Differences in Fusarium Species in brown midrib Sorghum and in Air Populations in Production Fields.

PubMed

Funnell-Harris, Deanna L; Scully, Erin D; Sattler, Scott E; French, Roy C; O'Neill, Patrick M; Pedersen, Jeffrey F

2017-11-01

Several Fusarium spp. cause sorghum (Sorghum bicolor) grain mold, resulting in deterioration and mycotoxin production in the field and during storage. Fungal isolates from the air (2005 to 2006) and from leaves and grain from wild-type and brown midrib (bmr)-6 and bmr12 plants (2002 to 2003) were collected from two locations. Compared with the wild type, bmr plants have reduced lignin content, altered cell wall composition, and different levels of phenolic intermediates. Multilocus maximum-likelihood analysis identified two Fusarium thapsinum operational taxonomic units (OTU). One was identified at greater frequency in grain and leaves of bmr and wild-type plants but was infrequently detected in air. Nine F. graminearum OTU were identified: one was detected at low levels in grain and leaves while the rest were only detected in air. Wright's F statistic (F ST ) indicated that Fusarium air populations differentiated between locations during crop anthesis but did not differ during vegetative growth, grain development, and maturity. F ST also indicated that Fusarium populations from wild-type grain were differentiated from those in bmr6 or bmr12 grain at one location but, at the second location, populations from wild-type and bmr6 grain were more similar. Thus, impairing monolignol biosynthesis substantially effected Fusarium populations but environment had a strong influence.

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.

Theoretical Analysis of Allosteric and Operator Binding for Cyclic-AMP Receptor Protein Mutants

NASA Astrophysics Data System (ADS)

Einav, Tal; Duque, Julia; Phillips, Rob

2018-02-01

Allosteric transcription factors undergo binding events both at their inducer binding sites as well as at distinct DNA binding domains, and it is often difficult to disentangle the structural and functional consequences of these two classes of interactions. In this work, we compare the ability of two statistical mechanical models - the Monod-Wyman-Changeux (MWC) and the Koshland-N\\'emethy-Filmer (KNF) models of protein conformational change - to characterize the multi-step activation mechanism of the broadly acting cyclic-AMP receptor protein (CRP). We first consider the allosteric transition resulting from cyclic-AMP binding to CRP, then analyze how CRP binds to its operator, and finally investigate the ability of CRP to activate gene expression. In light of these models, we examine data from a beautiful recent experiment that created a single-chain version of the CRP homodimer, thereby enabling each subunit to be mutated separately. Using this construct, six mutants were created using all possible combinations of the wild type subunit, a D53H mutant subunit, and an S62F mutant subunit. We demonstrate that both the MWC and KNF models can explain the behavior of all six mutants using a small, self-consistent set of parameters. In comparing the results, we find that the MWC model slightly outperforms the KNF model in the quality of its fits, but more importantly the parameters inferred by the MWC model are more in line with structural knowledge of CRP. In addition, we discuss how the conceptual framework developed here for CRP enables us to not merely analyze data retrospectively, but has the predictive power to determine how combinations of mutations will interact, how double mutants will behave, and how each construct would regulate gene expression.

A novel transformation system using a bleomycin resistance marker with chemosensitizers for Aspergillus oryzae.

PubMed

Suzuki, Satoshi; Tada, Sawaki; Fukuoka, Mari; Taketani, Hiroko; Tsukakoshi, Yoshiki; Matsushita, Mayumi; Oda, Kosuke; Kusumoto, Ken-Ichi; Kashiwagi, Yutaka; Sugiyama, Masanori

2009-05-22

Aspergillus oryzae is resistant to many kinds of antibiotics, which hampers their use to select transformants. In fact, the fungus is resistant to over 200microg/ml of bleomycin (Bm). By enhancing the susceptibility of A. oryzae to Bm using Triton X-100 as a detergent and an ATP-binding cassette (ABC) pump inhibitor, chlorpromazine, to the growing medium, we established a novel transformation system by Bm selection for A. oryzae. In a medium containing these reagents, A. oryzae showed little growth even in the presence of 30microg Bm/ml. Based on these findings, we constructed a Bm-resistance expression cassette (BmR), in which blmB encoding Bm N-acetyltransferase from Bm-producing Streptomyces verticillus was expressed under the control of a fungal promoter. We obtained a gene knockout mutant efficiently by Bm selection, i.e., the chromosomal ligD coding region was successfully replaced by BmR using ligD disruption cassette consisted of ligD flanking sequence and BmR through homologous recombination.

Evidence for co-operativity in coenzyme binding to tetrameric Sulfolobus solfataricus alcohol dehydrogenase and its structural basis: fluorescence, kinetic and structural studies of the wild-type enzyme and non-co-operative N249Y mutant

PubMed Central

2005-01-01

The interaction of coenzyme with thermostable homotetrameric NAD(H)-dependent alcohol dehydrogenase from the thermoacidophilic sulphur-dependent crenarchaeon Sulfolobus solfataricus (SsADH) and its N249Y (Asn-249→Tyr) mutant was studied using the high fluorescence sensitivity of its tryptophan residues Trp-95 and Trp-117 to the binding of coenzyme moieties. Fluorescence quenching studies performed at 25 °C show that SsADH exhibits linearity in the NAD(H) binding [the Hill coefficient (h)∼1) at pH 9.8 and at moderate ionic strength, in addition to positive co-operativity (h=2.0–2.4) at pH 7.8 and 6.8, and at pH 9.8 in the presence of salt. Furthermore, NADH binding is positively co-operative below 20 °C (h∼3) and negatively co-operative at 40–50 °C (h∼0.7), as determined at moderate ionic strength and pH 9.8. Steady-state kinetic measurements show that SsADH displays standard Michaelis–Menten kinetics between 35 and 45 °C, but exhibits positive and negative co-operativity for NADH oxidation below (h=3.3 at 20 °C) and above (h=0.7 at 70–80 °C) this range of temperatures respectively. However, N249Y SsADH displays non-co-operative behaviour in coenzyme binding under the same experimental conditions used for the wild-type enzyme. In loop 270–275 of the coenzyme domain and segments at the interface of dimer A–B, analyses of the wild-type and mutant SsADH structures identified the structural elements involved in the intersubunit communication and suggested a possible structural basis for co-operativity. This is the first report of co-operativity in a tetrameric ADH and of temperature-induced co-operativity in a thermophilic enzyme. PMID:15651978

Evidence for co-operativity in coenzyme binding to tetrameric Sulfolobus solfataricus alcohol dehydrogenase and its structural basis: fluorescence, kinetic and structural studies of the wild-type enzyme and non-co-operative N249Y mutant.

PubMed

Giordano, Antonietta; Febbraio, Ferdinando; Russo, Consiglia; Rossi, Mosè; Raia, Carlo A

2005-06-01

The interaction of coenzyme with thermostable homotetrameric NAD(H)-dependent alcohol dehydrogenase from the thermoacidophilic sulphur-dependent crenarchaeon Sulfolobus solfataricus (SsADH) and its N249Y (Asn-249-->Tyr) mutant was studied using the high fluorescence sensitivity of its tryptophan residues Trp-95 and Trp-117 to the binding of coenzyme moieties. Fluorescence quenching studies performed at 25 degrees C show that SsADH exhibits linearity in the NAD(H) binding [the Hill coefficient (h) approximately 1) at pH 9.8 and at moderate ionic strength, in addition to positive co-operativity (h=2.0-2.4) at pH 7.8 and 6.8, and at pH 9.8 in the presence of salt. Furthermore, NADH binding is positively co-operative below 20 degrees C (h approximately 3) and negatively co-operative at 40-50 degrees C (h approximately 0.7), as determined at moderate ionic strength and pH 9.8. Steady-state kinetic measurements show that SsADH displays standard Michaelis-Menten kinetics between 35 and 45 degrees C, but exhibits positive and negative co-operativity for NADH oxidation below (h=3.3 at 20 degrees C) and above (h=0.7 at 70-80 degrees C) this range of temperatures respectively. However, N249Y SsADH displays non-co-operative behaviour in coenzyme binding under the same experimental conditions used for the wild-type enzyme. In loop 270-275 of the coenzyme domain and segments at the interface of dimer A-B, analyses of the wild-type and mutant SsADH structures identified the structural elements involved in the intersubunit communication and suggested a possible structural basis for co-operativity. This is the first report of co-operativity in a tetrameric ADH and of temperature-induced co-operativity in a thermophilic enzyme.

Characterization of novel sorghum brown midrib mutants from an EMS-mutagenized population

DOE PAGES

Sattler, Scott E.; Saballos, Ana; Xin, Zhanguo; ...

2014-09-02

Reducing lignin concentration in lignocellulosic biomass can increase forage digestibility for ruminant livestock and saccharification yields of biomass for bioenergy. In sorghum ( Sorghum bicolor (L.) Moench) and several other C4 grasses, brown midrib ( bmr) mutants have been shown to reduce lignin concentration. Putative bmr mutants isolated from an EMS-mutagenized population were characterized and classified based on their leaf midrib phenotype and allelism tests with the previously described sorghum bmr mutants bmr2, bmr6, and bmr12. These tests resulted in the identification of additional alleles of bmr2, bmr6,and bmr12, and, in addition, six bmr mutants were identified that were notmore » allelic to these previously described loci. Further allelism testing among these six bmr mutants showed that they represented four novel bmr loci. Based on this study, the number of bmr loci uncovered in sorghum has doubled. The impact of these lines on agronomic traits and lignocellulosic composition was assessed in a 2-yr field study. Most of the identified bmr lines showed reduced lignin concentration of their biomass relative to wild-type (WT). Effects of the six new bmr mutants on enzymatic saccharification of lignocellulosic materials were determined, but the amount of glucose released from the stover was similar to WT in all cases. Like bmr2, bmr6, and bmr12, these mutants may affect monolignol biosynthesis and may be useful for bioenergy and forage improvement when stacked together or in combination with the three previously described bmr alleles.« less

Characterization of Novel Sorghum brown midrib Mutants from an EMS-Mutagenized Population

PubMed Central

Sattler, Scott E.; Saballos, Ana; Xin, Zhanguo; Funnell-Harris, Deanna L.; Vermerris, Wilfred; Pedersen, Jeffrey F.

2014-01-01

Reducing lignin concentration in lignocellulosic biomass can increase forage digestibility for ruminant livestock and saccharification yields of biomass for bioenergy. In sorghum (Sorghum bicolor (L.) Moench) and several other C4 grasses, brown midrib (bmr) mutants have been shown to reduce lignin concentration. Putative bmr mutants isolated from an EMS-mutagenized population were characterized and classified based on their leaf midrib phenotype and allelism tests with the previously described sorghum bmr mutants bmr2, bmr6, and bmr12. These tests resulted in the identification of additional alleles of bmr2, bmr6, and bmr12, and, in addition, six bmr mutants were identified that were not allelic to these previously described loci. Further allelism testing among these six bmr mutants showed that they represented four novel bmr loci. Based on this study, the number of bmr loci uncovered in sorghum has doubled. The impact of these lines on agronomic traits and lignocellulosic composition was assessed in a 2-yr field study. Overall, most of the identified bmr lines showed reduced lignin concentration of their biomass relative to wild-type (WT). Effects of the six new bmr mutants on enzymatic saccharification of lignocellulosic materials were determined, but the amount of glucose released from the stover was similar to WT in all cases. Like bmr2, bmr6, and bmr12, these mutants may affect monolignol biosynthesis and may be useful for bioenergy and forage improvement when stacked together or in combination with the three previously described bmr alleles. PMID:25187038

Peri-operative heart-type fatty acid binding protein is associated with acute kidney injury after cardiac surgery

PubMed Central

Schaub, Jennifer A.; Garg, Amit X.; Coca, Steven G.; Testani, Jeffrey M.; Shlipak, Michael G.; Eikelboom, John; Kavsak, Peter; McArthur, Eric; Shortt, Colleen; Whitlock, Richard; Parikh, Chirag R.

