction into lipid vesicles regained beta-glucoside-binding activity but with lower affinity (Kd = 130 nM). We conclude that the beta-glucan elicitor receptors of legumes are composed of the 75 kDa glucan-binding proteins as the critical components for ligand-recognition, and of an as yet unknown membrane anchor constituting the plasma membrane-associated receptor complex.

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity*

PubMed Central

Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L.

2015-01-01

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. PMID:25903123

DNA-binding studies of a tetraalkyl-substituted porphyrin and the mutually adaptive distortion principle.

PubMed

Ghimire, Srijana; Fanwick, Phillip E; McMillin, David R

2014-10-20

This investigation explores DNA-binding interactions of various forms of an alkyl-substituted cationic porphyrin, H2TC3 (5,10,15,20-tetra[3-(3'-methylimidazolium-1'-yl)]porphyrin). The motivating idea is that incorporating alkyl rather than aryl substituents in the meso positions will enhance the prospects for intercalative as well as external binding to DNA hosts. The ligands may also be applicable for photodynamic and/or anticancer therapy. Methods employed include absorbance, circular dichroism, and emission spectroscopies, as well as viscometry and X-ray crystallography. By comparison with the classical H2T4 system, H2TC3 exhibits a higher molar extinction coefficient but is more prone to self-association. Findings of note include that the copper(II)-containing form Cu(TC3) is adept at internalizing into single-stranded as well as B-form DNA, regardless of the base composition. Surprisingly, however, external binding of H2TC3 occurs within domains that are rich in adenine-thymine base pairs. The difference in the deformability of H2TC3 versus Cu(TC3) probably accounts for the reactivity difference. Finally, Zn(TC3) binds externally, as the metal center remains five-coordinate.

Microsolvation of Fluoromethane.

PubMed

Rosenberg, Robert E

2016-09-29

Fluorinated organic compounds are ubiquitous in the pharmaceutical and agricultural industries. To better discern the mode of action of these compounds, it is critical to understand the potential for and strength of hydrogen bonds involving fluorine. It is known that CH3F forms a hydrogen bond with H2O in the gas phase but does not dissolve in bulk water. This paper examines CH3F surrounded by one to six water molecules. For systems of similar topologies, CH3F formed hydrogen bonds of nearly the same strength as water. Although CH3F can bind to a second water cluster with only a modest loss in binding energy, it must bind to these clusters as a double hydrogen bond acceptor. This means that CH3F cannot form a low-energy cyclic 2D hydrogen bonding network with water molecules, which limits its solubility in bulk water. However, CH3F should be able to bind to the periphery of small hydrogen bonding networks. These conclusions were not appreciably altered by SMD calculations. A more complete consideration of solvation, especially entropic effects, was not undertaken. Data for geometries, population changes, and vibrational frequency shifts were also analyzed and compared to binding energies.

Iodine binding to explore the conformational state of internal chains of amylopectin.

PubMed

Shen, Xinyu; Bertoft, Eric; Zhang, Genyi; Hamaker, Bruce R

2013-10-15

Previous studies have found that the proportion of long chains of amylopectin correlates to its functional and nutritional properties. As a possible explanation of this correlation, the iodine binding property of amylopectin internal chains was investigated as an indirect evidence of their ability to form helices for intra- or inter-molecular interactions. Waxy and amylose-extender waxy corn starches were hydrolyzed by β-amylase for varying periods of time to incrementally remove the external chains, and the absorbance and the wavelength of maximum absorbance of iodine binding were examined. Experimental results suggest that iodine can bind with both external and internal chains; a significant amount of absorption comes from the latter, as stepwise removal of external chains only somewhat reduced absorption. Internal amylopectin chains, thus, were concluded to likely pre-exist in helical form, as opposed to a conformational change into helices facilitating iodine binding in the absence of external chains. Such internal chain helical structures possibly drive intermolecular interactions that would explain why amylopectin with high proportion of internal chains form harder gels, create pastes less prone to shear breakdown, and are more slowly digesting. Copyright © 2013 Elsevier Ltd. All rights reserved.

Molecular simulations and Markov state modeling reveal the structural diversity and dynamics of a theophylline-binding RNA aptamer in its unbound state

PubMed Central

Warfield, Becka M.

2017-01-01

RNA aptamers are oligonucleotides that bind with high specificity and affinity to target ligands. In the absence of bound ligand, secondary structures of RNA aptamers are generally stable, but single-stranded and loop regions, including ligand binding sites, lack defined structures and exist as ensembles of conformations. For example, the well-characterized theophylline-binding aptamer forms a highly stable binding site when bound to theophylline, but the binding site is unstable and disordered when theophylline is absent. Experimental methods have not revealed at atomic resolution the conformations that the theophylline aptamer explores in its unbound state. Consequently, in the present study we applied 21 microseconds of molecular dynamics simulations to structurally characterize the ensemble of conformations that the aptamer adopts in the absence of theophylline. Moreover, we apply Markov state modeling to predict the kinetics of transitions between unbound conformational states. Our simulation results agree with experimental observations that the theophylline binding site is found in many distinct binding-incompetent states and show that these states lack a binding pocket that can accommodate theophylline. The binding-incompetent states interconvert with binding-competent states through structural rearrangement of the binding site on the nanosecond to microsecond timescale. Moreover, we have simulated the complete theophylline binding pathway. Our binding simulations supplement prior experimental observations of slow theophylline binding kinetics by showing that the binding site must undergo a large conformational rearrangement after the aptamer and theophylline form an initial complex, most notably, a major rearrangement of the C27 base from a buried to solvent-exposed orientation. Theophylline appears to bind by a combination of conformational selection and induced fit mechanisms. Finally, our modeling indicates that when Mg2+ ions are present the population of binding-competent aptamer states increases more than twofold. This population change, rather than direct interactions between Mg2+ and theophylline, accounts for altered theophylline binding kinetics. PMID:28437473

Neuronal Calcium Sensor-1 Binds the D2 Dopamine Receptor and G-protein-coupled Receptor Kinase 1 (GRK1) Peptides Using Different Modes of Interactions.

PubMed

Pandalaneni, Sravan; Karuppiah, Vijaykumar; Saleem, Muhammad; Haynes, Lee P; Burgoyne, Robert D; Mayans, Olga; Derrick, Jeremy P; Lian, Lu-Yun

2015-07-24

Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca(2+)-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca(2+)/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca(2+)/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178-Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca(2+)/NCS-1·D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178-Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Neuronal Calcium Sensor-1 Binds the D2 Dopamine Receptor and G-protein-coupled Receptor Kinase 1 (GRK1) Peptides Using Different Modes of Interactions*

PubMed Central

Pandalaneni, Sravan; Karuppiah, Vijaykumar; Saleem, Muhammad; Haynes, Lee P.; Burgoyne, Robert D.; Mayans, Olga; Derrick, Jeremy P.; Lian, Lu-Yun

2015-01-01

Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca2+-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca2+/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca2+/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178–Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca2+/NCS-1·D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178–Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1. PMID:25979333

Two-Dimensional Wetting of a Stepped Copper Surface

NASA Astrophysics Data System (ADS)

Lin, C.; Avidor, N.; Corem, G.; Godsi, O.; Alexandrowicz, G.; Darling, G. R.; Hodgson, A.

2018-02-01

Highly corrugated, stepped surfaces present regular 1D arrays of binding sites, creating a complex, heterogeneous environment to water. Rather than decorating the hydrophilic step sites to form 1D chains, water on stepped Cu(511) forms an extended 2D network that binds strongly to the steps but bridges across the intervening hydrophobic Cu(100) terraces. The hydrogen-bonded network contains pentamer, hexamer, and octomer water rings that leave a third of the stable Cu step sites unoccupied in order to bind water H down close to the step dipole and complete three hydrogen bonds per molecule.

Analysis of RNA binding by the dengue virus NS5 RNA capping enzyme.

PubMed

Henderson, Brittney R; Saeedi, Bejan J; Campagnola, Grace; Geiss, Brian J

2011-01-01

Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5' end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K(D) for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5' phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM). Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5' di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.

Crystal Structure of the Arginine Repressor Protein in Complex With the DNA Operator From Mycobacterium Tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cherney, L.T.; Cherney, M.M.; Garen, C.R.

2009-05-12

The Mycobacterium tuberculosis (Mtb) gene product encoded by open reading frame Rv1657 is an arginine repressor (ArgR). All genes involved in the L-arginine (hereafter arginine) biosynthetic pathway are essential for optimal growth of the Mtb pathogen, thus making MtbArgR a potential target for drug design. The C-terminal domains of arginine repressors (CArgR) participate in oligomerization and arginine binding. Several crystal forms of CArgR from Mtb (MtbCArgR) have been obtained. The X-ray crystal structures of MtbCArgR were determined at 1.85 {angstrom} resolution with bound arginine and at 2.15 {angstrom} resolution in the unliganded form. These structures show that six molecules ofmore » MtbCArgR are arranged into a hexamer having approximate 32 point symmetry that is formed from two trimers. The trimers rotate relative to each other by about 11{sup o} upon binding arginine. All residues in MtbCArgR deemed to be important for hexamer formation and for arginine binding have been identified from the experimentally determined structures presented. The hexamer contains six regular sites in which the arginine molecules have one common binding mode and three sites in which the arginine molecules have two overlapping binding modes. The latter sites only bind the ligand at high (200 mM) arginine concentrations.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wurzburg, Beth A.; Kim, Beomkyu; Tarchevskaya, Svetlana S.

IgE antibodies interact with the high affinity IgE Fc receptor, FcϵRI, and activate inflammatory pathways associated with the allergic response. The IgE-Fc region, comprising the C-terminal domains of the IgE heavy chain, binds FcϵRI and can adopt different conformations ranging from a closed form incompatible with receptor binding to an open, receptor-bound state. A number of intermediate states are also observed in different IgE-Fc crystal forms. To further explore this apparent IgE-Fc conformational flexibility and to potentially trap a closed, inactive state, we generated a series of disulfide bond mutants. Here we describe the structure and biochemical properties of anmore » IgE-Fc mutant that is trapped in the closed, non-receptor binding state via an engineered disulfide at residue 335 (Cys-335). Reduction of the disulfide at Cys-335 restores the ability of IgE-Fc to bind to its high affinity receptor, FcϵRIα. The structure of the Cys-335 mutant shows that its conformation is within the range of previously observed, closed form IgE-Fc structures and that it retains the hydrophobic pocket found in the hinge region of the closed conformation. Locking the IgE-Fc into the closed state with the Cys-335 mutation does not affect binding of two other IgE-Fc ligands, omalizumab and DARPin E2_79, demonstrating selective blocking of the high affinity receptor binding.« less

Structural Insights into the Quadruplex-Duplex 3' Interface Formed from a Telomeric Repeat: A Potential Molecular Target.

PubMed

Russo Krauss, Irene; Ramaswamy, Sneha; Neidle, Stephen; Haider, Shozeb; Parkinson, Gary N

2016-02-03

We report here on an X-ray crystallographic and molecular modeling investigation into the complex 3' interface formed between putative parallel stranded G-quadruplexes and a duplex DNA sequence constructed from the human telomeric repeat sequence TTAGGG. Our crystallographic approach provides a detailed snapshot of a telomeric 3' quadruplex-duplex junction: a junction that appears to have the potential to form a unique molecular target for small molecule binding and interference with telomere-related functions. This unique target is particularly relevant as current high-affinity compounds that bind putative G-quadruplex forming sequences only rarely have a high degree of selectivity for a particular quadruplex. Here DNA junctions were assembled using different putative quadruplex-forming scaffolds linked at the 3' end to a telomeric duplex sequence and annealed to a complementary strand. We successfully generated a series of G-quadruplex-duplex containing crystals, both alone and in the presence of ligands. The structures demonstrate the formation of a parallel folded G-quadruplex and a B-form duplex DNA stacked coaxially. Most strikingly, structural data reveals the consistent formation of a TAT triad platform between the two motifs. This triad allows for a continuous stack of bases to link the quadruplex motif with the duplex region. For these crystal structures formed in the absence of ligands, the TAT triad interface occludes ligand binding at the 3' quadruplex-duplex interface, in agreement with in silico docking predictions. However, with the rearrangement of a single nucleotide, a stable pocket can be produced, thus providing an opportunity for the binding of selective molecules at the interface.