2015-01-01

Acute Kidney Injury (AKI) is a common complication after cardiac surgery and is associated with worse outcomes. Since heart fatty acid binding protein (H-FABP) is a myocardial protein that detects cardiac injury, we sought to determine if plasma H-FABP was associated with AKI in the TRIBE-AKI cohort; a multi-center cohort of 1219 patients at high risk for AKI who underwent cardiac surgery. The primary outcomes of interest were any AKI (Acute Kidney Injury Network (AKIN) stage 1 or higher) and severe AKI (AKIN stage 2 or higher). The secondary outcome was long-term mortality after discharge. Patients who developed AKI had higher levels of H-FABP pre- and post-operatively than patients who did not have AKI. In analyses adjusted for known AKI risk factors, first post-operative log(H-FABP) was associated with severe AKI (adjusted OR 5.39 [95% CI, 2.87-10.11] per unit increase), while pre-operative log(H-FABP) was associated with any AKI (2.07 [1.48-2.89]) and mortality (1.67 [1.17-2.37]). These relationships persisted after adjustment for change in serum creatinine (for first postoperative log(H-FABP)) and biomarkers of cardiac and kidney injury, including brain natriuretic peptide, cardiac troponin-I, interleukin-18, liver fatty acid binding protein, kidney injury molecule-1, and neutrophil gelatinase associated lipocalin. Thus, peri-operative plasma H-FABP levels may be used for risk-stratification of AKI and mortality following cardiac surgery. PMID:25830762

Evaluation of Brown Midrib Sorghum Mutants as a Potential Biomass Feedstock for 2,3-Butanediol Biosynthesis.

PubMed

Guragain, Yadhu N; Srinivasa Rao, P; Vara Prasad, P V; Vadlani, Praveen V

2017-11-01

Three sorghum backgrounds [Atlas, Early Hegari (EH), and Kansas Collier (KC)] and two bmr mutants (bmr6 and bmr12) of each line were evaluated and compared for grain and biomass yield, biomass composition, and 2,3-butanediol production from biomass. The data showed that the bmr6 mutation in EH background led to a significant decrease in stover yield and increase in grain yield, whereas the stover yield was increased by 64% without affecting grain yield in KC background. The bmr mutants had 10 to 25% and 2 to 9% less lignin and structural carbohydrate contents, respectively, and 24 to 93% more non-structural sugars than their parents in all sorghum lines, except EH bmr12. The total fermentable sugars released were 22 to 36% more in bmr mutants than in parents for Atlas and KC, but not for EH. The bmr6 mutation in KC background produced the most promising feedstock, among the evaluated bmr mutants, for 2,3-butanediol production without affecting grain yield, followed by KC bmr12 and Atlas bmr6, but the bmr mutation had an adverse effect in EH background. This indicated that the genetic background of the parent line and type of bmr mutation significantly affect the biomass quality as a feedstock for biochemical production.

Low Lignin (Brown Mid-rib) Sorghum Genotypes Restrict Growth of Fusarium Spp. as Compared with Near-Isogenic Wild-Type Sorghum

USDA-ARS?s Scientific Manuscript database

To increase usability of sorghum for bioenergy and forages, two different brown midrib (bmr) genes, bmr-6 and bmr-12, were backcrossed into five elite backgrounds, resulting in reduced lignin near-isogenic genotypes. Field-grown grain from bmr-6 and bmr-12 plants had significantly reduced colonizati...

Brown Midrib (Low Lignin) Sorghum Mutations Result in Restricted Growth of Fusarium and Alternaria spp

USDA-ARS?s Scientific Manuscript database

To increase usability of sorghum for bioenergy and forages, two different brown midrib (bmr) genes, bmr6 and bmr12, were backcrossed into five elite backgrounds, resulting in reduced lignin near-isogenic genotypes. When compared with wild-type, field-grown grain from bmr6 and bmr12 plants had signif...

A microcomputer controlled snow ski binding system--I. Instrumentation and field evaluation.

PubMed

MacGregor, D; Hull, M L; Dorius, L K

1985-01-01

This paper presents the design and field evaluation of the first microcomputer controlled ski binding system. This system incorporates an Intel 8086 microcomputer controller and an integral binding/dynamometer. This instrumentation system not only undertakes real time control, but also it records dynamometer data via a miniature digital cassette tape recorder. The integral binding/dynamometer offers the same operational and mounting convenience of commercially available mechanical bindings. The binding may be released either manually or electrically via the controller. Comprised of four octagonal half strain rings, the strain gage dynamometer measures the three moment load components at the boot. To enable the user to conveniently operate the computer, extensive operating software was developed. The operating software is discussed in relation to both the acquisition and storage of data from the dynamometer and the control of the electro-mechanical snow ski binding. The binding system has been used successfully to both record boot moment components and control ski binding release during actual skiing maneuvers. Moment histories typical of three common recreational skiing maneuvers are presented.

Environment, Migratory Tendency, Phylogeny and Basal Metabolic Rate in Birds

PubMed Central

Jetz, Walter; Freckleton, Robert P.; McKechnie, Andrew E.

2008-01-01

Basal metabolic rate (BMR) represents the minimum maintenance energy requirement of an endotherm and has far-reaching consequences for interactions between animals and their environments. Avian BMR exhibits considerable variation that is independent of body mass. Some long-distance migrants have been found to exhibit particularly high BMR, traditionally interpreted as being related to the energetic demands of long-distance migration. Here we use a global dataset to evaluate differences in BMR between migrants and non-migrants, and to examine the effects of environmental variables. The BMR of migrant species is significantly higher than that of non-migrants. Intriguingly, while the elevated BMR of migrants on their breeding grounds may reflect the metabolic machinery required for long-distance movements, an alternative (and statistically stronger) explanation is their occupation of predominantly cold high-latitude breeding areas. Among several environmental predictors, average annual temperature has the strongest effect on BMR, with a 50% reduction associated with a 20°C gradient. The negative effects of temperature variables on BMR hold separately for migrants and non-migrants and are not due their different climatic associations. BMR in migrants shows a much lower degree of phylogenetic inertia. Our findings indicate that migratory tendency need not necessarily be invoked to explain the higher BMR of migrants. A weaker phylogenetic signal observed in migrants supports the notion of strong phenotypic flexibility in this group which facilitates migration-related BMR adjustments that occur above and beyond environmental conditions. In contrast to the findings of previous analyses of mammalian BMR, primary productivity, aridity or precipitation variability do not appear to be important environmental correlates of avian BMR. The strong effects of temperature-related variables and varying phylogenetic effects reiterate the importance of addressing both broad-scale and

Comparison of brown midrib-6 and -18 forage sorghum with conventional sorghum and corn silage in diets of lactating dairy cows.

PubMed

Oliver, A L; Grant, R J; Pedersen, J F; O'Rear, J

2004-03-01

Total mixed rations containing conventional forage sorghum, brown midrib (bmr)-6 forage sorghum, bmr-18 forage sorghum, or corn silage were fed to Holstein dairy cows to determine the effect on lactation, ruminal fermentation, and total tract nutrient digestion. Sixteen multiparous cows (4 ruminally fistulated; 124 d in milk) were assigned to 1 of 4 diets in a replicated Latin square design with 4-wk periods (21-d adaptation and 7 d of collection). Diets consisted of 40% test silage, 10% alfalfa silage, and 50% concentrate mix (dry basis). Acid detergent lignin concentration was reduced by 21 and 13%, respectively, for the bmr-6 and bmr-18 sorghum silages when compared with the conventional sorghum. Dry matter intake was not affected by diet. Production of 4% fat-corrected milk was greatest for cows fed bmr-6 (33.7 kg/d) and corn silage (33.3 kg/d), was least for cows fed the conventional sorghum (29.1 kg/d), and was intermediate for cows fed the bmr-18 sorghum (31.2 kg/d), which did not differ from any other diet. Total tract neutral detergent fiber (NDF) digestibility was greatest for the bmr-6 sorghum (54.4%) and corn silage (54.1%) diets and was lower for the conventional (40.8%) and bmr-18 sorghum (47.9%) diets. In situ extent of NDF digestion was greatest for the bmr-6 sorghum (76.4%) and corn silage (79.0%) diets, least for the conventional sorghum diet (70.4%), and intermediate for the bmr-18 sorghum silage diet (73.1%), which was not different from the other diets. Results of this study indicate that the bmr-6 sorghum hybrid outperformed the conventional sorghum hybrid; the bmr-18 sorghum was intermediate between conventional and bmr-6 in most cases. Additionally, the bmr-6 hybrid resulted in lactational performance equivalent to the corn hybrid used in this study. There are important compositional differences among bmr forage sorghum hybrids that need to be characterized to predict animal response accurately.

Individual variation and repeatability of basal metabolism in the bank vole, Clethrionomys glareolus.

PubMed Central

Labocha, Marta K.; Sadowska, Edyta T.; Baliga, Katarzyna; Semer, Aleksandra K.; Koteja, Paweł

2004-01-01

Basal metabolic rate (BMR) is a fundamental energetic trait and has been measured in hundreds of birds and mammals. Nevertheless, little is known about the consistency of the population-average BMR or its repeatability at the level of individual variation. Here, we report that average mass-independent BMR did not differ between two generations of bank voles or between two trials separated by one month. Individual differences in BMR were highly repeatable across the one month interval: the coefficient of intraclass correlation was 0.70 for absolute log-transformed values and 0.56 for mass-independent values. Thus, BMR can be a meaningful measure of an individual physiological characteristic and can be used to test hypotheses concerning relationships between BMR and other traits. On the other hand, mass-independent BMR did not differ significantly across families, and the coefficient of intraclass correlation for full sibs did not differ from zero, which suggests that heritability of BMR in voles is not high. PMID:15101695

High basal metabolic rate does not elevate oxidative stress during reproduction in laboratory mice.

PubMed

Brzęk, Paweł; Książek, Aneta; Ołdakowski, Łukasz; Konarzewski, Marek

2014-05-01

Increased oxidative stress (OS) has been suggested as a physiological cost of reproduction. However, previous studies reported ambiguous results, with some even showing a reduction of oxidative damage during reproduction. We tested whether the link between reproduction and OS is mediated by basal metabolic rate (BMR), which has been hypothesized to affect both the rate of radical oxygen species production and antioxidative capacity. We studied the effect of reproduction on OS in females of laboratory mice divergently selected for high (H-BMR) and low (L-BMR) BMR, previously shown to differ with respect to parental investment. Non-reproducing L-BMR females showed higher oxidative damage to lipids (quantified as the level of malondialdehyde in internal organ tissues) and DNA (quantified as the level of 8-oxodG in blood serum) than H-BMR females. Reproduction did not affect oxidative damage to lipids in either line; however, it reduced damage to DNA in L-BMR females. Reproduction increased catalase activity in liver (significantly stronger in L-BMR females) and decreased it in kidneys. We conclude that the effect of reproduction on OS depends on the initial variation in BMR and varies between studied internal organs and markers of OS.

Basal metabolic rate studies in humans: measurement and development of new equations.

PubMed

Henry, C J K

2005-10-01

To facilitate the Food and Agriculture Organization/World Health Organization/United Nations University Joint (FAO/WHO/UNU) Expert Consultation on Energy and Protein Requirements which met in Rome in 1981, Schofield et al. reviewed the literature and produced predictive equations for both sexes for the following ages: 0-3, 3-10, 10-18, 18-30, 30-60 and >60 years. These formed the basis for the equations used in 1985 FAO/WHO/UNU document, Energy and Protein Requirements. While Schofield's analysis has served a significant role in re-establishing the importance of using basal metabolic rate (BMR) to predict human energy requirements, recent workers have subsequently queried the universal validity and application of these equations. A survey of the most recent studies (1980-2000) in BMR suggests that in most cases the current FAO/WHO/UNU predictive equations overestimate BMR in many communities. The FAO/WHO/UNU equations to predict BMR were developed using a database that contained a disproportionate number--3388 out of 7173 (47%)--of Italian subjects. The Schofield database contained relatively few subjects from the tropical region. The objective here is to review the historical development in the measurement and application of BMR and to critically review the Schofield et al. BMR database presenting a series of new equations to predict BMR. This division, while arbitrary, will enable readers who wish to omit the historical review of BMR to concentrate on the evolution of the new BMR equations. BMR data collected from published and measured values. A series of new equations (Oxford equations) have been developed using a data set of 10,552 BMR values that (1) excluded all the Italian subjects and (2) included a much larger number (4018) of people from the tropics. In general, the Oxford equations tend to produce lower BMR values than the current FAO/WHO/UNU equations in 18-30 and 30-60 year old males and in all females over 18 years of age. This is an opportune moment

Basal metabolism of the black-footed ferret (Mustela nigripes) and the Siberian polecat (M. eversmannii)

USGS Publications Warehouse

Harrington, L.A.; Biggins, D.E.; Alldredge, A.W.

2003-01-01

Black-footed ferrets (Mustela nigripes) and Siberian polecats (M. eversmannii) are medium-sized (about 1 kg) mustelids with similar ecological and morphological characteristics. We measured basal metabolic rates (BMR) for both species. In contrast with the commonly stated belief that mustelids have relatively high mass-specific BMR, neither the BMR of ferrets nor that of polecats in winter was greater than standard allometric predictions for all mammals. As suggested by previous authors, we believe that our relatively lower measurements for BMR are due to our efforts to minimize stress during the experimental procedure. These results support the contention that BMR in mustelids is no different from what is expected of mammals of this body mass. Seasonal differences were found in polecat BMR (higher in summer) but not in ferret BMR. Reasons for this interspecific difference may relate to differences in natural histories of these species.

Mechanism of Sulfide Binding by Ferric Hemeproteins.

PubMed

Boubeta, Fernando M; Bieza, Silvina A; Bringas, Mauro; Estrin, Darío A; Boechi, Leonardo; Bari, Sara E

2018-06-19

The reaction of hydrogen sulfide (H 2 S) with hemeproteins is a key physiological reaction; still, its mechanism and implications are not completely understood. In this work, we propose a combination of experimental and theoretical tools to shed light on the reaction in model system microperoxidase 11 (MP11-Fe III ) and myoglobin (Mb-Fe III ), from the estimation of the intrinsic binding constants of the species H 2 S and hydrosulfide (HS - ), and the computational description of the overall binding process. Our results show that H 2 S and HS - are the main reactive species in Mb-Fe III and MP11-Fe III , respectively, and that the magnitude of their intrinsic binding constants are similar to most of the binding constants reported so far for hemeproteins systems and model compounds. However, while the binding of HS - to Mb-Fe III was negligible, the binding of H 2 S to MP11-Fe III was significant, providing a frame for a discriminated analysis of both species and revealing differential mechanistic aspects. A joint inspection of the kinetic data and the free energy profiles of the binding processes suggests that a dissociative mechanism with the release of a coordinated water molecule as rate limiting step is operative in the binding of H 2 S to Mb-Fe III and that the binding of HS - is prevented in the access to the protein matrix. For the MP11-Fe III case, where no access restrictions for the ligands are present, an associative component in the mechanism seems to be operative. Overall, the results suggest that if accessing the active site then both H 2 S and HS - are capable of binding a ferric heme moiety.

Best Fitting Prediction Equations for Basal Metabolic Rate: Informing Obesity Interventions in Diverse Populations

PubMed Central

Sabounchi, Nasim S.; Rahmandad, Hazhir; Ammerman, Alice

2014-01-01

Basal Metabolic Rate (BMR) represents the largest component of total energy expenditure and is a major contributor to energy balance. Therefore, accurately estimating BMR is critical for developing rigorous obesity prevention and control strategies. Over the past several decades, numerous BMR formulas have been developed targeted to different population groups. A comprehensive literature search revealed 248 BMR estimation equations developed using diverse ranges of age, gender, race, fat free mass, fat mass, height, waist-to-hip ratio, body mass index, and weight. A subset of 47 studies included enough detail to allow for development of meta-regression equations. Utilizing these studies, meta-equations were developed targeted to twenty specific population groups. This review provides a comprehensive summary of available BMR equations and an estimate of their accuracy. An accompanying online BMR prediction tool (available at http://www.sdl.ise.vt.edu/tutorials.html) was developed to automatically estimate BMR based on the most appropriate equation after user-entry of individual age, race, gender, and weight. PMID:23318720

Dielectric response of molecules in empirical tight-binding theory

NASA Astrophysics Data System (ADS)

Boykin, Timothy B.; Vogl, P.

2002-01-01

In this paper we generalize our previous approach to electromagnetic interactions within empirical tight-binding theory to encompass molecular solids and isolated molecules. In order to guarantee physically meaningful results, we rederive the expressions for relevant observables using commutation relations appropriate to the finite tight-binding Hilbert space. In carrying out this generalization, we examine in detail the consequences of various prescriptions for the position and momentum operators in tight binding. We show that attempting to fit parameters of the momentum matrix directly generally results in a momentum operator which is incompatible with the underlying tight-binding model, while adding extra position parameters results in numerous difficulties, including the loss of gauge invariance. We have applied our scheme, which we term the Peierls-coupling tight-binding method, to the optical dielectric function of the molecular solid PPP, showing that this approach successfully predicts its known optical properties even in the limit of isolated molecules.

General transfer matrix formalism to calculate DNA–protein–drug binding in gene regulation: application to OR operator of phage λ

PubMed Central

Teif, Vladimir B.

2007-01-01

The transfer matrix methodology is proposed as a systematic tool for the statistical–mechanical description of DNA–protein–drug binding involved in gene regulation. We show that a genetic system of several cis-regulatory modules is calculable using this method, considering explicitly the site-overlapping, competitive, cooperative binding of regulatory proteins, their multilayer assembly and DNA looping. In the methodological section, the matrix models are solved for the basic types of short- and long-range interactions between DNA-bound proteins, drugs and nucleosomes. We apply the matrix method to gene regulation at the OR operator of phage λ. The transfer matrix formalism allowed the description of the λ-switch at a single-nucleotide resolution, taking into account the effects of a range of inter-protein distances. Our calculations confirm previously established roles of the contact CI–Cro–RNAP interactions. Concerning long-range interactions, we show that while the DNA loop between the OR and OL operators is important at the lysogenic CI concentrations, the interference between the adjacent promoters PR and PRM becomes more important at small CI concentrations. A large change in the expression pattern may arise in this regime due to anticooperative interactions between DNA-bound RNA polymerases. The applicability of the matrix method to more complex systems is discussed. PMID:17526526

Factors related to the formation of buccal mucosa ridging in university students

PubMed Central

Mizutani, Shinsuke; Tomofuji, Takaaki; Azuma, Tetsuji; Irie, Koichiro; Machida, Tatsuya; Yoneda, Toshiki; Iwasaki, Yoshiaki; Morita, Manabu

2014-01-01

Objective Buccal mucosa ridging (BMR) is known as a clinical sign of clenching, which is one of the major manifestations of bruxism. However, there are few reports about the formation of BMR and no data regarding the association between BMR and factors such as malocclusion. The purpose of the current study was to investigate the relationship between BMR and factors such as the number of teeth present, gender, body mass index (BMI), occlusion and clenching habit in university students. Materials and methods A total of 2101 students (1164 males, 937 females), aged 18–29 years old, were included in the study. BMR and the number of teeth present were recorded and malocclusion was defined using a modified version of the Index of Orthodontic Treatment Need. Additional information regarding gender, clenching habit and BMI was collected via a questionnaire. Results Forty-six per cent of the subjects had BMR and the prevalence of BMR in females was significantly higher than that of males (chi square test, p < 0.001). According to logistic regression analysis, the probability of BMR was significantly associated with female gender (OR = 1.501, 95% CI = 1.259–1.790, p < 0.001), crowding (OR = 2.102, 95% CI = 1.706–2.590, p < 0.001) and overjet (OR = 0.585, 95% CI = 0.418–0.818, p = 0.002). On the other hand, BMR was not associated with awareness of clenching habit and BMI. Conclusions Gender, crowding and overjet were related to the formation of BMR in university students. When evaluating BMR as a clinical sign of clenching, one might have to take factors such as gender and crowding into consideration. PMID:23692316

Isolation from genomic DNA of sequences binding specific regulatory proteins by the acceleration of protein electrophoretic mobility upon DNA binding.

PubMed

Subrahmanyam, S; Cronan, J E

1999-01-21

We report an efficient and flexible in vitro method for the isolation of genomic DNA sequences that are the binding targets of a given DNA binding protein. This method takes advantage of the fact that binding of a protein to a DNA molecule generally increases the rate of migration of the protein in nondenaturing gel electrophoresis. By the use of a radioactively labeled DNA-binding protein and nonradioactive DNA coupled with PCR amplification from gel slices, we show that specific binding sites can be isolated from Escherichia coli genomic DNA. We have applied this method to isolate a binding site for FadR, a global regulator of fatty acid metabolism in E. coli. We have also isolated a second binding site for BirA, the biotin operon repressor/biotin ligase, from the E. coli genome that has a very low binding efficiency compared with the bio operator region.

Predicting basal metabolic rates in Malaysian adult elite athletes.

PubMed

Wong, Jyh Eiin; Poh, Bee Koon; Nik Shanita, Safii; Izham, Mohd Mohamad; Chan, Kai Quin; Tai, Meng De; Ng, Wei Wei; Ismail, Mohd Noor

2012-11-01

This study aimed to measure the basal metabolic rate (BMR) of elite athletes and develop a gender specific predictive equation to estimate their energy requirements. 92 men and 33 women (aged 18-31 years) from 15 sports, who had been training six hours daily for at least one year, were included in the study. Body composition was measured using the bioimpedance technique, and BMR by indirect calorimetry. The differences between measured and estimated BMR using various predictive equations were calculated. The novel equation derived from stepwise multiple regression was evaluated using Bland and Altman analysis. The predictive equations of Cunningham and the Food and Agriculture Organization/World Health Organization/United Nations University either over- or underestimated the measured BMR by up to ± 6%, while the equations of Ismail et al, developed from the local non-athletic population, underestimated the measured BMR by 14%. The novel predictive equation for the BMR of athletes was BMR (kcal/day) = 669 + 13 (weight in kg) + 192 (gender: 1 for men and 0 for women) (R2 0.548; standard error of estimates 163 kcal). Predicted BMRs of elite athletes by this equation were within 1.2% ± 9.5% of the measured BMR values. The novel predictive equation presented in this study can be used to calculate BMR for adult Malaysian elite athletes. Further studies may be required to validate its predictive capabilities for other sports, nationalities and age groups.

Influence of binding pH and protein solubility on the dynamic binding capacity in hydrophobic interaction chromatography.

PubMed

Baumann, Pascal; Baumgartner, Kai; Hubbuch, Jürgen

2015-05-29

Hydrophobic interaction chromatography (HIC) is one of the most frequently used purification methods in biopharmaceutical industry. A major drawback of HIC, however, is the rather low dynamic binding capacity (DBC) obtained when compared to e.g. ion exchange chromatography (IEX). The typical purification procedure for HIC includes binding at neutral pH, independently of the proteins nature and isoelectric point. Most approaches to process intensification are based on resin and salt screenings. In this paper a combination of protein solubility data and varying binding pH leads to a clear enhancement of dynamic binding capacity. This is shown for three proteins of acidic, neutral, and alkaline isoelectric points. High-throughput solubility screenings as well as miniaturized and parallelized breakthrough curves on Media Scout RoboColumns (Atoll, Germany) were conducted at pH 3-10 on a fully automated robotic workstation. The screening results show a correlation between the DBC and the operational pH, the protein's isoelectric point and the overall solubility. Also, an inverse relationship of DBC in HIC and the binding kinetics was observed. By changing the operational pH, the DBC could be increased up to 30% compared to the standard purification procedure performed at neutral pH. As structural changes of the protein are reported during HIC processes, the applied samples and the elution fractions were proven not to be irreversibly unfolded. Copyright © 2015 Elsevier B.V. All rights reserved.

General transfer matrix formalism to calculate DNA-protein-drug binding in gene regulation: application to OR operator of phage lambda.

PubMed

Teif, Vladimir B

2007-01-01

The transfer matrix methodology is proposed as a systematic tool for the statistical-mechanical description of DNA-protein-drug binding involved in gene regulation. We show that a genetic system of several cis-regulatory modules is calculable using this method, considering explicitly the site-overlapping, competitive, cooperative binding of regulatory proteins, their multilayer assembly and DNA looping. In the methodological section, the matrix models are solved for the basic types of short- and long-range interactions between DNA-bound proteins, drugs and nucleosomes. We apply the matrix method to gene regulation at the O(R) operator of phage lambda. The transfer matrix formalism allowed the description of the lambda-switch at a single-nucleotide resolution, taking into account the effects of a range of inter-protein distances. Our calculations confirm previously established roles of the contact CI-Cro-RNAP interactions. Concerning long-range interactions, we show that while the DNA loop between the O(R) and O(L) operators is important at the lysogenic CI concentrations, the interference between the adjacent promoters P(R) and P(RM) becomes more important at small CI concentrations. A large change in the expression pattern may arise in this regime due to anticooperative interactions between DNA-bound RNA polymerases. The applicability of the matrix method to more complex systems is discussed.