Interaction between the C-terminal domains of measles virus nucleoprotein and phosphoprotein: a tight complex implying one binding site.

PubMed

Blocquel, David; Habchi, Johnny; Costanzo, Stéphanie; Doizy, Anthony; Oglesbee, Michael; Longhi, Sonia

2012-10-01

The intrinsically disordered C-terminal domain (N(TAIL) ) of the measles virus (MeV) nucleoprotein undergoes α-helical folding upon binding to the C-terminal X domain (XD) of the phosphoprotein. The N(TAIL) region involved in binding coupled to folding has been mapped to a conserved region (Box2) encompassing residues 489-506. In the previous studies published in this journal, we obtained experimental evidence supporting a K(D) for the N(TAIL) -XD binding reaction in the nM range and also showed that an additional N(TAIL) region (Box3, aa 517-525) plays a role in binding to XD. In striking contrast with these data, studies published in this journal by Kingston and coworkers pointed out a much less stable complex (K(D) in the μM range) and supported lack of involvement of Box3 in complex formation. The objective of this study was to critically re-evaluate the role of Box3 in N(TAIL) -XD binding. Since our previous studies relied on N(TAIL) -truncated forms possessing an irrelevant Flag sequence appended at their C-terminus, we, herein, generated an N(TAIL) devoid of Box3 and any additional C-terminal residues, as well as a form encompassing only residues 482-525. We then used isothermal titration calorimetry to characterize the binding reactions between XD and these N(TAIL) forms. Results effectively argue for the presence of a single XD-binding site located within Box2, in agreement with the results by Kingston et al., while providing clear experimental support for a high-affinity complex. Altogether, the present data provide mechanistic insights into the replicative machinery of MeV and clarify a hitherto highly debated point. Copyright © 2012 The Protein Society.

Interaction between the C-terminal domains of measles virus nucleoprotein and phosphoprotein: A tight complex implying one binding site

PubMed Central

Blocquel, David; Habchi, Johnny; Costanzo, Stéphanie; Doizy, Anthony; Oglesbee, Michael; Longhi, Sonia

2012-01-01

The intrinsically disordered C-terminal domain (NTAIL) of the measles virus (MeV) nucleoprotein undergoes α-helical folding upon binding to the C-terminal X domain (XD) of the phosphoprotein. The NTAIL region involved in binding coupled to folding has been mapped to a conserved region (Box2) encompassing residues 489–506. In the previous studies published in this journal, we obtained experimental evidence supporting a KD for the NTAIL–XD binding reaction in the nM range and also showed that an additional NTAIL region (Box3, aa 517–525) plays a role in binding to XD. In striking contrast with these data, studies published in this journal by Kingston and coworkers pointed out a much less stable complex (KD in the μM range) and supported lack of involvement of Box3 in complex formation. The objective of this study was to critically re-evaluate the role of Box3 in NTAIL–XD binding. Since our previous studies relied on NTAIL-truncated forms possessing an irrelevant Flag sequence appended at their C-terminus, we, herein, generated an NTAIL devoid of Box3 and any additional C-terminal residues, as well as a form encompassing only residues 482–525. We then used isothermal titration calorimetry to characterize the binding reactions between XD and these NTAIL forms. Results effectively argue for the presence of a single XD-binding site located within Box2, in agreement with the results by Kingston et al., while providing clear experimental support for a high-affinity complex. Altogether, the present data provide mechanistic insights into the replicative machinery of MeV and clarify a hitherto highly debated point. PMID:22887965

Structural Reorganization and the Cooperative Binding of Single-stranded Telomere DNA in Sterkiella nova*

PubMed Central

Buczek, Pawel; Horvath, Martin P.

2009-01-01

In Sterkiella nova, α and β telomere proteins bind cooperatively with single-stranded DNA to form a ternary α·β·DNA complex. Association of telomere protein subunits is DNA-dependent, and α-β association enhances DNA affinity. To further understand the molecular basis for binding cooperativity, we characterized several possible stepwise assembly pathways using isothermal titration calorimetry. In one path, α and DNA first form a stable α·DNA complex followed by addition of β in a second step. Binding energy accumulates with nearly equal free energy of association for each of these steps. Heat capacity is nonetheless dramatically different with ΔCp = −305 ± 3 cal mol−1 K−1 for α binding with DNA and ΔCp = −2010 ± 20 cal mol−1 K−1 for addition of β to complete the α·β·DNA complex. By examining alternate routes including titration of single-stranded DNA with a preformed α·β complex, a significant portion of binding energy and heat capacity could be assigned to structural reorganization involving protein-protein interactions and repositioning of the DNA. Structural reorganization probably affords a mechanism to regulate high affinity binding of telomere single-stranded DNA with important implications for telomere biology. Regulation of telomere complex dissociation is thought to involve post-translational modifications in the lysine-rich C-terminal portion of β. We observed no difference in binding energetics or crystal structure when comparing complexes prepared with full-length β or a C-terminally truncated form, supporting interesting parallels between the intrinsically disordered regions of histones and this portion of β. PMID:17082188

Dynamics of Fos-Jun-NFAT1 complexes

PubMed Central

Ramirez-Carrozzi, Vladimir R.; Kerppola, Tom K.

2001-01-01

Transcription initiation in eukaryotes is controlled by nucleoprotein complexes formed through cooperative interactions among multiple transcription regulatory proteins. These complexes may be assembled via stochastic collisions or defined pathways. We investigated the dynamics of Fos-Jun-NFAT1 complexes by using a multicolor fluorescence resonance energy transfer assay. Fos-Jun heterodimers can bind to AP-1 sites in two opposite orientations, only one of which is populated in mature Fos-Jun-NFAT1 complexes. We studied the reversal of Fos-Jun binding orientation in response to NFAT1 by measuring the efficiencies of energy transfer from donor fluorophores linked to opposite ends of an oligonucleotide to an acceptor fluorophore linked to one subunit of the heterodimer. The reorientation of Fos-Jun by NFAT1 was not inhibited by competitor oligonucleotides or heterodimers. The rate of Fos-Jun reorientation was faster than the rate of heterodimer dissociation at some binding sites. The facilitated reorientation of Fos-Jun heterodimers therefore can enhance the efficiency of Fos-Jun-NFAT1 complex formation. We also examined the influence of the preferred orientation of Fos-Jun binding on the stability and transcriptional activity of Fos-Jun-NFAT1 complexes. Complexes formed at sites where Fos-Jun favored the same binding orientation in the presence and absence of NFAT1 exhibited an 8-fold slower dissociation rate than complexes formed at sites where Fos-Jun favored the opposite binding orientation. Fos-Jun-NFAT1 complexes also exhibited greater transcription activation at promoter elements that favored the same orientation of Fos-Jun binding in the presence and absence of NFAT1. Thus, the orientation of heterodimer binding can influence both the dynamics and promoter selectivity of multiprotein transcription regulatory complexes. PMID:11320240

Dynamics of Fos-Jun-NFAT1 complexes.

PubMed

Ramirez-Carrozzi, V R; Kerppola, T K

2001-04-24

Transcription initiation in eukaryotes is controlled by nucleoprotein complexes formed through cooperative interactions among multiple transcription regulatory proteins. These complexes may be assembled via stochastic collisions or defined pathways. We investigated the dynamics of Fos-Jun-NFAT1 complexes by using a multicolor fluorescence resonance energy transfer assay. Fos-Jun heterodimers can bind to AP-1 sites in two opposite orientations, only one of which is populated in mature Fos-Jun-NFAT1 complexes. We studied the reversal of Fos-Jun binding orientation in response to NFAT1 by measuring the efficiencies of energy transfer from donor fluorophores linked to opposite ends of an oligonucleotide to an acceptor fluorophore linked to one subunit of the heterodimer. The reorientation of Fos-Jun by NFAT1 was not inhibited by competitor oligonucleotides or heterodimers. The rate of Fos-Jun reorientation was faster than the rate of heterodimer dissociation at some binding sites. The facilitated reorientation of Fos-Jun heterodimers therefore can enhance the efficiency of Fos-Jun-NFAT1 complex formation. We also examined the influence of the preferred orientation of Fos-Jun binding on the stability and transcriptional activity of Fos-Jun-NFAT1 complexes. Complexes formed at sites where Fos-Jun favored the same binding orientation in the presence and absence of NFAT1 exhibited an 8-fold slower dissociation rate than complexes formed at sites where Fos-Jun favored the opposite binding orientation. Fos-Jun-NFAT1 complexes also exhibited greater transcription activation at promoter elements that favored the same orientation of Fos-Jun binding in the presence and absence of NFAT1. Thus, the orientation of heterodimer binding can influence both the dynamics and promoter selectivity of multiprotein transcription regulatory complexes.

Interaction of human synovial phospholipase A2 with mixed lipid bilayers: a coarse-grain and all-atom molecular dynamics simulation study.

PubMed

Qin, Shan-Shan; Yu, Yang-Xin; Li, Qi-Kai; Yu, Zhi-Wu

2013-02-26

Human secreted phospholipase A2s have been shown to promote inflammation in mammals by catalyzing the first step of the arachidonic acid pathway by breaking down phospholipids, producing fatty acids, including arachidonic acid. They bind to the membrane water interface to access their phospholipid substrates from the membrane. Their binding modes on membrane surfaces are regulated by diverse factors, including membrane charge, fluidity, and heterogeneity. The influence of these factors on the binding modes of the enzymes is not well understood. Here we have studied several human synovial phospholipase A2 (hs-PLA2)/mixed bilayer systems through a combined coarse-grain and all-atom molecular dynamics simulation. It was found that hydrophobic residues Leu2, Val3, Ala18, Leu19, Phe23, Gly30, and Phe63 that form the edge of the entrance of the hydrophobic binding pocket in hs-PLA2 tend to penetrate into the hydrophobic area of lipid bilayers, and more than half of the total amino acid residues make contact with the lipid headgroups. Each enzyme molecule forms 19-38 hydrogen bonds with the bilayer to which it binds, most of which are with the phosphate groups. Analysis of the root-mean-square deviation (rmsd) shows that residues Val30-Thr40, Tyr66-Gln80, and Lys107-Arg118 have relatively large rmsds during all-atom molecular dynamics simulations, in accordance with the observation of an enlarged entrance region of the hydrophobic binding pocket. The amino acid sequences forming the entrance of the binding pocket prefer to interact with lipid molecules that are more fluid or negatively charged, and the opening of the binding pocket would be larger when the lipid components are more fluid.

Why Congo red binding is specific for amyloid proteins - model studies and a computer analysis approach.

PubMed

Roterman, I; KrUl, M; Nowak, M; Konieczny, L; Rybarska, J; Stopa, B; Piekarska, B; Zemanek, G

2001-01-01

The complexing of Congo red in two different ligand forms - unimolecular and supramolecular (seven molecules in a micelle) - with eight deca-peptides organized in a b-sheet was tested by computational analysis to identify its dye-binding preferences. Polyphenylananine and polylysine peptides were selected to represent the specific side chain interactions expected to ensure particularly the stabilization of the dye-protein complex. Polyalanine was used to verify the participation of non-specific backbone-derived interactions. The initial complexes for calculation were constructed by intercalating the dye between the peptides in the middle of the beta-sheet. The long axis of the dye molecule (in the case of unimolecular systems) or the long axis of the ribbon-like micelle (in the case of the supramolecular dye form) was oriented parallel to the peptide backbone. This positioning maximally reduced the exposure of the hydrophobic diphenyl (central dye fragment) to water. In general the complexes of supramolecular Congo red ligands appeared more stable than those formed by individual dye molecules. Specific interactions (electrostatic and/or ring stacking) dominated as binding forces in the case of the single molecule, while non-specific surface adsorption seemed decisive in complexing with the supramolecular ligand. Both the unimolecular and supramolecular versions of the dye ligand were found to be likely to form complexes of sufficient stability with peptides. The low stability of the protein and the gap accessible to penetration in the peptide sheet seem sufficient for supramolecular ligand binding, but the presence of positively charged or hydrophobic amino acids may strengthen binding significantly. The need for specific interaction makes single-molecule Congo red binding rather unusual as a general amyloid protein ligand. The structural feature of Congo red, which enables specific and common interaction with amyloid proteins, probably derives from the ribbon-like self-assembled form of the dye.