Ada/POSIX binding: A focused Ada investigation

NASA Technical Reports Server (NTRS)

Legrand, Sue

1988-01-01

NASA is seeking an operating system interface definition (OSID) for the Space Station Program (SSP) in order to take advantage of the commercial off-the-shelf (COTS) products available today and the many that are expected in the future. NASA would also like to avoid the reliance on any one source for operating systems, information system, communication system, or instruction set architecture. The use of the Portable Operating System Interface for Computer Environments (POSIX) is examined as a possible solution to this problem. Since Ada is already the language of choice for SSP, the question of an Ada/POSIX binding is addressed. The intent of the binding is to provide access to the POSIX standard operation system (OS) interface and environment, by which application portability of Ada applications will be supported at the source code level. A guiding principle of Ada/POSIX binding development is a clear conformance of the Ada interface with the functional definition of POSIX. The interface is intended to be used by both application developers and system implementors. The objective is to provide a standard that allows a strictly conforming application source program that can be compiled to execute on any conforming implementation. Special emphasis is placed on first providing those functions and facilities that are needed in a wide variety of commercial applications

Scanpath memory binding: multiple read-out experiments

NASA Astrophysics Data System (ADS)

Stark, Lawrence W.; Privitera, Claudio M.; Yang, Huiyang; Azzariti, Michela; Ho, Yeuk F.; Chan, Angie; Krischer, Christof; Weinberger, Adam

1999-05-01

The scanpath theory proposed that an internal spatial- cognitive model controls perception and the active looking eye movements, EMs, of the scanpath sequence. Evidence for this came from new quantitative methods, experiments with ambiguous figures and visual imagery and from MRI studies, all on cooperating human subjects. Besides recording EMs, we introduce other experimental techniques wherein the subject must depend upon memory bindings as in visual imagery, but may call upon other motor behaviors than EMs to read-out the remembered patterns. How is the internal model distributed and operationally assembled. The concept of binding speaks to the assigning of values for the model and its execution in various parts of the brain. Current neurological information helps to localize different aspects of the spatial-cognitive model in the brain. We suppose that there are several levels of 'binding' -- semantic or symbolic binding, structural binding for the spatial locations of the regions-of-interest and sequential binding for the dynamic execution program that yields the sequence of EMs. Our aim is to dissect out respective contributions of these different forms of binding.

The electrostatic role of the Zn-Cys2His2 complex in binding of operator DNA with transcription factors: mouse EGR-1 from the Cys2His2 family.

PubMed

Chirgadze, Y N; Boshkova, E A; Polozov, R V; Sivozhelezov, V S; Dzyabchenko, A V; Kuzminsky, M B; Stepanenko, V A; Ivanov, V V

2018-01-07

The mouse factor Zif268, known also as early growth response protein EGR-1, is a classical representative for the Cys2His2 transcription factor family. It is required for binding the RNA polymerase with operator dsDNA to initialize the transcription process. We have shown that only in this family of total six Zn-finger protein families the Zn complex plays a significant role in the protein-DNA binding. Electrostatic feature of this complex in the binding of factor Zif268 from Mus musculus with operator DNA has been considered. The factor consists of three similar Zn-finger units which bind with triplets of coding DNA. Essential contacts of the factor with the DNA phosphates are formed by three conservative His residues, one in each finger. We describe here the results of calculations of the electrostatic potentials for the Zn-Cys2His2 complex, Zn-finger unit 1, and the whole transcription factor. The potential of Zif268 has a positive area on the factor surface, and it corresponds exactly to the binding sites of each of Zn-finger units. The main part of these areas is determined by conservative His residues, which form contacts with the DNA phosphate groups. Our result shows that the electrostatic positive potential of this histidine residue is enhanced due to the Zn complex. The other contacts of the Zn-finger with DNA are related to nucleotide bases, and they are responsible for the sequence-specific binding with DNA. This result may be extended to all other members of the Cys2His2 transcription factor family.

Effects of behavioural exercise therapy on the effectiveness of a multidisciplinary rehabilitation for chronic non-specific low back pain: study protocol for a randomised controlled trial.

PubMed

Hofmann, Jana; Peters, Stefan; Geidl, Wolfgang; Hentschke, Christian; Pfeifer, Klaus

2013-03-11

In Germany, a multidisciplinary rehabilitation named "behavioural medical rehabilitation" (BMR) is available for treatment of chronic low back pain (clbp). A central component of BMR is standard exercise therapy (SET), which is directed mainly to improve physical fitness. There is a need to address psychosocial factors within SET and therefore to improve behavior change with a focus on the development of self-management skills in dealing with clbp. Furthermore, short-term effectiveness of BMR with a SET has been proven, but the impact of a behavioural exercise therapy (BET) for improvement of the long-term effectiveness of BMR is unclear. To compare the effectiveness of two exercise programs with different approaches within BMR on the effects of BMR a prospective randomized controlled trial (RCT) in two rehabilitation centres will be performed. 214 patients aged 18-65 with clbp will be, based on an "urn randomisation"-algorithm, randomly assigned to a BMR with SET (function-oriented, n=107) and BMR with BET (behaviour-oriented, n=107). Both exercise programs have a mean duration of 26 hours in three weeks and are delivered by a limited number of not-blinded study therapists in closed groups with six to twelve patients who will be masked regarding study group. The main differences of BET lie in its detailed manualised program with a theory-based, goal-orientated combination of exercise, education and behavioural elements, active participation of patients and consideration of their individual preferences and previous experiences with exercise. The primary outcome is functional ability assessed with the Hannover Functional Ability Questionnaire directly before and after the rehabilitation program, as well as a six and twelve-month follow-up. This RCT is designed to explore the effects of BET on the effectiveness of a BMR compared to a BMR with SET in the management of patients with clbp. Methodological challenges arise from conducting a RCT within routine health care

Basal metabolic rate is decreased in women with polycystic ovary syndrome and biochemical hyperandrogenemia and is associated with insulin resistance.

PubMed

Georgopoulos, Neoklis A; Saltamavros, Alexandros D; Vervita, Vasiliki; Karkoulias, Kyriakos; Adonakis, George; Decavalas, George; Kourounis, George; Markou, Kostas B; Kyriazopoulou, Venetsana

2009-07-01

To evaluate basal metabolic rate (BMR) in women with PCOS and to determine its association with insulin resistance (IR). Prospective assessment of BMR in women with PCOS. Outpatient clinic of the Division of Reproductive Endocrinology. The study included 91 Greek women with PCOS and biochemical hyperandrogenemia, with mean age 24.03 +/- 0.55 years and mean body mass index (BMI) 26.67 +/- 0.69 kg/m(2), and 48 matched regularly menstruating women, with mean age 26.33 +/- 0.93 years and mean BMI 23.35 +/- 0.85 kg/m(2), as control subjects. Assessment of BMR by indirect calorimetry, IR by HOMA and QUICKI indices, fasting insulin, and fasting glucose/insulin ratio. Reduced BMR in PCOS with or without IR. Adjusted BMR was 1,868 +/- 41 kcal/day in the control group, 1,445.57 +/- 76 in all PCOS women, 1,590 +/- 130 in PCOS women without IR and 1,116 +/- 106 in PCOS women with IR. Adjusted BMR showed a statistically significant difference between women with PCOS and control subjects, with lowest values in the group of PCOS women with IR, even after adjusting all groups for age and BMI. Women with PCOS, particularly those with IR, present a significantly decreased BMR.

Adaptation of metabolism and evaporative water loss along an aridity gradient.

PubMed Central

Tieleman, B Irene; Williams, Joseph B; Bloomer, Paulette

2003-01-01

Broad-scale comparisons of birds indicate the possibility of adaptive modification of basal metabolic rate (BMR) and total evaporative water loss (TEWL) in species from desert environments, but these might be confounded by phylogeny or phenotypic plasticity. This study relates variation in avian BMR and TEWL to a continuously varying measure of environment, aridity. We test the hypotheses that BMR and TEWL are reduced along an aridity gradient within the lark family (Alaudidae), and investigate the role of phylogenetic inertia. For 12 species of lark, BMR and TEWL decreased along a gradient of increasing aridity, a finding consistent with our proposals. We constructed a phylogeny for 22 species of lark based on sequences of two mitochondrial genes, and investigated whether phylogenetic affinity played a part in the correlation of phenotype and environment. A test for serial independence of the data for mass-corrected TEWL and aridity showed no influence of phylogeny on our findings. However, we did discover a significant phylogenetic effect in mass-corrected data for BMR, a result attributable to common phylogenetic history or to common ecological factors. A test of the relationship between BMR and aridity using phylogenetic independent constrasts was consistent with our previous analysis: BMR decreased with increasing aridity. PMID:12590762

Adaptation of metabolism and evaporative water loss along an aridity gradient.

PubMed

Tieleman, B Irene; Williams, Joseph B; Bloomer, Paulette

2003-01-22

Broad-scale comparisons of birds indicate the possibility of adaptive modification of basal metabolic rate (BMR) and total evaporative water loss (TEWL) in species from desert environments, but these might be confounded by phylogeny or phenotypic plasticity. This study relates variation in avian BMR and TEWL to a continuously varying measure of environment, aridity. We test the hypotheses that BMR and TEWL are reduced along an aridity gradient within the lark family (Alaudidae), and investigate the role of phylogenetic inertia. For 12 species of lark, BMR and TEWL decreased along a gradient of increasing aridity, a finding consistent with our proposals. We constructed a phylogeny for 22 species of lark based on sequences of two mitochondrial genes, and investigated whether phylogenetic affinity played a part in the correlation of phenotype and environment. A test for serial independence of the data for mass-corrected TEWL and aridity showed no influence of phylogeny on our findings. However, we did discover a significant phylogenetic effect in mass-corrected data for BMR, a result attributable to common phylogenetic history or to common ecological factors. A test of the relationship between BMR and aridity using phylogenetic independent constrasts was consistent with our previous analysis: BMR decreased with increasing aridity.

Phenotypic plasticity in the scaling of avian basal metabolic rate

PubMed Central

McKechnie, Andrew E; Freckleton, Robert P; Jetz, Walter

2006-01-01

Many birds exhibit short-term, reversible adjustments in basal metabolic rate (BMR), but the overall contribution of phenotypic plasticity to avian metabolic diversity remains unclear. The available BMR data include estimates from birds living in natural environments and captive-raised birds in more homogenous, artificial environments. All previous analyses of interspecific variation in BMR have pooled these data. We hypothesized that phenotypic plasticity is an important contributor to interspecific variation in avian BMR, and that captive-raised populations exhibit general differences in BMR compared to wild-caught populations. We tested this hypothesis by fitting general linear models to BMR data for 231 bird species, using the generalized least-squares approach to correct for phylogenetic relatedness when necessary. The scaling exponent relating BMR to body mass in captive-raised birds (0.670) was significantly shallower than in wild-caught birds (0.744). The differences in metabolic scaling between captive-raised and wild-caught birds persisted when migratory tendency and habitat aridity were controlled for. Our results reveal that phenotypic plasticity is a major contributor to avian interspecific metabolic variation. The finding that metabolic scaling in birds is partly determined by environmental factors provides further support for models that predict variation in scaling exponents, such as the allometric cascade model. PMID:16627278

Comparative analyses of basal rate of metabolism in mammals: data selection does matter.

PubMed

Genoud, Michel; Isler, Karin; Martin, Robert D

2018-02-01

Basal rate of metabolism (BMR) is a physiological parameter that should be measured under strictly defined experimental conditions. In comparative analyses among mammals BMR is widely used as an index of the intensity of the metabolic machinery or as a proxy for energy expenditure. Many databases with BMR values for mammals are available, but the criteria used to select metabolic data as BMR estimates have often varied and the potential effect of this variability has rarely been questioned. We provide a new, expanded BMR database reflecting compliance with standard criteria (resting, postabsorptive state; thermal neutrality; adult, non-reproductive status for females) and examine potential effects of differential selectivity on the results of comparative analyses. The database includes 1739 different entries for 817 species of mammals, compiled from the original sources. It provides information permitting assessment of the validity of each estimate and presents the value closest to a proper BMR for each entry. Using different selection criteria, several alternative data sets were extracted and used in comparative analyses of (i) the scaling of BMR to body mass and (ii) the relationship between brain mass and BMR. It was expected that results would be especially dependent on selection criteria with small sample sizes and with relatively weak relationships. Phylogenetically informed regression (phylogenetic generalized least squares, PGLS) was applied to the alternative data sets for several different clades (Mammalia, Eutheria, Metatheria, or individual orders). For Mammalia, a 'subsampling procedure' was also applied, in which random subsamples of different sample sizes were taken from each original data set and successively analysed. In each case, two data sets with identical sample size and species, but comprising BMR data with different degrees of reliability, were compared. Selection criteria had minor effects on scaling equations computed for large clades

On binding energy of trions in bulk materials

NASA Astrophysics Data System (ADS)

Filikhin, Igor; Kezerashvili, Roman Ya.; Vlahovic, Branislav

2018-03-01

We study the negatively T- and positively T+ charged trions in bulk materials in the effective mass approximation within the framework of a potential model. The binding energies of trions in various semiconductors are calculated by employing Faddeev equation in configuration space. Results of calculations of the binding energies for T- are consistent with previous computational studies and are in reasonable agreement with experimental measurements, while the T+ is unbound for all considered cases. The mechanism of formation of the binding energy of trions is analyzed by comparing contributions of a mass-polarization term related to kinetic energy operators and a term related to the Coulomb repulsion of identical particles.

Basal metabolic rate in free-living tropical birds: the influence of phylogenetic, behavioral, and ecological factors

PubMed Central

Tolstenkov, Oleg; Zubkova, Ekaterina; Solovyeva, Eugenia; Kerimov, Anvar

2018-01-01

Abstract The majority of our knowledge of avian energetics is based on studies of birds from temperate and high latitudes. Using the largest existing sample of wild-caught Old World tropical species, we showed that birds from Southern Vietnam had lower basal metabolic rate (BMR) than temperate species. The strongest dissimilarity between tropical and temperate species was the low scaling exponent in the allometric relation between BMR and body mass in tropical birds (the regression slope was 0.573). The passerine migrants to temperate and high latitudes had higher BMR than tropical sedentary passerines. Body mass alone accounted for 93% of the variation in BMR (body mass ranged from 5 to 252 g). Contrary to some other studies, we did not find evidence besides the above mentioned that phylogeny, taxonomy, behavior, or ecology have a significant influence on BMR variation among tropical birds. PMID:29492036

Effect of corn silage hybrids differing in starch and neutral detergent fiber digestibility on lactation performance and total-tract nutrient digestibility by dairy cows.

PubMed

Ferraretto, L F; Fonseca, A C; Sniffen, C J; Formigoni, A; Shaver, R D

2015-01-01

Selection for hybrids with greater starch and NDF digestibility may be beneficial for dairy producers. The objective of this study was to determine the effect of feeding a TMR containing a floury-leafy corn silage hybrid (LFY) compared with a brown midrib corn silage hybrid (BMR) for intake, lactation performance, and total-tract nutrient digestibility in dairy cows. Ninety-six multiparous Holstein cows, 105±31d in milk at trial initiation, were stratified by DIM and randomly assigned to 12 pens of 8 cows each. Pens were randomly assigned to 1 of 2 treatments, BMR or LFY, in a completely randomized design; a 2-wk covariate period with cows fed a common diet followed by a 14-wk treatment period with cows fed their assigned treatment diet. Starch digestibilities, in situ, in vitro, and in vivo, were greater for LFY compared with BMR; the opposite was observed for NDF digestibility. Cows fed BMR consumed 1.7kg/d more dry matter than LFY. Although, actual-, energy-, and solids-corrected milk yields were greater for BMR than LFY, feed conversions (kg of milk or component-corrected milk per kg of DMI) did not differ. Fat-corrected milk and milk fat yield were similar, as milk fat content was greater for cows fed LFY (4.05%) than BMR (3.83%). Cows fed BMR had lower milk urea nitrogen concentration, but greater milk protein and lactose yields compared with LFY. Body weight change and condition score were unaffected by treatment. Total-tract starch digestibility was greater for cows fed the LFY corn silage; however, dry matter intake and milk and protein yields were greater for cows fed the BMR corn silage. Although total-tract starch digestibility was greater for cows fed the LFY corn silage, feed efficiency was not affected by hybrid type due to greater dry matter intake and milk and protein yields by cows fed the BMR corn silage. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The Case against Binding Interest Arbitration.

ERIC Educational Resources Information Center

Ecker, Charles I.

1984-01-01

The author contends that districts should reject binding interest arbitration as a means of resolving an impasse in contract negotiations, charging that it hampers good faith bargaining, adversely affects fiscal and operational management of the school system, and diminishes the governing role of the board of education. (MJL)

The effect of long term combined yoga practice on the basal metabolic rate of healthy adults.

PubMed

Chaya, M S; Kurpad, A V; Nagendra, H R; Nagarathna, R

2006-08-31

Different procedures practiced in yoga have stimulatory or inhibitory effects on the basal metabolic rate when studied acutely. In daily life however, these procedures are usually practiced in combination. The purpose of the present study was to investigate the net change in the basal metabolic rate (BMR) of individuals actively engaging in a combination of yoga practices (asana or yogic postures, meditation and pranayama or breathing exercises) for a minimum period of six months, at a residential yoga education and research center at Bangalore. The measured BMR of individuals practicing yoga through a combination of practices was compared with that of control subjects who did not practice yoga but led similar lifestyles. The BMR of the yoga practitioners was significantly lower than that of the non-yoga group, and was lower by about 13 % when adjusted for body weight (P < 0.001). This difference persisted when the groups were stratified by gender; however, the difference in BMR adjusted for body weight was greater in women than men (about 8 and 18% respectively). In addition, the mean BMR of the yoga group was significantly lower than their predicted values, while the mean BMR of non-yoga group was comparable with their predicted values derived from 1985 WHO/FAO/UNU predictive equations. This study shows that there is a significantly reduced BMR, probably linked to reduced arousal, with the long term practice of yoga using a combination of stimulatory and inhibitory yogic practices.

The effect of long term combined yoga practice on the basal metabolic rate of healthy adults

PubMed Central

Chaya, MS; Kurpad, AV; Nagendra, HR; Nagarathna, R

2006-01-01

Background Different procedures practiced in yoga have stimulatory or inhibitory effects on the basal metabolic rate when studied acutely. In daily life however, these procedures are usually practiced in combination. The purpose of the present study was to investigate the net change in the basal metabolic rate (BMR) of individuals actively engaging in a combination of yoga practices (asana or yogic postures, meditation and pranayama or breathing exercises) for a minimum period of six months, at a residential yoga education and research center at Bangalore. Methods The measured BMR of individuals practicing yoga through a combination of practices was compared with that of control subjects who did not practice yoga but led similar lifestyles. Results The BMR of the yoga practitioners was significantly lower than that of the non-yoga group, and was lower by about 13 % when adjusted for body weight (P < 0.001). This difference persisted when the groups were stratified by gender; however, the difference in BMR adjusted for body weight was greater in women than men (about 8 and 18% respectively). In addition, the mean BMR of the yoga group was significantly lower than their predicted values, while the mean BMR of non-yoga group was comparable with their predicted values derived from 1985 WHO/FAO/UNU predictive equations. Conclusion This study shows that there is a significantly reduced BMR, probably linked to reduced arousal, with the long term practice of yoga using a combination of stimulatory and inhibitory yogic practices. PMID:16945127

[Study of Basal metabolic rate of 81 young adults aged 20-29 years old in Changsha].

PubMed

Zhou, X; Mao, D Q; Luo, J Y; Wu, J H; Zhuo, Q; Li, Y M

2017-07-06

Objective: To determine the basal metabolic rate (BMR) of young adults aged between 20-29 years old in Changsha. Methods: We recruited volunteers to join in our research project from April to May, 2015. All recruited volunteers must meet the inclusion criteria: aged 20-29 years old, height between 164-180 centimeters in males and 154-167 centimeters in females, in good health condition, and with no habit of regular physical exercise in last year. Finally, 81 qualified volunteers were selected as research objects, including 43 males and 38 females. The BMR, resting lying metabolism rate and resting sitting metabolism rate of the subjects were detected, and the determined BMR was compared with the calculated results: from the adjusted Schofield equation. Results The BMR, resting lying metabolism rate and resting sitting metabolism rate among males were (166.10±22.09), (174.22±24.56), and (179.54±23.35) kJ·m(-2)·h(-1), respectively, which were all higher than those among females were (137.70±20.04), (149.79±19.25), and (167.78±26.02) kJ·m(-2)·h(-1), respectively, ( P< 0.001). The BMR of males and females calculated from the adjusted Schofield equation were (160.83±3.93), and (140.29±4.18) kJ·m(-2)·h(-1), respectively, and there was no significantly statistical difference found between the determined BMR and the calculated results from Schofield equation (adjusted) classified by sex, all P values >0.05. Conclusion: The BMR of young adults aged 20-29 years old in Changsha was in the national average level, and the adjusted Schofield equation displayed fine accuracy in predicting BMR of young adults aged 20-29 years old in Changsha.

An explanation of the relationship between mass, metabolic rate and characteristic length for placental mammals

PubMed Central

2015-01-01

The Mass, Metabolism and Length Explanation (MMLE) was advanced in 1984 to explain the relationship between metabolic rate and body mass for birds and mammals. This paper reports on a modernized version of MMLE. MMLE deterministically computes the absolute value of Basal Metabolic Rate (BMR) and body mass for individual animals. MMLE is thus distinct from other examinations of these topics that use species-averaged data to estimate the parameters in a statistically best fit power law relationship such as BMR = a(bodymass)b. Beginning with the proposition that BMR is proportional to the number of mitochondria in an animal, two primary equations are derived that compute BMR and body mass as functions of an individual animal’s characteristic length and sturdiness factor. The characteristic length is a measureable skeletal length associated with an animal’s means of propulsion. The sturdiness factor expresses how sturdy or gracile an animal is. Eight other parameters occur in the equations that vary little among animals in the same phylogenetic group. The present paper modernizes MMLE by explicitly treating Froude and Strouhal dynamic similarity of mammals’ skeletal musculature, revising the treatment of BMR and using new data to estimate numerical values for the parameters that occur in the equations. A mass and length data set with 575 entries from the orders Rodentia, Chiroptera, Artiodactyla, Carnivora, Perissodactyla and Proboscidea is used. A BMR and mass data set with 436 entries from the orders Rodentia, Chiroptera, Artiodactyla and Carnivora is also used. With the estimated parameter values MMLE can calculate characteristic length and sturdiness factor values so that every BMR and mass datum from the BMR and mass data set can be computed exactly. Furthermore MMLE can calculate characteristic length and sturdiness factor values so that every body mass and length datum from the mass and length data set can be computed exactly. Whether or not MMLE can

Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding by Streptomyces Antibiotic Regulatory Protein-Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators in Streptomyces natalensis.

PubMed

Barreales, Eva G; Vicente, Cláudia M; de Pedro, Antonio; Santos-Aberturas, Javier; Aparicio, Jesús F

2018-05-15

The biosynthesis of small-size polyene macrolides is ultimately controlled by a couple of transcriptional regulators that act in a hierarchical way. A Streptomyces antibiotic regulatory protein-large ATP-binding regulator of the LuxR family (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encoding gene and activates its transcription, and in turn, the gene product of the latter activates transcription from various promoters of the polyene gene cluster directly. The primary operator of PimR, the archetype of SARP-LAL regulators, contains three heptameric direct repeats separated by four-nucleotide spacers, but the regulator can also bind a secondary operator with only two direct repeats separated by a 3-nucleotide spacer, both located in the promoter region of its unique target gene, pimM A similar arrangement of operators has been identified for PimR counterparts encoded by gene clusters for different antifungal secondary metabolites, including not only polyene macrolides but peptidyl nucleosides, phoslactomycins, or cycloheximide. Here, we used promoter engineering and quantitative transcriptional analyses to determine the contributions of the different heptameric repeats to transcriptional activation and final polyene production. Optimized promoters have thus been developed. Deletion studies and electrophoretic mobility assays were used for the definition of DNA-binding boxes formed by 22-nucleotide sequences comprising two conserved heptameric direct repeats separated by four-nucleotide less conserved spacers. The cooperative binding of PimR SARP appears to be the mechanism involved in the binding of regulator monomers to operators, and at least two protein monomers are required for efficient binding. IMPORTANCE Here, we have shown that a modulation of the production of the antifungal pimaricin in Streptomyces natalensis can be accomplished via promoter engineering of the PAS-LuxR transcriptional activator pimM The expression of this gene is

Total Body Capacitance for Estimating Human Basal Metabolic Rate in an Egyptian Population

PubMed Central

M. Abdel-Mageed, Samir; I. Mohamed, Ehab

2016-01-01

Determining basal metabolic rate (BMR) is important for estimating total energy needs in the human being yet, concerns have been raised regarding the suitability of sex-specific equations based on age and weight for its calculation on an individual or population basis. It has been shown that body cell mass (BCM) is the body compartment responsible for BMR. The objectives of this study were to investigate the relationship between total body capacitance (TBC), which is considered as an expression for BCM, and BMR and to develop a formula for calculating BMR in comparison with widely used equations. Fifty healthy nonsmoking male volunteers [mean age (± SD): 24.93 ± 4.15 year and body mass index (BMI): 25.63 ± 3.59 kg/m2] and an equal number of healthy nonsmoking females matched for age and BMI were recruited for the study. TBC and BMR were measured for all participants using octopolar bioelectric impedance analysis and indirect calorimetry techniques, respectively. A significant regressing equation based on the covariates: sex, weight, and TBC for estimating BMR was derived (R=0.96, SEE=48.59 kcal, and P<0.0001), which will be useful for nutritional and health status assessment for both individuals and populations. PMID:27127453

Exploring individual quality: Basal metabolic rate and reproductive performance in storm-petrels

USGS Publications Warehouse

Blackmer, A.L.; Mauck, R.A.; Ackerman, Joshua T.; Huntington, C.E.; Nevitt, G.A.; Williams, J.B.