Integration of G protein α (Gα) signaling by the regulator of G protein signaling 14 (RGS14).

PubMed

Brown, Nicole E; Goswami, Devrishi; Branch, Mary Rose; Ramineni, Suneela; Ortlund, Eric A; Griffin, Patrick R; Hepler, John R

2015-04-03

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gαi/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gαi1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gαi1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gαo-AlF4(-). Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gαi1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gαo-AlF4(-) and an AlF4(-)-insensitive mutant (G42R) of Gαi1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gαi1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gαo-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional magnetic microspheres

NASA Technical Reports Server (NTRS)

Rembaum, Alan (Inventor); Landel, Robert F. (Inventor); Yen, Shiao-Ping S. (Inventor)

1981-01-01

Functional magnetic particles are formed by dissolving a mucopolysaccharide such as chitosan in acidified aqueous solution containing a mixture of ferrous chloride and ferric chloride. As the pH of the solution is raised magnetite is formed in situ in the solution by raising the pH. The dissolved chitosan is a polyelectrolyte and forms micelles surrounding the granules at pH of 8-9. The chitosan precipitates on the granules to form microspheres containing the magnetic granules. On addition of the microspheres to waste aqueous streams containing dissolved ions, the hydroxyl and amine functionality of the chitosan forms chelates binding heavy metal cations such as lead, copper, and mercury and the chelates in turn bind anions such as nitrate, fluoride, phosphate and borate.

B 36N 36 fullerene-like nanocages: A novel material for drug delivery

NASA Astrophysics Data System (ADS)

Ganji, M. D.; Yazdani, H.; Mirnejad, A.

2010-07-01

We study interaction between B 36N 36 fullerene-like nanocage and glycine amino acid from the first- principles. Binding energy is calculated and glycine binding to the pure C 60 fullerene is compared. We also analyze the electronic structure and charge Mulliken population for the energetically most favorable complexes. Our results indicate that glycine can form stable bindings with B 36N 36 nanocage via their carbonyl oxygen (O) active site while, the C 60 fullerene might be unable to form stable bindings to glycine amino acid via their active sites, which is consistence with recent experimental and theoretical investigations. Thus, we arrive at the prediction that the B 36N 36 nanocage can be implemented as a novel material for drug delivery applications.

The transition state structure for binding between TAZ1 of CBP and the disordered Hif-1α CAD.

PubMed

Lindström, Ida; Andersson, Eva; Dogan, Jakob

2018-05-18

Intrinsically disordered proteins (IDPs) are common in eukaryotes. However, relatively few experimental studies have addressed the nature of the rate-limiting transition state for the coupled binding and folding reactions involving IDPs. By using site-directed mutagenesis in combination with kinetics measurements we have here characterized the transition state for binding between the globular TAZ1 domain of CREB binding protein and the intrinsically disordered C-terminal activation domain of Hif-1α (Hif-1α CAD). A total of 17 Hif-1α CAD point-mutations were generated and a Φ-value binding analysis was carried out. We found that native hydrophobic binding interactions are not formed at the transition state. We also investigated the effect the biologically important Hif-1α CAD Asn-803 hydroxylation has on the binding kinetics, and found that the whole destabilization effect due the hydroxylation is within the dissociation rate constant. Thus, the rate-limiting transition state is "disordered-like", with native hydrophobic binding contacts being formed cooperatively after the rate-limiting barrier, which is clearly shown by linear free energy relationships. The same behavior was observed in a previously characterized TAZ1/IDP interaction, which may suggest common features for the rate-limiting transition state for TAZ1/IDP interactions.

Probing the human estrogen receptor-α binding requirements for phenolic mono- and di-hydroxyl compounds: A combined synthesis, binding and docking study

PubMed Central

McCullough, Christopher; Neumann, Terrence S.; Gone, Jayapal Reddy; He, Zhengjie; Herrild, Christian; Wondergem, Julie; Pandey, Rajesh K.; Donaldson, William A.; Sem, Daniel S.

2014-01-01

Various estrogen analogs were synthesized and tested for binding to human ERα using a fluorescence polarization displacement assay. Binding affinity and orientation were also predicted using docking calculations. Docking was able to accurately predict relative binding affinity and orientation for estradiol, but only if a tightly bound water molecule bridging Arg394/Glu353 is present. Di-hydroxyl compounds sometimes bind in two orientations, which are flipped in terms of relative positioning of their hydroxyl groups. Di-hydroxyl compounds were predicted to bind with their aliphatic hydroxyl group interacting with His524 in ERα. One nonsteroid-based dihdroxyl compound was 1000-fold specific for ERβ over ERα, and was also 25-fold specific for agonist ERβ versus antagonist activity. Docking predictions suggest this specificity may be due to interaction of the aliphatic hydroxyl with His475 in the agonist form of ERβ, versus with Thr299 in the antagonist form. But, the presence of this aliphatic hydroxyl is not required in all compounds, since mono-hydroxyl (phenolic) compounds bind ERα with high affinity, via hydroxyl hydrogen bonding interactions with the ERα Arg394/Glu353/water triad, and van der Waals interactions with the rest of the molecule. PMID:24315190

Structural and functional analysis of the YAP-binding domain of human TEAD2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tian, Wei; Yu, Jianzhong; Tomchick, Diana R.

2010-06-15

The Hippo pathway controls organ size and suppresses tumorigenesis in metazoans by blocking cell proliferation and promoting apoptosis. The TEAD1-4 proteins (which contain a DNA-binding domain but lack an activation domain) interact with YAP (which lacks a DNA-binding domain but contains an activation domain) to form functional heterodimeric transcription factors that activate proliferative and prosurvival gene expression programs. The Hippo pathway inhibits the YAP-TEAD hybrid transcription factors by phosphorylating and promoting cytoplasmic retention of YAP. Here we report the crystal structure of the YAP-binding domain (YBD) of human TEAD2. TEAD2 YBD adopts an immunoglobulin-like {beta}-sandwich fold with two extra helix-turn-helixmore » inserts. NMR studies reveal that the TEAD-binding domain of YAP is natively unfolded and that TEAD binding causes localized conformational changes in YAP. In vitro binding and in vivo functional assays define an extensive conserved surface of TEAD2 YBD as the YAP-binding site. Therefore, our studies suggest that a short segment of YAP adopts an extended conformation and forms extensive contacts with a rigid surface of TEAD. Targeting a surface-exposed pocket of TEAD might be an effective strategy to disrupt the YAP-TEAD interaction and to reduce the oncogenic potential of YAP.« less

Holoclone Forming Cells from Pancreatic Cancer Cells Enrich Tumor Initiating Cells and Represent a Novel Model for Study of Cancer Stem Cells

PubMed Central

Tan, Lei; Sui, Xin; Deng, Hongkui; Ding, Mingxiao

2011-01-01

Background Pancreatic cancer is one of the direct causes of cancer-related death. High level of chemoresistance is one of the major obstacles of clinical treatment. In recent years, cancer stem cells have been widely identified and indicated as the origin of chemoresistance in multi-types of solid tumors. Increasing evidences suggest that cancer stem cells reside in the cells capable of forming holoclones continuously. However, in pancreatic cancer, holoclone-forming cells have not been characterized yet. Therefore, the goal of our present study was to indentify the holoclone-forming pancreatic cancer stem cells and develop an in vitro continuous colony formation system, which will greatly facilitate the study of pancreatic cancer stem cells. Methodology/Principal Findings Pancreatic cancer cell line BxPC3 was submitted to monoclonal cultivation to generate colonies. Based on the morphologies, colonies were classified and analyzed for their capacities of secondary colony formation, long-term survival in vitro, tumor formation in vivo, and drug resistance. Flowcytometry and quantitative RT-PCR were performed to detect the expression level of cancer stem cells associated cell surface markers, regulatory genes and microRNAs in distinct types of colonies. Three types of colonies with distinct morphologies were identified and termed as holo-, mero-, and paraclones, in which only holoclones generated descendant colonies of all three types in further passages. Compared to mero- and paraclones, holoclones possessed higher capacities of long-term survival, tumor initiation, and chemoresistance. The preferential expression of cancer stem cells related marker (CXCR4), regulatory genes (BMI1, GLI1, and GLI2) and microRNAs (miR-214, miR-21, miR-221, miR-222 and miR-155) in holoclones were also highlighted. Conclusions/Significance Our results indicate that the pancreatic tumor-initiating cells with high level of chemoresistance were enriched in holoclones derived from BxPC3 cell line. Generation of holoclones can serve as a novel model for studying cancer stem cells, and attribute to developing new anti-cancer drugs. PMID:21826251

Thermodynamics of the GTP-GDP-operated Conformational Switch of Selenocysteine-specific Translation Factor SelB*

PubMed Central

Paleskava, Alena; Konevega, Andrey L.; Rodnina, Marina V.

2012-01-01

SelB is a specialized translation factor that binds GTP and GDP and delivers selenocysteyl-tRNA (Sec-tRNASec) to the ribosome. By analogy to elongation factor Tu (EF-Tu), SelB is expected to control the delivery and release of Sec-tRNASec to the ribosome by the structural switch between GTP- and GDP-bound conformations. However, crystal structures of SelB suggested a similar domain arrangement in the apo form and GDP- and GTP-bound forms of the factor, raising the question of how SelB can fulfill its delivery function. Here, we studied the thermodynamics of guanine nucleotide binding to SelB by isothermal titration calorimetry in the temperature range between 10 and 25 °C using GTP, GDP, and two nonhydrolyzable GTP analogs, guanosine 5′-O-(γ-thio)triphosphate (GTPγS) and guanosine 5′-(β,γ-imido)-triphosphate (GDPNP). The binding of SelB to either guanine nucleotide is characterized by a large heat capacity change (−621, −467, −235, and −275 cal × mol−1 × K−1, with GTP, GTPγS, GDPNP, and GDP, respectively), associated with compensatory changes in binding entropy and enthalpy. Changes in heat capacity indicate a large decrease of the solvent-accessible surface area in SelB, amounting to 43 or 32 amino acids buried upon binding of GTP or GTPγS, respectively, and 15–19 amino acids upon binding GDP or GDPNP. The similarity of the GTP and GDP forms in the crystal structures can be attributed to the use of GDPNP, which appears to induce a structure of SelB that is more similar to the GDP than to the GTP-bound form. PMID:22740700

Interaction of acute-phase-inducible and liver-enriched nuclear factors with the promoter region of the mouse alpha sub 1 -acid glycoprotein gene-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alam, T.; Papaconstantinou, J.

1992-02-25

The synthesis and secretion of several acute-phase proteins increases markedly following physiological stress. {alpha}{sub 1}-Acid glycoprotein (AGP), a major acute-phase reactant made by the liver, is strongly induced by inflammatory agents such as lipopolysaccharide (LPS). Nuclear run-on assay showed a 17-fold increase in the rate of AGP transcription 4 h following LPS injection. DNase I footprinting assays revealed multiple protein binding domains in the mouse AGP-1 promoter region. Region B ({minus}104 to {minus}91) is protected by a liver-enriched transcription factor that is heat labile and in limiting quantity. An adjacent region, C ({minus}125 to {minus}104), is well-protected by nuclear extractsmore » from hepatocytes. Electrophoretic mobility shift assays indicated that only one DNA-protein complex can form with an oligonucleotide corresponding to region B. However, nuclear proteins from untreated mouse liver can form three strong complexes (C1, C2, and C3) and a weak one (C4) with oligonucleotide C. An acute-phase-inducible DNA-binding protein (AP-DBP) forms complex 4. A dramatic increase (over 11-fold) in AP-DBP binding activity is seen with nuclear proteins from LPS-stimulated animals. Interestingly, AP-DBP, a heat-stable factor, can form heterodimers with the transcription factor CCAAT/enhancer binding protein (C/EBP). Furthermore, purified C/EBP also binds avidly to region C. The studies indicate that several liver-enriched nuclear factors can interact with AGP-1 promoter and that AP-DBP binds to the AGP-1 promoter with high affinity only during the acute-phase induction.« less

Thermodynamics of the GTP-GDP-operated conformational switch of selenocysteine-specific translation factor SelB.