2005-01-01

Despite evidence that some individuals achieve both superior reproductive performance and high survivorship, the factors underlying variation in individual quality are not well understood. The compensation and increased-intake hypotheses predict that basal metabolic rate (BMR) influences reproductive performance; if so, variation in BMR may be related to differences in individual quality. We evaluated whether BMR measured during the incubation period provides a proximate explanation for variation in individual quality by measuring the BMRs and reproductive performance of Leach's storm-petrels (Oceanodroma leucorhoa) breeding on Kent Island, New Brunswick, Canada, during 2000 and 2001. We statistically controlled for internal (body mass, breeding age, sex) and external (year, date, time of day) effects on BMR. We found that males with relatively low BMRs hatched their eggs earlier in the season and that their chicks' wing growth rates were faster compared to males with relatively high BMRs. Conversely, BMR was not related to egg volume, hatching date, or chick growth rate for females or to lifetime (???23 years) hatching success for either sex. Thus, for males but not for females, our results support the compensation hypothesis. This hypothesis predicts that animals with low BMRs will achieve better reproductive performance than animals with high BMRs because they have lower self-maintenance costs and therefore can apportion more energy to reproduction. These results provide evidence that intraspecific variation in reproductive performance is related to BMR and suggest that BMR may influence individual quality in males. ?? The Author 2005. Published by Oxford University Press on behalf of the International Society for Behavioral Ecology. All rights reserved.

Basal metabolic rate in women with PCOS compared to eumenorrheic controls.

PubMed

Churchill, Sara J; Wang, Erica T; Bhasin, Gaisu; Alexander, Carolyn; Bresee, Catherine; Pall, Marita; Azziz, Ricardo; Mathur, Ruchi; Pisarska, Margareta D

2015-09-01

PCOS is associated with obesity and insulin resistance. Efforts have focused on whether an abnormal energy homeostasis contributes to the development of obesity in these patients. There are conflicting results in the literature regarding whether women with PCOS have an altered basal metabolic rate (BMR), thereby leading to difficulties in weight loss. The objective of this study is to compare basal metabolic rate (BMR) in women with PCOS and controls. Cross-sectional study. One hundred and twenty-eight PCOS patients diagnosed by original NIH consensus criteria and 72 eumenorrheic, non-hirsute controls were recruited from an academic medical centre. Assessment of BMR using the InBody portable bioelectrical impedance analysis (BIA) device and insulin resistance by HOMA-IR indices. PCOS women were younger than controls. As expected, PCOS subjects had higher body mass index (BMI), serum androgens and estimated insulin resistance. After adjusting for age and BMI, there was no significant difference in BMR between PCOS subjects (adjusted mean 5807 kJ/day, 95% CI 5715-5899) and controls (adjusted mean 5916 kJ/day, 95% CI 5786-6046) (P = 0·193). BMR was also comparable in a secondary analysis comparing PCOS women with and without insulin resistance. After adjusting for age and BMI, there was no difference in BMR between PCOS women and controls. © 2015 John Wiley & Sons Ltd.

Optimized Standard Operating Procedures for the Analysis of Cerebrospinal Fluid Aβ42 and the Ratios of Aβ Isoforms Using Low Protein Binding Tubes.

PubMed

Vanderstichele, Hugo Marcel Johan; Janelidze, Shorena; Demeyer, Leentje; Coart, Els; Stoops, Erik; Herbst, Victor; Mauroo, Kimberley; Brix, Britta; Hansson, Oskar

2016-05-31

Reduced cerebrospinal fluid (CSF) concentration of amyloid-β1-42 (Aβ1-42) reflects the presence of amyloidopathy in brains of subjects with Alzheimer's disease (AD). To qualify the use of Aβ1-42/Aβ1-40 for improvement of standard operating procedures (SOP) for measurement of CSF Aβ with a focus on CSF collection, storage, and analysis. Euroimmun ELISAs for CSF Aβ isoforms were used to set up a SOP with respect to recipient properties (low binding, polypropylene), volume of tubes, freeze/thaw cycles, addition of detergents (Triton X-100, Tween-20) in collection or storage tubes or during CSF analysis. Data were analyzed with linear repeated measures and mixed effects models. Optimization of CSF analysis included a pre-wash of recipients (e.g., tubes, 96-well plates) before sample analysis. Using the Aβ1-42/Aβ1-40 ratio, in contrast to Aβ1-42, eliminated effects of tube type, additional freeze/thaw cycles, or effect of CSF volumes for polypropylene storage tubes. 'Low binding' tubes reduced the loss of Aβ when aliquoting CSF or in function of additional freeze/thaw cycles. Addition of detergent in CSF collection tubes resulted in an almost complete absence of variation in function of collection procedures, but affected the concentration of Aβ isoforms in the immunoassay. The ratio of Aβ1-42/Aβ1-40 is a more robust biomarker than Aβ1-42 toward (pre-) analytical interfering factors. Further, 'low binding' recipients and addition of detergent in collection tubes are able to remove effects of SOP-related confounding factors. Integration of the Aβ1-42/Aβ1-40 ratio and 'low-binding tubes' into guidance criteria may speed up worldwide standardization of CSF biomarker analysis.

Estimation of basal metabolic rate in Chinese: are the current prediction equations applicable?

PubMed

Camps, Stefan G; Wang, Nan Xin; Tan, Wei Shuan Kimberly; Henry, C Jeyakumar

2016-08-31

Measurement of basal metabolic rate (BMR) is suggested as a tool to estimate energy requirements. Therefore, BMR prediction equations have been developed in multiple populations because indirect calorimetry is not always feasible. However, there is a paucity of data on BMR measured in overweight and obese adults living in Asia and equations developed for this group of interest. The aim of this study was to develop a new BMR prediction equation for Chinese adults applicable for a large BMI range and compare it with commonly used prediction equations. Subjects were 121 men and 111 women (age: 21-67 years, BMI: 16-41 kg/m(2)). Height, weight, and BMR were measured. Continuous open-circuit indirect calorimetry using a ventilated hood system for 30 min was used to measure BMR. A regression equation was derived using stepwise regression and accuracy was compared to 6 existing equations (Harris-Benedict, Henry, Liu, Yang, Owen and Mifflin). Additionally, the newly derived equation was cross-validated in a separate group of 70 Chinese subjects (26 men and 44 women, age: 21-69 years, BMI: 17-39 kg/m(2)). The equation developed from our data was: BMR (kJ/d) = 52.6 x weight (kg) + 828 x gender + 1960 (women = 0, men = 1; R(2) = 0.81). The accuracy rate (within 10 % accurate) was 78 % which compared well to Owen (70 %), Henry (67 %), Mifflin (67 %), Liu (58 %), Harris-Benedict (45 %) and Yang (37 %) for the whole range of BMI. For a BMI greater than 23, the Singapore equation reached an accuracy rate of 76 %. Cross-validation proved an accuracy rate of 80 %. To date, the newly developed Singapore equation is the most accurate BMR prediction equation in Chinese and is applicable for use in a large BMI range including those overweight and obese.

Antibody binding in altered gravity: implications for immunosorbent assay during space flight

NASA Technical Reports Server (NTRS)

Maule, Jake; Fogel, Marilyn; Steele, Andrew; Wainwright, Norman; Pierson, Duane L.; McKay, David S.

2003-01-01

A single antibody-incubation step of an indirect, enzyme-linked immunosorbent assay (ELISA) was performed during microgravity, Martian gravity (0.38 G) and hypergravity (1.8 G) phases of parabolic flight, onboard the NASA KC-135 aircraft. Antibody-antigen binding occurred within 15 seconds; the level of binding did not differ between microgravity, Martian gravity and 1 G (Earth's gravity) conditions. During hypergravity and 1 G, antibody binding was directly proportional to the fluid volume (per microtiter well) used for incubation; this pattern was not observed during microgravity. These effects in microgravity may be due to "fluid spread" within the chamber (observed during microgravity with digital photography), leading to greater fluid-surface contact and subsequently antibody-antigen contact. In summary, these results demonstrate that: i) ELISA antibody-incubation and washing steps can be successfully performed by human operators during microgravity, Martian gravity and hypergravity; ii) there is no significant difference in antibody binding between microgravity, Martian gravity and 1 G conditions; and iii) a smaller fluid volume/well (and therefore less antibody) was required for a given level of binding during microgravity. These conclusions indicate that reduced gravity would not present a barrier to successful operation of immunosorbent assays during spaceflight.

AtSPX1 affects the AtPHR1-DNA-binding equilibrium by binding monomeric AtPHR1 in solution.

PubMed

Qi, Wanjun; Manfield, Iain W; Muench, Stephen P; Baker, Alison

2017-10-23

Phosphorus is an essential macronutrient for plant growth and is deficient in ∼50% of agricultural soils. The transcription factor phosphate starvation response 1 (PHR1) plays a central role in regulating the expression of a subset of phosphate starvation-induced (PSI) genes through binding to a cis -acting DNA element termed P1BS (PHR1-binding sequences). In Arabidopsis and rice, activity of AtPHR1/OsPHR2 is regulated in part by their downstream target SPX ( S yg1, P ho81, X pr1) proteins through protein-protein interaction. Here, we provide kinetic and affinity data for interaction between AtPHR1 and P1BS sites. Using surface plasmon resonance, a tandem P1BS sequence showed ∼50-fold higher affinity for MBPAtdPHR1 (a fusion protein comprising the DNA-binding domain and coiled-coil domain of AtPHR1 fused to maltose-binding protein) than a single site. The affinity difference was largely reflected in a much slower dissociation rate from the 2× P1BS-binding site, suggesting an important role for protein co-operativity. Injection of AtSPX1 in the presence of phosphate or inositol hexakisphosphate (InsP6) failed to alter the MBPAtdPHR1-P1BS dissociation rate, while pre-mixing of these two proteins in the presence of either 5 mM Pi or 500 µM InsP6 resulted in a much lower DNA-binding signal from MBPAtdPHR1. These data suggest that, in the Pi-restored condition, AtSPX1 can bind to monomeric AtPHR1 in solution and therefore regulate PSI gene expression by tuning the AtPHR1-DNA-binding equilibrium. This Pi-dependent regulation of AtPHR1-DNA-binding equilibrium also generates a negative feedback loop on the expression of AtSPX1 itself, providing a tight control of PSI gene expression. © 2017 The Author(s).

Detergent-mediated incorporation of transmembrane proteins in giant unilamellar vesicles with controlled physiological contents

PubMed Central

Dezi, Manuela; Di Cicco, Aurelie; Bassereau, Patricia; Lévy, Daniel

2013-01-01

Giant unilamellar vesicles (GUVs) are convenient biomimetic systems of the same size as cells that are increasingly used to quantitatively address biophysical and biochemical processes related to cell functions. However, current approaches to incorporate transmembrane proteins in the membrane of GUVs are limited by the amphiphilic nature or proteins. Here, we report a method to incorporate transmembrane proteins in GUVs, based on concepts developed for detergent-mediated reconstitution in large unilamellar vesicles. Reconstitution is performed either by direct incorporation from proteins purified in detergent micelles or by fusion of purified native vesicles or proteoliposomes in preformed GUVs. Lipid compositions of the membrane and the ionic, protein, or DNA compositions in the internal and external volumes of GUVs can be controlled. Using confocal microscopy and functional assays, we show that proteins are unidirectionally incorporated in the GUVs and keep their functionality. We have successfully tested our method with three types of transmembrane proteins. GUVs containing bacteriorhodopsin, a photoactivable proton pump, can generate large transmembrane pH and potential gradients that are light-switchable and stable for hours. GUVs with FhuA, a bacterial porin, were used to follow the DNA injection by T5 phage upon binding to its transmembrane receptor. GUVs incorporating BmrC/BmrD, a bacterial heterodimeric ATP-binding cassette efflux transporter, were used to demonstrate the protein-dependent translocation of drugs and their interactions with encapsulated DNA. Our method should thus apply to a wide variety of membrane or peripheral proteins for producing more complex biomimetic GUVs. PMID:23589883

The Effect of "Rogue" Active Regions on the Solar Cycle

NASA Astrophysics Data System (ADS)

Nagy, Melinda; Lemerle, Alexandre; Labonville, François; Petrovay, Kristóf; Charbonneau, Paul

2017-11-01

The origin of cycle-to-cycle variations in solar activity is currently the focus of much interest. It has recently been pointed out that large individual active regions with atypical properties can have a significant impact on the long-term behavior of solar activity. We investigate this possibility in more detail using a recently developed 2×2D dynamo model of the solar magnetic cycle. We find that even a single "rogue" bipolar magnetic region (BMR) in the simulations can have a major effect on the further development of solar activity cycles, boosting or suppressing the amplitude of subsequent cycles. In extreme cases, an individual BMR can completely halt the dynamo, triggering a grand minimum. Rogue BMRs also have the potential to induce significant hemispheric asymmetries in the solar cycle. To study the effect of rogue BMRs in a more systematic manner, a series of dynamo simulations were conducted, in which a large test BMR was manually introduced in the model at various phases of cycles of different amplitudes. BMRs emerging in the rising phase of a cycle can modify the amplitude of the ongoing cycle, while BMRs emerging in later phases will only affect subsequent cycles. In this model, the strongest effect on the subsequent cycle occurs when the rogue BMR emerges around cycle maximum at low latitudes, but the BMR does not need to be strictly cross-equatorial. Active regions emerging as far as 20° from the equator can still have a significant effect. We demonstrate that the combined effect of the magnetic flux, tilt angle, and polarity separation of the BMR on the dynamo is via their contribution to the dipole moment, δ D_{BMR}. Our results indicate that prediction of the amplitude, starting epoch, and duration of a cycle requires an accurate accounting of a broad range of active regions emerging in the previous cycle.

Surface flux transport simulations: Effect of inflows toward active regions and random velocities on the evolution of the Sun's large-scale magnetic field

NASA Astrophysics Data System (ADS)

Martin-Belda, D.; Cameron, R. H.

2016-02-01

Aims: We aim to determine the effect of converging flows on the evolution of a bipolar magnetic region (BMR), and to investigate the role of these inflows in the generation of poloidal flux. We also discuss whether the flux dispersal due to turbulent flows can be described as a diffusion process. Methods: We developed a simple surface flux transport model based on point-like magnetic concentrations. We tracked the tilt angle, the magnetic flux and the axial dipole moment of a BMR in simulations with and without inflows and compared the results. To test the diffusion approximation, simulations of random walk dispersal of magnetic features were compared against the predictions of the diffusion treatment. Results: We confirm the validity of the diffusion approximation to describe flux dispersal on large scales. We find that the inflows enhance flux cancellation, but at the same time affect the latitudinal separation of the polarities of the bipolar region. In most cases the latitudinal separation is limited by the inflows, resulting in a reduction of the axial dipole moment of the BMR. However, when the initial tilt angle of the BMR is small, the inflows produce an increase in latitudinal separation that leads to an increase in the axial dipole moment in spite of the enhanced flux destruction. This can give rise to a tilt of the BMR even when the BMR was originally aligned parallel to the equator.

A 'slow pace of life' in Australian old-endemic passerine birds is not accompanied by low basal metabolic rates.

PubMed

Bech, Claus; Chappell, Mark A; Astheimer, Lee B; Londoño, Gustavo A; Buttemer, William A

2016-05-01

Life history theory suggests that species experiencing high extrinsic mortality rates allocate more resources toward reproduction relative to self-maintenance and reach maturity earlier ('fast pace of life') than those having greater life expectancy and reproducing at a lower rate ('slow pace of life'). Among birds, many studies have shown that tropical species have a slower pace of life than temperate-breeding species. The pace of life has been hypothesized to affect metabolism and, as predicted, tropical birds have lower basal metabolic rates (BMR) than temperate-breeding birds. However, many temperate-breeding Australian passerines belong to lineages that evolved in Australia and share 'slow' life-history traits that are typical of tropical birds. We obtained BMR from 30 of these 'old-endemics' and ten sympatric species of more recently arrived passerine lineages (derived from Afro-Asian origins or introduced by Europeans) with 'faster' life histories. The BMR of 'slow' temperate-breeding old-endemics was indistinguishable from that of new-arrivals and was not lower than the BMR of 'fast' temperate-breeding non-Australian passerines. Old-endemics had substantially smaller clutches and longer maximal life spans in the wild than new arrivals, but neither clutch size nor maximum life span was correlated with BMR. Our results suggest that low BMR in tropical birds is not functionally linked to their 'slow pace of life' and instead may be a consequence of differences in annual thermal conditions experienced by tropical versus temperate species.

Reversibly bound chloride in the atrial natriuretic peptide receptor hormone-binding domain: possible allosteric regulation and a conserved structural motif for the chloride-binding site.

PubMed

Ogawa, Haruo; Qiu, Yue; Philo, John S; Arakawa, Tsutomu; Ogata, Craig M; Misono, Kunio S

2010-03-01

The binding of atrial natriuretic peptide (ANP) to its receptor requires chloride, and it is chloride concentration dependent. The extracellular domain (ECD) of the ANP receptor (ANPR) contains a chloride near the ANP-binding site, suggesting a possible regulatory role. The bound chloride, however, is completely buried in the polypeptide fold, and its functional role has remained unclear. Here, we have confirmed that chloride is necessary for ANP binding to the recombinant ECD or the full-length ANPR expressed in CHO cells. ECD without chloride (ECD(-)) did not bind ANP. Its binding activity was fully restored by bromide or chloride addition. A new X-ray structure of the bromide-bound ECD is essentially identical to that of the chloride-bound ECD. Furthermore, bromide atoms are localized at the same positions as chloride atoms both in the apo and in the ANP-bound structures, indicating exchangeable and reversible halide binding. Far-UV CD and thermal unfolding data show that ECD(-) largely retains the native structure. Sedimentation equilibrium in the absence of chloride shows that ECD(-) forms a strongly associated dimer, possibly preventing the structural rearrangement of the two monomers that is necessary for ANP binding. The primary and tertiary structures of the chloride-binding site in ANPR are highly conserved among receptor-guanylate cyclases and metabotropic glutamate receptors. The chloride-dependent ANP binding, reversible chloride binding, and the highly conserved chloride-binding site motif suggest a regulatory role for the receptor bound chloride. Chloride-dependent regulation of ANPR may operate in the kidney, modulating ANP-induced natriuresis.

Reversibly Bound Chloride in the Atrial Natriuretic Peptide Receptor Hormone Binding Domain: Possible Allosteric Regulation and a Conserved Structural Motif for the Chloride-binding Site

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

Ogawa, H.; Qiu, Y; Philo, J

2010-01-01

The binding of atrial natriuretic peptide (ANP) to its receptor requires chloride, and it is chloride concentration dependent. The extracellular domain (ECD) of the ANP receptor (ANPR) contains a chloride near the ANP-binding site, suggesting a possible regulatory role. The bound chloride, however, is completely buried in the polypeptide fold, and its functional role has remained unclear. Here, we have confirmed that chloride is necessary for ANP binding to the recombinant ECD or the full-length ANPR expressed in CHO cells. ECD without chloride (ECD(-)) did not bind ANP. Its binding activity was fully restored by bromide or chloride addition. Amore » new X-ray structure of the bromide-bound ECD is essentially identical to that of the chloride-bound ECD. Furthermore, bromide atoms are localized at the same positions as chloride atoms both in the apo and in the ANP-bound structures, indicating exchangeable and reversible halide binding. Far-UV CD and thermal unfolding data show that ECD(-) largely retains the native structure. Sedimentation equilibrium in the absence of chloride shows that ECD(-) forms a strongly associated dimer, possibly preventing the structural rearrangement of the two monomers that is necessary for ANP binding. The primary and tertiary structures of the chloride-binding site in ANPR are highly conserved among receptor-guanylate cyclases and metabotropic glutamate receptors. The chloride-dependent ANP binding, reversible chloride binding, and the highly conserved chloride-binding site motif suggest a regulatory role for the receptor bound chloride. Chloride-dependent regulation of ANPR may operate in the kidney, modulating ANP-induced natriuresis.« less

Reversibly bound chloride in the atrial natriuretic peptide receptor hormone-binding domain: Possible allosteric regulation and a conserved structural motif for the chloride-binding site

PubMed Central

Ogawa, Haruo; Qiu, Yue; Philo, John S; Arakawa, Tsutomu; Ogata, Craig M; Misono, Kunio S

2010-01-01

The binding of atrial natriuretic peptide (ANP) to its receptor requires chloride, and it is chloride concentration dependent. The extracellular domain (ECD) of the ANP receptor (ANPR) contains a chloride near the ANP-binding site, suggesting a possible regulatory role. The bound chloride, however, is completely buried in the polypeptide fold, and its functional role has remained unclear. Here, we have confirmed that chloride is necessary for ANP binding to the recombinant ECD or the full-length ANPR expressed in CHO cells. ECD without chloride (ECD(−)) did not bind ANP. Its binding activity was fully restored by bromide or chloride addition. A new X-ray structure of the bromide-bound ECD is essentially identical to that of the chloride-bound ECD. Furthermore, bromide atoms are localized at the same positions as chloride atoms both in the apo and in the ANP-bound structures, indicating exchangeable and reversible halide binding. Far-UV CD and thermal unfolding data show that ECD(−) largely retains the native structure. Sedimentation equilibrium in the absence of chloride shows that ECD(−) forms a strongly associated dimer, possibly preventing the structural rearrangement of the two monomers that is necessary for ANP binding. The primary and tertiary structures of the chloride-binding site in ANPR are highly conserved among receptor-guanylate cyclases and metabotropic glutamate receptors. The chloride-dependent ANP binding, reversible chloride binding, and the highly conserved chloride-binding site motif suggest a regulatory role for the receptor bound chloride. Chloride-dependent regulation of ANPR may operate in the kidney, modulating ANP-induced natriuresis. PMID:20066666

Negative inotropic effect of carbachol and interaction between acetylcholine receptor-operated potassium channel (K.ACh channel) and GTP binding protein in mouse isolated atrium--a novel methodological trial.

PubMed

Okada, Muneyoshi; Noma, Chihiro; Yamawaki, Hideyuki; Hara, Yukio

2013-01-01

Interaction between acetylcholine receptor-operated potassium channel (K.ACh channel) and GTP binding protein was examined by an immunoprecipitation-Western blotting system in mouse isolated atrium. The carbachol-induced negative inotropic action in indomethacin-pretreated mouse atrium was significantly inhibited by a K.ACh channel blocker, tertiapin or atropine. Kir3.1 K.ACh channel (Kir3.1) was immunoprecipitated with a mouse anti-Kir3.1 antibody. Coprecipitating Gβ with Kir3.1, detected by Western blotting, was significantly augmented by carbachol. Atropine, but not tertiapin, significantly inhibited the carbachol-induced coprecipitating Gβ with Kir3.1. The data indicate that immunoprecipitation with Kir3.1 and Western blotting of Gβ system is a useful method for assessing interaction between K.ACh channel and GTP binding protein in mouse atrium.

Architecture of a Fur Binding Site: a Comparative Analysis

PubMed Central

Lavrrar, Jennifer L.; McIntosh, Mark A.