PubMed

Paleskava, Alena; Konevega, Andrey L; Rodnina, Marina V

2012-08-10

SelB is a specialized translation factor that binds GTP and GDP and delivers selenocysteyl-tRNA (Sec-tRNA(Sec)) to the ribosome. By analogy to elongation factor Tu (EF-Tu), SelB is expected to control the delivery and release of Sec-tRNA(Sec) to the ribosome by the structural switch between GTP- and GDP-bound conformations. However, crystal structures of SelB suggested a similar domain arrangement in the apo form and GDP- and GTP-bound forms of the factor, raising the question of how SelB can fulfill its delivery function. Here, we studied the thermodynamics of guanine nucleotide binding to SelB by isothermal titration calorimetry in the temperature range between 10 and 25 °C using GTP, GDP, and two nonhydrolyzable GTP analogs, guanosine 5'-O-(γ-thio)triphosphate (GTPγS) and guanosine 5'-(β,γ-imido)-triphosphate (GDPNP). The binding of SelB to either guanine nucleotide is characterized by a large heat capacity change (-621, -467, -235, and -275 cal × mol(-1) × K(-1), with GTP, GTPγS, GDPNP, and GDP, respectively), associated with compensatory changes in binding entropy and enthalpy. Changes in heat capacity indicate a large decrease of the solvent-accessible surface area in SelB, amounting to 43 or 32 amino acids buried upon binding of GTP or GTPγS, respectively, and 15-19 amino acids upon binding GDP or GDPNP. The similarity of the GTP and GDP forms in the crystal structures can be attributed to the use of GDPNP, which appears to induce a structure of SelB that is more similar to the GDP than to the GTP-bound form.

An insect-inspired model for visual binding II: functional analysis and visual attention.

PubMed

Northcutt, Brandon D; Higgins, Charles M

2017-04-01

We have developed a neural network model capable of performing visual binding inspired by neuronal circuitry in the optic glomeruli of flies: a brain area that lies just downstream of the optic lobes where early visual processing is performed. This visual binding model is able to detect objects in dynamic image sequences and bind together their respective characteristic visual features-such as color, motion, and orientation-by taking advantage of their common temporal fluctuations. Visual binding is represented in the form of an inhibitory weight matrix which learns over time which features originate from a given visual object. In the present work, we show that information represented implicitly in this weight matrix can be used to explicitly count the number of objects present in the visual image, to enumerate their specific visual characteristics, and even to create an enhanced image in which one particular object is emphasized over others, thus implementing a simple form of visual attention. Further, we present a detailed analysis which reveals the function and theoretical limitations of the visual binding network and in this context describe a novel network learning rule which is optimized for visual binding.

Structural basis underlying CAC RNA recognition by the RRM domain of dimeric RNA-binding protein RBPMS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Teplova, Marianna; Farazi, Thalia A.; Tuschl, Thomas

Abstract RNA-binding protein with multiple splicing (designated RBPMS) is a higher vertebrate mRNA-binding protein containing a single RNA recognition motif (RRM). RBPMS has been shown to be involved in mRNA transport, localization and stability, with key roles in axon guidance, smooth muscle plasticity, as well as regulation of cancer cell proliferation and migration. We report on structure-function studies of the RRM domain of RBPMS bound to a CAC-containing single-stranded RNA. These results provide insights into potential topologies of complexes formed by the RBPMS RRM domain and the tandem CAC repeat binding sites as detected by photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation. Thesemore » studies establish that the RRM domain of RBPMS forms a symmetrical dimer in the free state, with each monomer binding sequence-specifically to all three nucleotides of a CAC segment in the RNA bound state. Structure-guided mutations within the dimerization and RNA-binding interfaces of RBPMS RRM on RNA complex formation resulted in both disruption of dimerization and a decrease in RNA-binding affinity as observed by size exclusion chromatography and isothermal titration calorimetry. As anticipated from biochemical binding studies, over-expression of dimerization or RNA-binding mutants of Flag-HA-tagged RBPMS were no longer able to track with stress granules in HEK293 cells, thereby documenting the deleterious effects of such mutationsin vivo.« less

Molecular docking studies of selected tricyclic and quinone derivatives on trypanothione reductase of Leishmania infantum.

PubMed

Venkatesan, Santhosh Kannan; Shukla, Anil Kumar; Dubey, Vikash Kumar

2010-10-01

Visceral leishmaniasis, most lethal form of Leishmaniasis, is caused by Leishmania infantum in the Old world. Current therapeutics for the disease is associated with a risk of high toxicity and development of drug resistant strains. Thiol-redox metabolism involving trypanothione and trypanothione reductase, key for survival of Leishmania, is a validated target for rational drug design. Recently published structure of trypanothione reductase (TryR) from L. infantum, in oxidized and reduced form along with Sb(III), provides vital clues on active site of the enzyme. In continuation with our attempts to identify potent inhibitors of TryR, we have modeled binding modes of selected tricyclic compounds and quinone derivatives, using AutoDock4. Here, we report a unique binding mode for quinone derivatives and 9-aminoacridine derivatives, at the FAD binding domain. A conserved hydrogen bonding pattern was observed in all these compounds with residues Thr335, Lys60, His461. With the fact that these residues aid in the orientation of FAD towards the active site forming the core of the FAD binding domain, designing selective and potent compounds that could replace FAD in vivo during the synthesis of Trypanothione reductase can be deployed as an effective strategy in designing new drugs towards Leishmaniasis. We also report the binding of Phenothiazine and 9-aminoacridine derivatives at the Z site of the protein. The biological significance and possible mode of inhibition by quinone derivatives, which binds to FAD binding domain, along with other compounds are discussed. (c) 2010 Wiley Periodicals, Inc.

Stability and Sugar Recognition Ability of Ricin-Like Carbohydrate Binding Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yao, Jianzhuang; Nellas, Ricky B; Glover, Mary M

2011-01-01

Lectins are a class of proteins known for their novel binding to saccharides. Understanding this sugar recognition process can be crucial in creating structure-based designs of proteins with various biological roles. We focus on the sugar binding of a particular lectin, ricin, which has two -trefoil carbohydrate-binding domains (CRDs) found in several plant protein toxins. The binding ability of possible sites of ricin-like CRD has been puzzling. The apo and various (multiple) ligand-bound forms of the sugar-binding domains of ricin were studied by molecular dynamics simulations. By evaluating structural stability, hydrogen bond dynamics, flexibility, and binding energy, we obtained amore » detailed picture of the sugar recognition of the ricin-like CRD. Unlike what was previously believed, we found that the binding abilities of the two known sites are not independent of each other. The binding ability of one site is positively affected by the other site. While the mean positions of different binding scenarios are not altered significantly, the flexibility of the binding pockets visibly decreases upon multiple ligand binding. This change in flexibility seems to be the origin of the binding cooperativity. All the hydrogen bonds that are strong in the monoligand state are also strong in the double-ligand complex, although the stability is much higher in the latter form due to cooperativity. These strong hydrogen bonds in a monoligand state are deemed to be the essential hydrogen bonds. Furthermore, by examining the structural correlation matrix, the two domains are structurally one entity. Galactose hydroxyl groups, OH4 and OH3, are the most critical parts in both site 1 and site 2 recognition.« less

Functional identification and characterization of sodium binding sites in Na symporters

PubMed Central

Loo, Donald D. F.; Jiang, Xuan; Gorraitz, Edurne; Hirayama, Bruce A.; Wright, Ernest M.

2013-01-01

Sodium cotransporters from several different gene families belong to the leucine transporter (LeuT) structural family. Although the identification of Na+ in binding sites is beyond the resolution of the structures, two Na+ binding sites (Na1 and Na2) have been proposed in LeuT. Na2 is conserved in the LeuT family but Na1 is not. A biophysical method has been used to measure sodium dissociation constants (Kd) of wild-type and mutant human sodium glucose cotransport (hSGLT1) proteins to identify the Na+ binding sites in hSGLT1. The Na1 site is formed by residues in the sugar binding pocket, and their mutation influences sodium binding to Na1 but not to Na2. For the canonical Na2 site formed by two –OH side chains, S392 and S393, and three backbone carbonyls, mutation of S392 to cysteine increased the sodium Kd by sixfold. This was accompanied by a dramatic reduction in the apparent sugar and phlorizin affinities. We suggest that mutation of S392 in the Na2 site produces a structural rearrangement of the sugar binding pocket to disrupt both the binding of the second Na+ and the binding of sugar. In contrast, the S393 mutations produce no significant changes in sodium, sugar, and phlorizin affinities. We conclude that the Na2 site is conserved in hSGLT1, the side chain of S392 and the backbone carbonyl of S393 are important in the first Na+ binding, and that Na+ binding to Na2 promotes binding to Na1 and also sugar binding. PMID:24191006

S-Layer Nanosheet Binding of Zn and Gd

DOE Data Explorer

Ajo-Franklin, Caroline (ORCID:0000000189096712); Charrier, Marimikel; Yang, Li

2016-04-15

This data characterizes binding of Zn2+ and Gd3+ to engineered nanosheets at 40C and in a brine solution. The engineered nanosheets are composed of surface-layer (S-layer) proteins which form 2 D crystalline sheets and display Zn2+- or Gd3+-binding domains on these sheets. Their ability to bind Zn2+ is compared to S-layer nanosheets that do not contain Zn2+-binding domains. We found that the purification method of these nanosheets was a critical determinant of their function and thus have provided data on the binding from two different purification methods. A key distinction of this dataset from other datasets is that the engineered nanosheets were expressed and purified from E. coli grown at 37C as described in (Kinns, 2010; Howorka, 2000), Kinns, H., et al. Identifying assembly-inhibiting and assembly-tolerant sites in the SbsB S-layer protein from Geobacillus stearothermophilus. Journal of Molecular Biology, 2010. 395(4): p. 742-753. Howorka, S., et al. Surface-accessible residues in the monomeric and assembled forms of a bacterial surface layer protein. Journal of Biological Chemistry, 2000. 275(48): p. 37876-37886.

Thermodynamics and Mechanism of the Interaction of Willardiine Partial Agonists with a Glutamate Receptor: Implications for Drug Development

PubMed Central

2015-01-01

Understanding the thermodynamics of binding of a lead compound to a receptor can provide valuable information for drug design. The binding of compounds, particularly partial agonists, to subtypes of the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor is, in some cases, driven by increases in entropy. Using a series of partial agonists based on the structure of the natural product, willardiine, we show that the charged state of the ligand determines the enthalpic contribution to binding. Willardiines have uracil rings with pKa values ranging from 5.5 to 10. The binding of the charged form is largely driven by enthalpy, while that of the uncharged form is largely driven by entropy. This is due at least in part to changes in the hydrogen bonding network within the binding site involving one water molecule. This work illustrates the importance of charge to the thermodynamics of binding of agonists and antagonists to AMPA receptors and provides clues for further drug discovery. PMID:24850223

The Phosphatidic Acid Binding Site of the Arabidopsis Trigalactosyldiacylglycerol 4 (TGD4) Protein Required for Lipid Import into Chloroplasts*

PubMed Central

Wang, Zhen; Anderson, Nicholas Scott; Benning, Christoph

2013-01-01

Chloroplast membrane lipid synthesis relies on the import of glycerolipids from the ER. The TGD (TriGalactosylDiacylglycerol) proteins are required for this lipid transfer process. The TGD1, -2, and -3 proteins form a putative ABC (ATP-binding cassette) transporter transporting ER-derived lipids through the inner envelope membrane of the chloroplast, while TGD4 binds phosphatidic acid (PtdOH) and resides in the outer chloroplast envelope. We identified two sequences in TGD4, amino acids 1–80 and 110–145, which are necessary and sufficient for PtdOH binding. Deletion of both sequences abolished PtdOH binding activity. We also found that TGD4 from 18:3 plants bound specifically and with increased affinity PtdOH. TGD4 did not interact with other proteins and formed a homodimer both in vitro and in vivo. Our results suggest that TGD4 is an integral dimeric β-barrel lipid transfer protein that binds PtdOH with its N terminus and contains dimerization domains at its C terminus. PMID:23297418

A molecular dynamics simulation study of the association of 1,1";-binaphthyl-2,2";-diyl hydrogenphosphate enantiomers with a chiral molecular micelle

NASA Astrophysics Data System (ADS)

Morris, Kevin F.; Billiot, Eugene J.; Billiot, Fereshteh H.; Gladis, Ashley A.; Lipkowitz, Kenny B.; Southerland, William M.; Fang, Yayin

2014-08-01

Molecular dynamics (MD) simulations were used to investigate the binding of 1,1";-binaphthyl-2,2";-diyl hydrogenphosphate (BNP) enantiomers to the molecular micelle poly-(sodium undecyl-(L,L)-leucine-valine) (poly(SULV)). Poly(SULV) is used as a chiral selector in capillary electrophoresis separations. Four poly(SULV) binding pockets were identified and either (R)-BNP or (S)-BNP were docked into each pocket. MD simulations were then used to identify the preferred BNP binding site. Within the preferred site, both enantiomers formed hydrogen bonds with poly(SULV) and penetrated into the poly(SULV) core. Comparisons of BNP enantiomer binding to the preferred poly(SULV) pocket showed that (S)-BNP formed stronger hydrogen bonds, moved deeper into the binding site, and had a lower poly(SULV) binding free energy than the (R) enantiomer. Finally, MD simulation results were in agreement with capillary electrophoresis and NMR experiments. Each technique showed (S)-BNP interacted more strongly with poly(SULV) than (R)-BNP and that the site of chiral recognition was near the poly(SULV) leucine chiral center.

The biochemistry and immunology of non-canonical forms of HLA-B27.

PubMed

Shaw, Jacqueline; Hatano, Hiroko; Kollnberger, Simon

2014-01-01

HLA-B27 (B27) is strongly associated with the spondyloarthritides. B27 is expressed at the cell surface of antigen presenting cells (APC) both as canonical β2m-associated and non-canonical β2m-free heavy chain (FHC) forms which include B27 dimers (termed B272). B27 FHC forms arise in an endosomal compartment from recycling β2m-associated B27. Formation of cell surface FHC dimers is critically dependent on an unpaired reactive cysteine 67 in the α1 helix of the class I heavy chain. HLA-B27 also form redox-inducible β2m-associated dimers on exosomes and apoptosing cells. By contrast with cell surface expressed cysteine 67-dependent heavy chain dimers these dimers are dependent on a cytoplasmic cysteine 325 for their formation. HLA-B27 binds to immunoregulatory receptors including members of the Killer cell Immunoglobulin-like (KIR) and Leukocyte Immunoglobulin-like receptor family. B27 FHC bind to different but overlapping sets of these immunoreceptors compared to classical β2m-associated HLA-B27. B27 FHC bind more strongly to KIR3DL2 and LILRB2 immune receptor than other β2m-associated HLA-class I ligands. Genetic studies have implicated genes which control production of the important proinflammatory cytokine IL-17 in the pathogenesis of spondyloarthritis. Cell surface HLA-B27 FHC binding to these immune receptors or acting through other mechanisms could impact on the pathogenesis of spondyloarthritis by promoting immune cell production of IL-17. Here we review the literature on these non-canonical forms of HLA-B27 and the immune receptors they bind to and discuss the possible relevance of these interactions to the pathogenesis of spondyloarthropathy. Copyright © 2013 Elsevier Ltd. All rights reserved.

The green hemoproteins of bovine erythrocytes. II. Spectral, ligand-binding, and electrochemical properties.

PubMed

DeFilippi, L J; Hultquist, D E

1978-05-10

The two green hemoproteins isolated from bovine erythrocytes (form I and form II) have been characterized as to spectral, electrochemical, and chemical properties. The absorption spectra of the isolated hemoproteins are typical of high spin ferric states. Reduction of the hemoproteins yields high spin ferrohemoproteins. Complexation of the ferrohemoproteins with CO and the ferrihemoproteins with cyanide yields low spin complexes, demonstrating the presence of an exchangeable weak field ligand in both the ferrous and ferric states of the hemoproteins. The differences in position and intensity of the absorption peaks of the visible spectra allow the two forms to be distinguished from one another. The midpoint potential of forms I and II were found to be +0.075 and +0.019 V, respectively, at pH 6.4 and +0.038 and -0.005 V, respectively, at pH 7.0. This is consistent with the gaining of 1 proton/electron during the reduction. The Nernst plot reveals an unusual 0.5-electron transfer, whereas a quantitative titration demonstrates a 1-electron transfer. Form I binds cyanide more tightly than form II (KD of 84 and 252 micrometer, respectively). The observed spectral, electrochemical, and ligand-binding differences between forms I and II can be explained in terms of a greater electron-withdrawing ability of the side chains of the heme of form I relative to the heme of form II.

Silver(I) complexes with DNA and RNA studied by Fourier transform infrared spectroscopy and capillary electrophoresis.

PubMed Central

Arakawa, H; Neault, J F; Tajmir-Riahi, H A

2001-01-01

Ag(I) is a strong nucleic acids binder and forms several complexes with DNA such as types I, II, and III. However, the details of the binding mode of silver(I) in the Ag-polynucleotides remains unknown. Therefore, it was of interest to examine the binding of Ag(I) with calf-thymus DNA and bakers yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentration of DNA or RNA and various concentrations of Ag(I). Fourier transform infrared spectroscopy and capillary electrophoresis were used to analyze the Ag(I) binding mode, the binding constant, and the polynucleotides' structural changes in the Ag-DNA and Ag-RNA complexes. The spectroscopic results showed that in the type I complex formed with DNA, Ag(I) binds to guanine N7 at low cation concentration (r = 1/80) and adenine N7 site at higher concentrations (r = 1/20 to 1/10), but not to the backbone phosphate group. At r = 1/2, type II complexes formed with DNA in which Ag(I) binds to the G-C and A-T base pairs. On the other hand, Ag(I) binds to the guanine N7 atom but not to the adenine and the backbone phosphate group in the Ag-RNA complexes. Although a minor alteration of the sugar-phosphate geometry was observed, DNA remained in the B-family structure, whereas RNA retained its A conformation. Scatchard analysis following capillary electrophoresis showed two binding sites for the Ag-DNA complexes with K(1) = 8.3 x 10(4) M(-1) for the guanine and K(2) = 1.5 x 10(4) M(-1) for the adenine bases. On the other hand, Ag-RNA adducts showed one binding site with K = 1.5 x 10(5) M(-1) for the guanine bases. PMID:11509371

Multiple genes contribute to anhydrobiosis (tolerance to extreme desiccation) in the nematode Panagrolaimus superbus

PubMed Central

Evangelista, Cláudia Carolina Silva; Guidelli, Giovanna Vieira; Borges, Gustavo; Araujo, Thais Fenz; de Souza, Tiago Alves Jorge; Neves, Ubiraci Pereira da Costa; Tunnacliffe, Alan; Pereira, Tiago Campos

2017-01-01

Abstract The molecular basis of anhydrobiosis, the state of suspended animation entered by some species during extreme desiccation, is still poorly understood despite a number of transcriptome and proteome studies. We therefore conducted functional screening by RNA interference (RNAi) for genes involved in anhydrobiosis in the holo-anhydrobiotic nematode Panagrolaimus superbus. A new method of survival analysis, based on staining, and proof-of-principle RNAi experiments confirmed a role for genes involved in oxidative stress tolerance, while a novel medium-scale RNAi workflow identified a further 40 anhydrobiosis-associated genes, including several involved in proteostasis, DNA repair and signal transduction pathways. This suggests that multiple genes contribute to anhydrobiosis in P. superbus. PMID:29111563

The Escherichia coli cAMP receptor protein bound at a single target can activate transcription initiation at divergent promoters: a systematic study that exploits new promoter probe plasmids.

PubMed Central

El-Robh, Mohamed Samir; Busby, Stephen J W

2002-01-01

We report the first detailed quantitative study of divergent promoters dependent on the Escherichia coli cAMP receptor protein (CRP), a factor known to activate transcription initiation at target promoters by making direct interactions with the RNA polymerase holoenzyme. In this work, we show that CRP bound at a single target site is able to activate transcription at two divergently organized promoters. Experiments using promoter probe plasmids, designed to study divergent promoters in vivo and in vitro, show that the divergent promoters function independently. Further in vitro experiments show that two holo RNA polymerase molecules cannot be accommodated simultaneously at the divergent promoters. PMID:12350222

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding sitemore » are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pack, Chan-Gi, E-mail: changipack@amc.seoul.kr; Ahn, Sang-Gun

The cellular response to stress is primarily controlled in cells via transcriptional activation by heat shock factor 1 (HSF1). HSF1 is well-known to form homotrimers for activation upon heat shock and subsequently bind to target DNAs, such as heat-shock elements, by forming stress granules. A previous study demonstrated that nuclear HSF1 and HSF2 molecules in live cells interacted with target DNAs on the stress granules. However, the process underlying the binding interactions of HSF family in cells upon heat shock remains unclear. This study demonstrate for the first time that the interaction kinetics among nuclear HSF1, HSF2, and HSF4 uponmore » heat shock can be detected directly in live cells using dual color fluorescence cross-correlation spectroscopy (FCCS). FCCS analyses indicated that the binding between HSFs was dramatically changed by heat shock. Interestingly, the recovery kinetics of interaction between HSF1 molecules after heat shock could be represented by changes in the relative interaction amplitude and mobility. - Highlights: • The binding interactions among nuclear HSFs were successfully detected. • The binding kinetics between HSF1s during recovery was quantified. • HSF2 and HSF4 strongly formed hetero-complex, even before heat shock. • Nuclear HSF2 and HSF4 bound to HSF1 only after heat shock.« less

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity.

PubMed

Petzold, Christine; Marceau, Aimee H; Miller, Katherine H; Marqusee, Susan; Keck, James L

2015-06-05

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor alpha subunit form the strychnine-binding site.

PubMed Central

Vandenberg, R J; French, C R; Barry, P H; Shine, J; Schofield, P R

1992-01-01

The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. Glycine activation of the receptor is antagonized by the convulsant alkaloid strychnine. Using in vitro mutagenesis and functional analysis of the cDNA encoding the alpha 1 subunit of the human GlyR, we have identified several amino acid residues that form the strychnine-binding site. These residues were identified by transient expression of mutated cDNAs in mammalian (293) cells and examination of resultant [3H]strychnine binding, glycine displacement of [3H]strychnine, and electrophysiological responses to the application of glycine and strychnine. This mutational analysis revealed that residues from two separate domains within the alpha 1 subunit form the binding site for the antagonist strychnine. The first domain includes the amino acid residues Gly-160 and Tyr-161, and the second domain includes the residues Lys-200 and Tyr-202. These results, combined with analyses of other ligand-gated ion channel receptors, suggest a conserved tertiary structure and a common mechanism for antagonism in this receptor superfamily. PMID:1311851

Rapid, High Affinity Binding by a Fluorescein Templated Copolymer Combining Covalent, Hydrophobic, and Acid–Base Noncovalent Crosslinks

PubMed Central

Timberman, Anthony; Yang, Rongfang; Papantones, Alex; Seitz, W. Rudolf

2018-01-01

A new type of biomimetic templated copolymer has been prepared by reverse addition fragmentation chain transfer polymerization (RAFT) in dioxane. The initial formulation includes the template fluorescein, N-isopropylacrylamide (NIPAM, 84 mol %), methacrylic acid (MAA, 5-mol %), 4-vinylpyridine (4-VP, 9 mmol %), and N,N′-methylenebis(acrylamide) (MBA, 2 mol %). PolyNIPAM is a thermosensitive polymer that comes out of aqueous solution above its lower critical solution temperature forming hydrophobic ‘crosslinks’. MAA and 4-VP interact in dioxane forming acid–base crosslinks. The excess 4-VP serves as a recognition monomer organizing around the template fluorescein to form a binding site that is held in place by the noncovalent and covalent crosslinks. The MBA is a covalent crosslinker. The RAFT agent in the resulting copolylmer was reduced to a thiol and attached to gold nanoparticles. The gold nanoparticle bound copolymer binds fluorescein completely in less than two seconds with an affinity constant greater than 108 M−1. A reference copolymer prepared with the same monomers by the same procedure binds fluorescein much more weakly. PMID:29693601

An RNA-Binding Multimer Specifies Nematode Sperm Fate.

PubMed

Aoki, Scott T; Porter, Douglas F; Prasad, Aman; Wickens, Marvin; Bingman, Craig A; Kimble, Judith

2018-06-26

FOG-3 is a master regulator of sperm fate in Caenorhabditis elegans and homologous to Tob/BTG proteins, which in mammals are monomeric adaptors that recruit enzymes to RNA binding proteins. Here, we determine the FOG-3 crystal structure and in vitro demonstrate that FOG-3 forms dimers that can multimerize. The FOG-3 multimeric structure has a basic surface potential, suggestive of binding nucleic acid. Consistent with that prediction, FOG-3 binds directly to nearly 1,000 RNAs in nematode spermatogenic germ cells. Most binding is to the 3' UTR, and most targets (94%) are oogenic mRNAs, even though assayed in spermatogenic cells. When tethered to a reporter mRNA, FOG-3 represses its expression. Together these findings elucidate the molecular mechanism of sperm fate specification and reveal the evolution of a protein from monomeric to multimeric form with acquisition of a distinct mode of mRNA repression. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Ribosome protection by antibiotic resistance ATP-binding cassette protein.

PubMed

Su, Weixin; Kumar, Veerendra; Ding, Yichen; Ero, Rya; Serra, Aida; Lee, Benjamin Sian Teck; Wong, Andrew See Weng; Shi, Jian; Sze, Siu Kwan; Yang, Liang; Gao, Yong-Gui

2018-05-15

The ribosome is one of the richest targets for antibiotics. Unfortunately, antibiotic resistance is an urgent issue in clinical practice. Several ATP-binding cassette family proteins confer resistance to ribosome-targeting antibiotics through a yet unknown mechanism. Among them, MsrE has been implicated in macrolide resistance. Here, we report the cryo-EM structure of ATP form MsrE bound to the ribosome. Unlike previously characterized ribosomal protection proteins, MsrE is shown to bind to ribosomal exit site. Our structure reveals that the domain linker forms a unique needle-like arrangement with two crossed helices connected by an extended loop projecting into the peptidyl-transferase center and the nascent peptide exit tunnel, where numerous antibiotics bind. In combination with biochemical assays, our structure provides insight into how MsrE binding leads to conformational changes, which results in the release of the drug. This mechanism appears to be universal for the ABC-F type ribosome protection proteins. Copyright © 2018 the Author(s). Published by PNAS.

Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Neto, J.L. Siqueira; Instituto de Biologia, UNICAMP, Campinas, SP; Lira, C.B.B.

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 ismore » a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres.« less

MicroRNAs form triplexes with double stranded DNA at sequence-specific binding sites; a eukaryotic mechanism via which microRNAs could directly alter gene expression

DOE PAGES

Paugh, Steven W.; Coss, David R.; Bao, Ju; ...

2016-02-04

MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA). Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence that microRNAs form triple-helical structures with duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show thatmore » several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 x 10 -16) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. As a result, this work has thus revealed a new mechanism by which microRNAs can interact with gene promoter regions to modify gene transcription.« less

MicroRNAs form triplexes with double stranded DNA at sequence-specific binding sites; a eukaryotic mechanism via which microRNAs could directly alter gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paugh, Steven W.; Coss, David R.; Bao, Ju

MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA). Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence that microRNAs form triple-helical structures with duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show thatmore » several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 x 10 -16) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. As a result, this work has thus revealed a new mechanism by which microRNAs can interact with gene promoter regions to modify gene transcription.« less

Polyethylene glycol binding alters human telomere G-quadruplex structure by conformational selection

PubMed Central

Buscaglia, Robert; Miller, M. Clarke; Dean, William L.; Gray, Robert D.; Lane, Andrew N.; Trent, John O.; Chaires, Jonathan B.

2013-01-01

Polyethylene glycols (PEGs) are widely used to perturb the conformations of nucleic acids, including G-quadruplexes. The mechanism by which PEG alters G-quadruplex conformation is poorly understood. We describe here studies designed to determine how PEG and other co-solutes affect the conformation of the human telomeric quadruplex. Osmotic stress studies using acetonitrile and ethylene glycol show that conversion of the ‘hybrid’ conformation to an all-parallel ‘propeller’ conformation is accompanied by the release of about 17 water molecules per quadruplex and is energetically unfavorable in pure aqueous solutions. Sedimentation velocity experiments show that the propeller form is hydrodynamically larger than hybrid forms, ruling out a crowding mechanism for the conversion by PEG. PEGs do not alter water activity sufficiently to perturb quadruplex hydration by osmotic stress. PEG titration experiments are most consistent with a conformational selection mechanism in which PEG binds more strongly to the propeller conformation, and binding is coupled to the conformational transition between forms. Molecular dynamics simulations show that PEG binding to the propeller form is sterically feasible and energetically favorable. We conclude that PEG does not act by crowding and is a poor mimic of the intranuclear environment, keeping open the question of the physiologically relevant quadruplex conformation. PMID:23804761

Computational study of Gleevec and G6G reveals molecular determinants of kinase inhibitor selectivity

DOE PAGES

Lin, Yen -Lin; Meng, Yilin; Huang, Lei; ...

2014-10-22

Gleevec is a potent inhibitor of Abl tyrosine kinase but not of the highly homologous c-Src kinase. Because the ligand binds to an inactive form of the protein in which an Asp-Phe-Gly structural motif along the activation loop adopts a so-called DFG-out conformation, it was suggested that binding specificity was controlled by a “conformational selection” mechanism. In this context, the binding affinity displayed by the kinase inhibitor G6G poses an intriguing challenge. Although it possesses a chemical core very similar to that of Gleevec, G6G is a potent inhibitor of both Abl and c-Src kinases. Both inhibitors bind to themore » DFG-out conformation of the kinases, which seems to be in contradiction with the conformational selection mechanism. To address this issue and display the hidden thermodynamic contributions affecting the binding selectivity, molecular dynamics free energy simulations with explicit solvent molecules were carried out. Relative to Gleevec, G6G forms highly favorable van der Waals dispersive interactions upon binding to the kinases via its triazine functional group, which is considerably larger than the corresponding pyridine moiety in Gleevec. Upon binding of G6G to c-Src, these interactions offset the unfavorable free energy cost of the DFG-out conformation. When binding to Abl, however, G6G experiences an unfavorable free energy penalty due to steric clashes with the phosphate-binding loop, yielding an overall binding affinity that is similar to that of Gleevec. Such steric clashes are absent when G6G binds to c-Src, due to the extended conformation of the phosphate-binding loop.« less

Morphology manipulation of M13 bacteriophage template for nanostructure assembly

NASA Astrophysics Data System (ADS)

Ngo-Duc, Tam-Triet; Zaman, Mohammed S.; Moon, Chung-Hee; Haberer, Elaine D.

2014-08-01

A gold-binding M13 bacteriophage was used as a model system to explore templating of inorganic material on geometrically transformed viruses . Gold-binding filamentous phage were converted to spheroid form with a short chloroform treatment, and the resulting morphology was investigated with electron microscopy. Binding studies revealed that spheroid-shaped gold-binding phage preserved its affinity for gold. Spheroids adhered to a planar substrate assembled clusters or rings of gold nanoparticles. This gold-binding phage served as a demonstration of a highly shape-modifiable viral-template for inorganic materials.

Modeling covalent-modifier drugs.

PubMed

Awoonor-Williams, Ernest; Walsh, Andrew G; Rowley, Christopher N

2017-11-01

In this review, we present a summary of how computer modeling has been used in the development of covalent-modifier drugs. Covalent-modifier drugs bind by forming a chemical bond with their target. This covalent binding can improve the selectivity of the drug for a target with complementary reactivity and result in increased binding affinities due to the strength of the covalent bond formed. In some cases, this results in irreversible inhibition of the target, but some targeted covalent inhibitor (TCI) drugs bind covalently but reversibly. Computer modeling is widely used in drug discovery, but different computational methods must be used to model covalent modifiers because of the chemical bonds formed. Structural and bioinformatic analysis has identified sites of modification that could yield selectivity for a chosen target. Docking methods, which are used to rank binding poses of large sets of inhibitors, have been augmented to support the formation of protein-ligand bonds and are now capable of predicting the binding pose of covalent modifiers accurately. The pK a 's of amino acids can be calculated in order to assess their reactivity towards electrophiles. QM/MM methods have been used to model the reaction mechanisms of covalent modification. The continued development of these tools will allow computation to aid in the development of new covalent-modifier drugs. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces

PubMed Central

Szent-Gyorgyi, Chris; Stanfield, Robyn L.; Andreko, Susan; Dempsey, Alison; Ahmed, Mushtaq; Capek, Sara; Waggoner, Alan; Wilson, Ian A.; Bruchez, Marcel P.

2013-01-01

We report that a symmetric small molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* Fluorogen Activating Protein (FAP) is a VL domain that binds malachite green dye (MG) to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two VL domains. Unliganded L5* VL domains crystallize as a similar antiparallel VL/VL homodimer. The complementarity determining regions (CDRs) are spatially oriented to form novel VL/VL and VL/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable VL/MG/VL complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two VL domains into a single protein tightens MG binding over 1,000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high affinity protein tags and capture reagents. PMID:23978698

Pyromellitamide aggregates and their response to anion stimuli.

PubMed

Webb, James E A; Crossley, Maxwell J; Turner, Peter; Thordarson, Pall

2007-06-06

The N,N',N'',N'''-1,2,4,5-tetra(ethylhexanoate) pyromellitamide is found to be capable of both intermolecular aggregation and binding to small anions. It is synthesized by aminolysis of pyromellitic anhydride with ethanolamine, followed by a reaction with hexanoyl chloride. The single-crystal X-ray structure of the pyromellitamide shows that it forms one-dimensional columnar stacks through an intermolecular hydrogen-bonding network. It also forms self-assembled gels in nonpolar solvents, presumably by a hydrogen-bonding network similar to the solid-state structure as shown by IR and XRD studies. Aggregation by intermolecular hydrogen bonding of the pyromellitamide is also observed by NMR and IR in solution. Fitting of NMR dilution data for pyromellitamide in d6-acetone to a cooperative aggregation model gave KE=232 M-1 and positive cooperativity of aggregation (rho=0.22). The pyromellitamide binds to a range of small anions with the binding strength decreasing in the order chloride>acetate>bromide>nitrate approximately iodide. The data indicate that the pyromellitamide binds two anions and that it displays negative cooperativity. The intermolecular aggregation of the pyromellitamide can also be altered using small anion stimuli; anion addition to preformed self-assembled pyromellitamide gels causes their collapse. The kinetics of anion-induced gel collapse are qualitatively correlated to the binding affinities of the same anions in solution. The cooperative anion binding properties and the sensitivity of the self-assembled gels formed by pyromellitamide toward anions could be useful in the development of sensors and switching/releasing devices.

Out of Place, Out of Mind: Schema-Driven False Memory Effects for Object-Location Bindings

ERIC Educational Resources Information Center

Lew, Adina R.; Howe, Mark L.

2017-01-01

Events consist of diverse elements, each processed in specialized neocortical networks, with temporal lobe memory systems binding these elements to form coherent event memories. We provide a novel theoretical analysis of an unexplored consequence of the independence of memory systems for elements and their bindings, 1 that raises the paradoxical…

Hyper-Binding across Time: Age Differences in the Effect of Temporal Proximity on Paired-Associate Learning

ERIC Educational Resources Information Center

Campbell, Karen L.; Trelle, Alexandra; Hasher, Lynn

2014-01-01

Older adults show hyper- (or excessive) binding effects for simultaneously and sequentially presented distraction. Here, we addressed the potential role of hyper-binding in paired-associate learning. Older and younger adults learned a list of word pairs and then received an associative recognition task in which rearranged pairs were formed from…

Coupling ligand recognition to protein folding in an engineered variant of rabbit ileal lipid binding protein.

PubMed

Kouvatsos, Nikolaos; Meldrum, Jill K; Searle, Mark S; Thomas, Neil R

2006-11-28

We have engineered a variant of the beta-clam shell protein ILBP which lacks the alpha-helical motif that caps the central binding cavity; the mutant protein is sufficiently destabilised that it is unfolded under physiological conditions, however, it unexpectedly binds its natural bile acid substrates with high affinity forming a native-like beta-sheet rich structure and demonstrating strong thermodynamic coupling between ligand binding and protein folding.

Complementation analysis of mutants of nitric oxide synthase reveals that the active site requires two hemes.

PubMed Central

Xie, Q W; Leung, M; Fuortes, M; Sassa, S; Nathan, C

1996-01-01

For catalytic activity, nitric oxide synthases (NOSs) must be dimeric. Previous work revealed that the requirements for stable dimerization included binding of tetrahydrobiopterin (BH4), arginine, and heme. Here we asked what function is served by dimerization. We assessed the ability of individually inactive mutants of mouse inducible NOS (iNOS; NOS2), each deficient in binding a particular cofactor or cosubstrate, to complement each other by generating NO upon cotransfection into human epithelial cells. The ability of the mutants to homodimerize was gauged by gel filtration and/or PAGE under partially denaturing conditions, both followed by immunoblot. Their ability to heterodimerize was assessed by coimmunoprecipitation. Heterodimers that contained only one COOH-terminal hemimer and only one BH4-binding site could both form and function, even though the NADPH-, FAD-, and FMN-binding domains (in the COOH-terminal hemimer) and the BH4-binding sites (in the NH2-terminal hemimer) were contributed by opposite chains. Heterodimers that contained only one heme-binding site (Cys-194) could also form, either in cis or in trans to the nucleotide-binding domains. However, for NO production, both chains had to bind heme. Thus, NO production by iNOS requires dimerization because the active site requires two hemes. Images Fig. 2 Fig. 3 Fig. 4 Fig. 7 PMID:8643499

Evidence that Chemical Chaperone 4-Phenylbutyric Acid Binds to Human Serum Albumin at Fatty Acid Binding Sites

PubMed Central

James, Joel; Shihabudeen, Mohamed Sham; Kulshrestha, Shweta; Goel, Varun; Thirumurugan, Kavitha

2015-01-01

Endoplasmic reticulum stress elicits unfolded protein response to counteract the accumulating unfolded protein load inside a cell. The chemical chaperone, 4-Phenylbutyric acid (4-PBA) is a FDA approved drug that alleviates endoplasmic reticulum stress by assisting protein folding. It is found efficacious to augment pathological conditions like type 2 diabetes, obesity and neurodegeneration. This study explores the binding nature of 4-PBA with human serum albumin (HSA) through spectroscopic and molecular dynamics approaches, and the results show that 4-PBA has high binding specificity to Sudlow Site II (Fatty acid binding site 3, subdomain IIIA). Ligand displacement studies, RMSD stabilization profiles and MM-PBSA binding free energy calculation confirm the same. The binding constant as calculated from fluorescence spectroscopic studies was found to be kPBA = 2.69 x 105 M-1. Like long chain fatty acids, 4-PBA induces conformational changes on HSA as shown by circular dichroism, and it elicits stable binding at Sudlow Site II (fatty acid binding site 3) by forming strong hydrogen bonding and a salt bridge between domain II and III of HSA. This minimizes the fluctuation of HSA backbone as shown by limited conformational space occupancy in the principal component analysis. The overall hydrophobicity of W214 pocket (located at subdomain IIA), increases upon occupancy of 4-PBA at any FA site. Descriptors of this pocket formed by residues from other subdomains largely play a role in compensating the dynamic movement of W214. PMID:26181488

Dissecting the Catalytic Mechanism of Betaine-Homocysteine S-Methyltransferase Using Intrinsic Tryptophan Fluorescence and Site-Directed Mutagenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Castro, C.; Gratson, A.A.; Evans, J.C.

2010-03-05

Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent enzyme that catalyzes the transfer of a methyl group from glycine betaine (Bet) to homocysteine (Hcy) to form dimethylglycine (DMG) and methionine (Met). Previous studies in other laboratories have indicated that catalysis proceeds through the formation of a ternary complex, with a transition state mimicked by the inhibitor S-({delta}-carboxybutyl)-l-homocysteine (CBHcy). Using changes in intrinsic tryptophan fluorescence to determine the affinity of human BHMT for substrates, products, or CBHcy, we now demonstrate that the enzyme-substrate complex reaches its transition state through an ordered bi-bi mechanism in which Hcy is the first substrate to bind andmore » Met is the last product released. Hcy, Met, and CBHcy bind to the enzyme to form binary complexes with K{sub d} values of 7.9, 6.9, and 0.28 {micro}M, respectively. Binary complexes with Bet and DMG cannot be detected with fluorescence as a probe, but Bet and DMG bind tightly to BHMT-Hcy to form ternary complexes with K{sub d} values of 1.1 and 0.73 {micro}M, respectively. Mutation of each of the seven tryptophan residues in human BHMT provides evidence that the enzyme undergoes two distinct conformational changes that are reflected in the fluorescence of the enzyme. The first is induced when Hcy binds, and the second, when Bet binds. As predicted by the crystal structure of BHMT, the amino acids Trp44 and Tyr160 are involved in binding Bet, and Glu159 in binding Hcy. Replacing these residues by site-directed mutagenesis significantly reduces the catalytic efficiency (V{sub max}/K{sub m}) of the enzyme. Replacing Tyr77 with Phe abolishes enzyme activity.« less

Structure of the C-terminal domain of the arginine repressor protein from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cherney, Leonid T.; Cherney, Maia M.; Garen, Craig R.

2008-09-01

The structure of the core domain of the arginine repressor protein from M. tuberculosis has been determined with (1.85 Å resolution) and without (2.15 Å resolution) the arginine corepressor bound. Three additional arginine molecules have been found to bind to the core domain hexamer at high (0.2 M) arginine concentration. The Mycobacterium tuberculosis (Mtb) gene product encoded by open reading frame Rv1657 is an arginine repressor (ArgR). All genes involved in the l-arginine (hereafter arginine) biosynthetic pathway are essential for optimal growth of the Mtb pathogen, thus making MtbArgR a potential target for drug design. The C-terminal domains of argininemore » repressors (CArgR) participate in oligomerization and arginine binding. Several crystal forms of CArgR from Mtb (MtbCArgR) have been obtained. The X-ray crystal structures of MtbCArgR were determined at 1.85 Å resolution with bound arginine and at 2.15 Å resolution in the unliganded form. These structures show that six molecules of MtbCArgR are arranged into a hexamer having approximate 32 point symmetry that is formed from two trimers. The trimers rotate relative to each other by about 11° upon binding arginine. All residues in MtbCArgR deemed to be important for hexamer formation and for arginine binding have been identified from the experimentally determined structures presented. The hexamer contains six regular sites in which the arginine molecules have one common binding mode and three sites in which the arginine molecules have two overlapping binding modes. The latter sites only bind the ligand at high (200 mM) arginine concentrations.« less

Identification and application of self-binding zipper-like sequences in SARS-CoV spike protein.

PubMed

Zhang, Si Min; Liao, Ying; Neo, Tuan Ling; Lu, Yanning; Liu, Ding Xiang; Vahlne, Anders; Tam, James P

2018-05-22

Self-binding peptides containing zipper-like sequences, such as the Leu/Ile zipper sequence within the coiled coil regions of proteins and the cross-β spine steric zippers within the amyloid-like fibrils, could bind to the protein-of-origin through homophilic sequence-specific zipper motifs. These self-binding sequences represent opportunities for the development of biochemical tools and/or therapeutics. Here, we report on the identification of a putative self-binding β-zipper-forming peptide within the severe acute respiratory syndrome-associated coronavirus spike (S) protein and its application in viral detection. Peptide array scanning of overlapping peptides covering the entire length of S protein identified 34 putative self-binding peptides of six clusters, five of which contained octapeptide core consensus sequences. The Cluster I consensus octapeptide sequence GINITNFR was predicted by the Eisenberg's 3D profile method to have high amyloid-like fibrillation potential through steric β-zipper formation. Peptide C6 containing the Cluster I consensus sequence was shown to oligomerize and form amyloid-like fibrils. Taking advantage of this, C6 was further applied to detect the S protein expression in vitro by fluorescence staining. Meanwhile, the coiled-coil-forming Leu/Ile heptad repeat sequences within the S protein were under-represented during peptide array scanning, in agreement with that long peptide lengths were required to attain high helix-mediated interaction avidity. The data suggest that short β-zipper-like self-binding peptides within the S protein could be identified through combining the peptide scanning and predictive methods, and could be exploited as biochemical detection reagents for viral infection. Copyright © 2018. Published by Elsevier Ltd.

Replication initiator protein RepE of mini-F plasmid: functional differentiation between monomers (initiator) and dimers (autogenous repressor).

PubMed Central

Ishiai, M; Wada, C; Kawasaki, Y; Yura, T

1994-01-01

Replication of mini-F plasmid requires the plasmid-encoded RepE initiator protein and several host factors including DnaJ, DnaK, and GrpE, heat shock proteins of Escherichia coli. The RepE protein plays a crucial role in replication and exhibits two major functions: initiation of replication from the origin, ori2, and autogenous repression of repE transcription. One of the mini-F plasmid mutants that can replicate in the dnaJ-defective host produces an altered RepE (RepE54) with a markedly enhanced initiator activity but little or no repressor activity. RepE54 has been purified from cell extracts primarily in monomeric form, unlike the wild-type RepE that is recovered in dimeric form. Gel-retardation assays revealed that RepE54 monomers bind to ori2 (direct repeats) with a very high efficiency but hardly bind to the repE operator (inverted repeat), in accordance with the properties of RepE54 in vivo. Furthermore, the treatment of wild-type RepE dimers with protein denaturants enhanced their binding to ori2 but reduced binding to the operator: RepE dimers were partially converted to monomers, and the ori2 binding activity was uniquely associated with monomers. These results strongly suggest that RepE monomers represent an active form by binding to ori2 to initiate replication, whereas dimers act as an autogenous repressor by binding to the operator. We propose that RepE is structurally and functionally differentiated and that monomerization of RepE dimers, presumably mediated by heat shock protein(s), activates the initiator function and participates in regulation of mini-F DNA replication. Images PMID:8170998

Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hou, Feng; Miyakawa, Takuya; Kataoka, Michihiko

2014-04-18

Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystalmore » structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.« less

Cloning of Novel Isoforms of the Human Gli2 Oncogene and Their Activities To Enhance Tax-Dependent Transcription of the Human T-Cell Leukemia Virus Type 1 Genome

PubMed Central

Tanimura, Akira; Dan, Shingo; Yoshida, Mitsuaki

1998-01-01

The expression of human T-cell leukemia virus type 1 (HTLV-1) is activated by interaction of a viral transactivator protein, Tax, and cellular transcription factor, CREB (cyclic AMP response element binding protein), which bind to a 21-bp enhancer in the long terminal repeats (LTR). THP (Tax-helping protein) was previously determined to enhance the transactivation by Tax protein. Here we report novel forms of the human homolog of a member of the Gli oncogene family, Gli2 (also termed Gli2/THP), an extended form of a zinc finger protein, THP, which was described previously. Four possible isoforms (hGli2 α, β, γ, and δ) are formed by combinations of two independent alternative splicings, and all the isoforms could bind to a DNA motif, TRE2S, in the LTR. The longer isoforms, α and β, were abundantly expressed in various cell lines including HTLV-1-infected T-cell lines. Fusion proteins of the hGli2 isoforms with the DNA-binding domain of Gal4 activated transcription when the reporter contained a Gal4-binding site and one copy of the 21-bp sequence, to which CREB binds. This activation was observed only in the presence of Tax. The 21-bp sequence in the reporter was also essential for the activation. These results suggest that simultaneous binding of hGli2 and CREB to the respective sites in the reporter seems to be critical for Tax protein to activate transcription. Consequently, it is probable that the LTR can be regulated by two independent signals through hGli2 and CREB, since the LTR contains the 21-bp and TRE2S sequences in the vicinity. PMID:9557682

Effect of binding in cyclic phosphorylation-dephosphorylation process and in energy transformation.

PubMed

Sarkar, A; Beard, D A; Franza, B R

2006-07-01

The effects of binding on the phosphorylation-dephosphorylation cycle (PDPC) - one of the key components of the signal transduction processes - is analyzed based on a mathematical model. The model shows that binding of proteins, forming a complex, diminishes the ultrasensitivity of the PDPC to the differences in activity between kinase and phosphatase in the cycle. It is also found that signal amplification depends upon the strength of the binding affinity of the protein (phosphorylated or dephosphorylated) to other proteins . It is also observed that the amplification of signal is not only dependent on phosphorylation potential but also on binding properties and resulting adjustments in binding energies.

Architecture of the human regulatory network derived from ENCODE data.

PubMed

Gerstein, Mark B; Kundaje, Anshul; Hariharan, Manoj; Landt, Stephen G; Yan, Koon-Kiu; Cheng, Chao; Mu, Xinmeng Jasmine; Khurana, Ekta; Rozowsky, Joel; Alexander, Roger; Min, Renqiang; Alves, Pedro; Abyzov, Alexej; Addleman, Nick; Bhardwaj, Nitin; Boyle, Alan P; Cayting, Philip; Charos, Alexandra; Chen, David Z; Cheng, Yong; Clarke, Declan; Eastman, Catharine; Euskirchen, Ghia; Frietze, Seth; Fu, Yao; Gertz, Jason; Grubert, Fabian; Harmanci, Arif; Jain, Preti; Kasowski, Maya; Lacroute, Phil; Leng, Jing Jane; Lian, Jin; Monahan, Hannah; O'Geen, Henriette; Ouyang, Zhengqing; Partridge, E Christopher; Patacsil, Dorrelyn; Pauli, Florencia; Raha, Debasish; Ramirez, Lucia; Reddy, Timothy E; Reed, Brian; Shi, Minyi; Slifer, Teri; Wang, Jing; Wu, Linfeng; Yang, Xinqiong; Yip, Kevin Y; Zilberman-Schapira, Gili; Batzoglou, Serafim; Sidow, Arend; Farnham, Peggy J; Myers, Richard M; Weissman, Sherman M; Snyder, Michael

2012-09-06

Transcription factors bind in a combinatorial fashion to specify the on-and-off states of genes; the ensemble of these binding events forms a regulatory network, constituting the wiring diagram for a cell. To examine the principles of the human transcriptional regulatory network, we determined the genomic binding information of 119 transcription-related factors in over 450 distinct experiments. We found the combinatorial, co-association of transcription factors to be highly context specific: distinct combinations of factors bind at specific genomic locations. In particular, there are significant differences in the binding proximal and distal to genes. We organized all the transcription factor binding into a hierarchy and integrated it with other genomic information (for example, microRNA regulation), forming a dense meta-network. Factors at different levels have different properties; for instance, top-level transcription factors more strongly influence expression and middle-level ones co-regulate targets to mitigate information-flow bottlenecks. Moreover, these co-regulations give rise to many enriched network motifs (for example, noise-buffering feed-forward loops). Finally, more connected network components are under stronger selection and exhibit a greater degree of allele-specific activity (that is, differential binding to the two parental alleles). The regulatory information obtained in this study will be crucial for interpreting personal genome sequences and understanding basic principles of human biology and disease.

Possible Insecticidal Mechanisms Mediated by Immune-Response-Related Cry-Binding Proteins in the Midgut Juice of Plutella xylostella and Spodoptera exigua.

PubMed

Lu, Keyu; Gu, Yuqing; Liu, Xiaoping; Lin, Yi; Yu, Xiao-Qiang

2017-03-15

Cry toxins are insecticidal toxin proteins produced by a spore-forming Gram-positive bacterium Bacillus thuringiensis. Interactions between the Cry toxins and the receptors from midgut brush border membrane vesicles (BBMVs), such as cadherin, alkaline phosphatase, and aminopeptidase, are key steps for the specificity and insecticidal activity of Cry proteins. However, little is known about the midgut juice proteins that may interfere with Cry binding to the receptors. To validate the hypothesis that there exist Cry-binding proteins that can interfere with the insecticidal process of Cry toxins, we applied Cry1Ab1-coupled Sepharose beads to isolate Cry-binding proteins form midgut juice of Plutella xylostella and Spodoptera exigua. Trypsin-like serine proteases and Dorsal were found to be Cry1Ab1-binding proteins in the midgut juice of P. xylostella. Peroxidase-C (POX-C) was found to be the Cry1Ab1-binding protein in the midgut juice of S. exigua. We proposed possible insecticidal mechanisms of Cry1Ab1 mediated by the two immune-related proteins: Dorsal and POX-C. Our results suggested that there exist, in the midgut juice, Cry-binding proteins, which are different from BBMV-specific receptors.

Large-Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin α.

PubMed

Delaforge, Elise; Milles, Sigrid; Bouvignies, Guillaume; Bouvier, Denis; Boivin, Stephane; Salvi, Nicola; Maurin, Damien; Martel, Anne; Round, Adam; Lemke, Edward A; Jensen, Malene Ringkjøbing; Hart, Darren J; Blackledge, Martin

2015-12-09

Influenza A RNA polymerase complex is formed from three components, PA, PB1, and PB2. PB2 is independently imported into the nucleus prior to polymerase reconstitution. All crystallographic structures of the PB2 C-terminus (residues 536-759) reveal two globular domains, 627 and NLS, that form a tightly packed heterodimer. The molecular basis of the affinity of 627-NLS for importins remained unclear from these structures, apparently requiring large-scale conformational changes prior to importin binding. Using a combination of solution-state NMR, small-angle neutron scattering, small-angle X-ray scattering (SAXS), and Förster resonance energy transfer (FRET), we show that 627-NLS populates a temperature-dependent dynamic equilibrium between closed and open states. The closed state is stabilized by a tripartite salt bridge involving the 627-NLS interface and the linker, that becomes flexible in the open state, with 627 and NLS dislocating into a highly dynamic ensemble. Activation enthalpies and entropies associated with the rupture of this interface were derived from simultaneous analysis of temperature-dependent chemical exchange saturation transfer measurements, revealing a strong temperature dependence of both open-state population and exchange rate. Single-molecule FRET and SAXS demonstrate that only the open-form is capable of binding to importin α and that, upon binding, the 627 domain samples a dynamic conformational equilibrium in the vicinity of the C-terminus of importin α. This intrinsic large-scale conformational flexibility therefore enables 627-NLS to bind importin through conformational selection from a temperature-dependent equilibrium comprising both functional forms of the protein.

Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo.

PubMed

Anikaev, A Y; Korepanov, A P; Korobeinikova, A V; Kljashtorny, V G; Piendl, W; Nikonov, S V; Garber, M B; Gongadze, G M

2014-08-01

5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.

Edible oil structures at low and intermediate concentrations. I. Modeling, computer simulation, and predictions for X ray scattering

NASA Astrophysics Data System (ADS)

Pink, David A.; Quinn, Bonnie; Peyronel, Fernanda; Marangoni, Alejandro G.

2013-12-01

Triacylglycerols (TAGs) are biologically important molecules which form the recently discovered highly anisotropic crystalline nanoplatelets (CNPs) and, ultimately, the large-scale fat crystal networks in edible oils. Identifying the hierarchies of these networks and how they spontaneously self-assemble is important to understanding their functionality and oil binding capacity. We have modelled CNPs and studied how they aggregate under the assumption that all CNPs are present before aggregation begins and that their solubility in the liquid oil is very low. We represented CNPs as rigid planar arrays of spheres with diameter ≈50 nm and defined the interaction between spheres in terms of a Hamaker coefficient, A, and a binding energy, VB. We studied three cases: weak binding, |VB|/kBT ≪ 1, physically realistic binding, VB = Vd(R, Δ), so that |VB|/kBT ≈ 1, and Strong binding with |VB|/kBT ≫ 1. We divided the concentration of CNPs, ϕ, with 0≤ϕ= 10-2 (solid fat content) ≤1, into two regions: Low and intermediate concentrations with 0<ϕ<0.25 and high concentrations with 0.25 < ϕ and considered only the first case. We employed Monte Carlo computer simulation to model CNP aggregation and analyzed them using static structure functions, S(q). We found that strong binding cases formed aggregates with fractal dimension, D, 1.7≤D ≤1.8, in accord with diffusion limited cluster-cluster aggregation (DLCA) and weak binding formed aggregates with D =3, indicating a random distribution of CNPs. We found that models with physically realistic intermediate binding energies formed linear multilayer stacks of CNPs (TAGwoods) with fractal dimension D =1 for ϕ =0.06,0.13, and 0.22. TAGwood lengths were greater at lower ϕ than at higher ϕ, where some of the aggregates appeared as thick CNPs. We increased the spatial scale and modelled the TAGwoods as rigid linear arrays of spheres of diameter ≈500 nm, interacting via the attractive van der Waals interaction. We found that TAGwoods aggregated via DLCA into clusters with fractal dimension D =1.7-1.8. As the simulations were run further, TAGwoods relaxed their positions in order to maximize the attractive interaction making the process look like reaction limited cluster-cluster aggregation with the fractal dimension increasing to D =2.0-2.1. For higher concentrations of CNPs, many TAGwood clusters were formed and, because of their weak interactions, were distributed randomly with D =3.0. We summarize the hierarchy of structures and make predictions for X-ray scattering.

Positive selection moments identify potential functional residues in human olfactory receptors

NASA Technical Reports Server (NTRS)

Singer, M. S.; Weisinger-Lewin, Y.; Lancet, D.; Shepherd, G. M.

1996-01-01

Correlated mutation analysis and molecular models of olfactory receptors have provided evidence that residues in the transmembrane domains form a binding pocket for odor ligands. As an independent test of these results, we have calculated positive selection moments for the alpha-helical sixth transmembrane domain (TM6) of human olfactory receptors. The moments can be used to identify residues that have been preferentially affected by positive selection and are thus likely to interact with odor ligands. The results suggest that residue 622, which is commonly a serine or threonine, could form critical H-bonds. In some receptors a dual-serine subsite, formed by residues 622 and 625, could bind hydroxyl determinants on odor ligands. The potential importance of these residues is further supported by site-directed mutagenesis in the beta-adrenergic receptor. The findings should be of practical value for future physiological studies, binding assays, and site-directed mutagenesis.

Investigation of uranium binding forms in selected German mineral waters.

PubMed

Osman, Alfatih A A; Geipel, Gerhard; Bernhard, Gert; Worch, Eckhard

2013-12-01

Cryogenic time-resolved laser-induced fluorescence spectroscopy was successfully used to identify uranium binding forms in selected German mineral waters of extremely low uranium concentrations (<2.0 μg/L). The measurements were performed at a low temperature of 153 K. The spectroscopic data showed a prevalence of aquatic species Ca2UO2(CO3)3 in all investigated waters, while other uranyl-carbonate complexes, viz, UO2CO3(aq) and UO2(CO3)2 (2-), only existed as minor species. The pH value, alkalinity (CO3 (2-)), and the main water inorganic constituents, specifically the Ca(2+) concentration, showed a clear influence on uranium speciation. Speciation modeling was performed using the most recent thermodynamic data for aqueous complexes of uranium. The modeling results for the main uranium binding form in the investigated waters indicated a good agreement with the spectroscopy measurements.

Glycan microarray analysis of the carbohydrate-recognition specificity of native and recombinant forms of the lectin ArtinM.

PubMed

Liu, Y; Cecílio, N T; Carvalho, F C; Roque-Barreira, M C; Feizi, T

2015-12-01

This article contains data related to the researc.h article entitled "Yeast-derived ArtinM shares structure, carbohydrate recognition, and biological effects with native ArtinM" by Cecílio et al. (2015) [1]. ArtinM, a D-mannose-binding lectin isolated from the seeds of Artocarpus heterophyllus, exerts immunomodulatory and regenerative activities through its Carbohydrate Recognition Domain (CRD) (Souza et al., 2013; Mariano et al., 2014 [2], [3]). The limited availability of the native lectin (n-ArtinM) led us to characterize a recombinant form of the protein, obtained by expression in Saccharomyces cerevisiae (y-ArtinM). We compared the carbohydrate-binding specificities of y-ArtinM and n-ArtinM by analyzing the binding of biotinylated preparations of the two lectin forms using a neoglycolipid (NGL)-based glycan microarray. Data showed that y-ArtinM mirrored the specificity exhibited by n-ArtinM.