2003-01-01

Fur is an iron-binding transcriptional repressor that recognizes a 19-bp consensus site of the sequence 5′-GATAATGATAATCATTATC-3′. This site can be defined as three adjacent hexamers of the sequence 5′-GATAAT-3′, with the third being slightly imperfect (an F-F-F configuration), or as two hexamers in the forward orientation separated by one base pair from a third hexamer in the reverse orientation (an F-F-x-R configuration). Although Fur can bind synthetic DNA sequences containing the F-F-F arrangement, most natural binding sites are variations of the F-F-x-R arrangement. The studies presented here compared the ability of Fur to recognize synthetic DNA sequences containing two to four adjacent hexamers with binding to sequences containing variations of the F-F-x-R arrangement (including natural operator sequences from the entS and fepB promoter regions of Escherichia coli). Gel retardation assays showed that the F-F-x-R architecture was necessary for high-affinity Fur-DNA interactions and that contiguous hexamers were not recognized as effectively. In addition, the stoichiometry of Fur at each binding site was determined, showing that Fur interacted with its minimal 19-bp binding site as two overlapping dimers. These data confirm the proposed overlapping-dimer binding model, where the unit of interaction with a single Fur dimer is two inverted hexamers separated by a C:G base pair, with two overlapping units comprising the 19-bp consensus binding site required for the high-affinity interaction with two Fur dimers. PMID:12644489

Detecting cis-regulatory binding sites for cooperatively binding proteins

PubMed Central

van Oeffelen, Liesbeth; Cornelis, Pierre; Van Delm, Wouter; De Ridder, Fedor; De Moor, Bart; Moreau, Yves

2008-01-01

Several methods are available to predict cis-regulatory modules in DNA based on position weight matrices. However, the performance of these methods generally depends on a number of additional parameters that cannot be derived from sequences and are difficult to estimate because they have no physical meaning. As the best way to detect cis-regulatory modules is the way in which the proteins recognize them, we developed a new scoring method that utilizes the underlying physical binding model. This method requires no additional parameter to account for multiple binding sites; and the only necessary parameters to model homotypic cooperative interactions are the distances between adjacent protein binding sites in basepairs, and the corresponding cooperative binding constants. The heterotypic cooperative binding model requires one more parameter per cooperatively binding protein, which is the concentration multiplied by the partition function of this protein. In a case study on the bacterial ferric uptake regulator, we show that our scoring method for homotypic cooperatively binding proteins significantly outperforms other PWM-based methods where biophysical cooperativity is not taken into account. PMID:18400778

Saccharin and Cyclamate Inhibit Binding of Epidermal Growth Factor

NASA Astrophysics Data System (ADS)

Lee, L. S.

1981-02-01

The binding of 125I-labeled mouse epidermal growth factor (EGF) to 18 cell lines, including HeLa (human carcinoma), MDCK (dog kidney cells), HTC (rat hepatoma), K22 (rat liver), HF (human foreskin), GM17 (human skin fibroblasts), XP (human xeroderma pigmentosum fibroblasts), and 3T3-L1 (mouse fibroblasts), was inhibited by saccharin and cyclamate. The human cells were more sensitive to inhibition by these sweeteners than mouse or rat cells. EGF at doses far above the physiological levels reversed the inhibition in rodent cells but not in HeLa cells. In HeLa cells, the doses of saccharin and cyclamate needed for 50% inhibition were 3.5 and 9.3 mg/ml, respectively. Glucose, 2-deoxyglucose, sucrose, and xylitol did not inhibit EGF binding. Previous studies have shown that phorbol esters, strongly potent tumor promoters, also inhibit EGF binding to tissue culture cells. To explain the EGF binding inhibition by such greatly dissimilar molecules as phorbol esters, saccharin, and cyclamate, it is suggested that they operate through the activation of a hormone response control unit.

Substrate-Triggered Exosite Binding: Synergistic Dendrimer/Folic Acid Action for Achieving Specific, Tight-Binding to Folate Binding Protein.

PubMed

Chen, Junjie; van Dongen, Mallory A; Merzel, Rachel L; Dougherty, Casey A; Orr, Bradford G; Kanduluru, Ananda Kumar; Low, Philip S; Marsh, E Neil G; Banaszak Holl, Mark M

2016-03-14

Polymer-ligand conjugates are designed to bind proteins for applications as drugs, imaging agents, and transport scaffolds. In this work, we demonstrate a folic acid (FA)-triggered exosite binding of a generation five poly(amidoamine) (G5 PAMAM) dendrimer scaffold to bovine folate binding protein (bFBP). The protein exosite is a secondary binding site on the protein surface, separate from the FA binding pocket, to which the dendrimer binds. Exosite binding is required to achieve the greatly enhanced binding constants and protein structural change observed in this study. The G5Ac-COG-FA1.0 conjugate bound tightly to bFBP, was not displaced by a 28-fold excess of FA, and quenched roughly 80% of the initial fluorescence. Two-step binding kinetics were measured using the intrinsic fluorescence of the FBP tryptophan residues to give a KD in the low nanomolar range for formation of the initial G5Ac-COG-FA1.0/FBP* complex, and a slow conversion to the tight complex formed between the dendrimer and the FBP exosite. The extent of quenching was sensitive to the choice of FA-dendrimer linker chemistry. Direct amide conjugation of FA to G5-PAMAM resulted in roughly 50% fluorescence quenching of the FBP. The G5Ac-COG-FA, which has a longer linker containing a 1,2,3-triazole ring, exhibited an ∼80% fluorescence quenching. The binding of the G5Ac-COG-FA1.0 conjugate was compared to poly(ethylene glycol) (PEG) conjugates of FA (PEGn-FA). PEG2k-FA had a binding strength similar to that of FA, whereas other PEG conjugates with higher molecular weight showed weaker binding. However, no PEG conjugates gave an increased degree of total fluorescence quenching.

A high-resolution structure of the DNA-binding domain of AhrC, the arginine repressor/activator protein from Bacillus subtilis

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

Garnett, James A.; Baumberg, Simon; Stockley, Peter G.

2007-11-01

The structure of the winged helix–turn–helix DNA-binding domain of AhrC has been determined at 1.0 Å resolution. The largely hydrophobic β-wing shows high B factors and may mediate the dimer interface in operator complexes. In Bacillus subtilis the concentration of l-arginine is controlled by the transcriptional regulator AhrC, which interacts with 18 bp DNA operator sites called ARG boxes in the promoters of arginine biosynthetic and catabolic operons. AhrC is a 100 kDa homohexamer, with each subunit having two domains. The C-terminal domains form the core, mediating intersubunit interactions and binding of the co-repressor l-arginine, whilst the N-terminal domains containmore » a winged helix–turn–helix DNA-binding motif and are arranged around the periphery. The N-terminal domain of AhrC has been expressed, purified and characterized and it has been shown that the fragment still binds DNA operators as a recombinant monomer. The DNA-binding domain has also been crystallized and the crystal structure refined to 1.0 Å resolution is presented.« less

An Investigation of the Relative Effectiveness of the Basic Mathematics Review Program at Essex Community College.

ERIC Educational Resources Information Center

Bloomberg, Jerome

Basic Mathematics Review (BMR) is a remedial non-credit course at Essex Community College (Maryland) being taught on an individualized basis. Following diagnostic testing and placement, instruction utilizes programmed materials, tutors, and self-tests. Evaluation of the new individualized BMR and comparison with the traditional remedial course…

Hoxa2 Selectively Enhances Meis Binding to Change a Branchial Arch Ground State

PubMed Central

Amin, Shilu; Donaldson, Ian J.; Zannino, Denise A.; Hensman, James; Rattray, Magnus; Losa, Marta; Spitz, François; Ladam, Franck; Sagerström, Charles; Bobola, Nicoletta

2015-01-01

Summary Hox transcription factors (TFs) are essential for vertebrate development, but how these evolutionary conserved proteins function in vivo remains unclear. Because Hox proteins have notoriously low binding specificity, they are believed to bind with cofactors, mainly homeodomain TFs Pbx and Meis, to select their specific targets. We mapped binding of Meis, Pbx, and Hoxa2 in the branchial arches, a series of segments in the developing vertebrate head. Meis occupancy is largely similar in Hox-positive and -negative arches. Hoxa2, which specifies second arch (IIBA) identity, recognizes a subset of Meis prebound sites that contain Hox motifs. Importantly, at these sites Meis binding is strongly increased. This enhanced Meis binding coincides with active enhancers, which are linked to genes highly expressed in the IIBA and regulated by Hoxa2. These findings show that Hoxa2 operates as a tissue-specific cofactor, enhancing Meis binding to specific sites that provide the IIBA with its anatomical identity. PMID:25640223

Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

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

Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya

2014-07-01

Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which oftenmore » takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.« less

Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales

NASA Astrophysics Data System (ADS)

Qian, Long; Kussell, Edo

The composition of genomes with respect to short DNA motifs impacts the ability of DNA binding proteins to locate and bind their target sites. Since nonfunctional DNA binding can be detrimental to cellular functions and ultimately to organismal fitness, organisms could benefit from reducing the number of nonfunctional binding sites genome wide. Using in vitro measurements of binding affinities for a large collection of DNA binding proteins, in multiple species, we detect a significant global avoidance of weak binding sites in genomes. The underlying evolutionary process leaves a distinct genomic hallmark in that similar words have correlated frequencies, which we detect in all species across domains of life. We hypothesize that natural selection against weak binding sites contributes to this process, and using an evolutionary model we show that the strength of selection needed to maintain global word compositions is on the order of point mutation rates. Alternative contributions may come from interference of protein-DNA binding with replication and mutational repair processes, which operates with similar rates. We conclude that genome-wide word compositions have been molded by DNA binding proteins through tiny evolutionary steps over timescales spanning millions of generations.

Mammalian basal metabolic rate is proportional to body mass2/3

PubMed Central

White, Craig R.; Seymour, Roger S.

2003-01-01

The relationship between mammalian basal metabolic rate (BMR, ml of O2 per h) and body mass (M, g) has been the subject of regular investigation for over a century. Typically, the relationship is expressed as an allometric equation of the form BMR = aMb. The scaling exponent (b) is a point of contention throughout this body of literature, within which arguments for and against geometric (b = 2/3) and quarter-power (b = 3/4) scaling are made and rebutted. Recently, interest in the topic has been revived by published explanations for quarter-power scaling based on fractal nutrient supply networks and four-dimensional biology. Here, a new analysis of the allometry of mammalian BMR that accounts for variation associated with body temperature, digestive state, and phylogeny finds no support for a metabolic scaling exponent of 3/4. Data encompassing five orders of magnitude variation in M and featuring 619 species from 19 mammalian orders show that BMR ∝ M2/3. PMID:12637681

Conformational and thermodynamic hallmarks of DNA operator site specificity in the copper sensitive operon repressor from Streptomyces lividans

PubMed Central

Tan, Benedict G.; Vijgenboom, Erik; Worrall, Jonathan A. R.

2014-01-01

Metal ion homeostasis in bacteria relies on metalloregulatory proteins to upregulate metal resistance genes and enable the organism to preclude metal toxicity. The copper sensitive operon repressor (CsoR) family is widely distributed in bacteria and controls the expression of copper efflux systems. CsoR operator sites consist of G-tract containing pseudopalindromes of which the mechanism of operator binding is poorly understood. Here, we use a structurally characterized CsoR from Streptomyces lividans (CsoRSl) together with three specific operator targets to reveal the salient features pertaining to the mechanism of DNA binding. We reveal that CsoRSl binds to its operator site through a 2-fold axis of symmetry centred on a conserved 5′-TAC/GTA-3′ inverted repeat. Operator recognition is stringently dependent not only on electropositive residues but also on a conserved polar glutamine residue. Thermodynamic and circular dichroic signatures of the CsoRSl–DNA interaction suggest selectivity towards the A-DNA-like topology of the G-tracts at the operator site. Such properties are enhanced on protein binding thus enabling the symmetrical binding of two CsoRSl tetramers. Finally, differential binding modes may exist in operator sites having more than one 5′-TAC/GTA-3′ inverted repeat with implications in vivo for a mechanism of modular control. PMID:24121681

Probing the binding of fluoxetine hydrochloride to human serum albumin by multispectroscopic techniques

NASA Astrophysics Data System (ADS)

Katrahalli, Umesha; Jaldappagari, Seetharamappa; Kalanur, Shankara S.

2010-01-01

The interaction between human serum albumin (HSA) and fluoxetine hydrochloride (FLX) have been studied by using different spectroscopic techniques viz., fluorescence, UV-vis absorption, circular dichroism and FTIR under simulated physiological conditions. Fluorescence results revealed the presence of static type of quenching mechanism in the binding of FLX to HSA. The values of binding constant, K of FLX-HSA were evaluated at 289, 300 and 310 K and were found to be 1.90 × 10 3, 1.68 × 10 3 and 1.45 × 10 3 M -1, respectively. The number of binding sites, n was noticed to be almost equal to unity thereby indicating the presence of a single class of binding site for FLX on HSA. Based on the thermodynamic parameters, Δ H0 and Δ S0 nature of binding forces operating between HSA and FLX were proposed. Spectral results revealed the conformational changes in protein upon interaction. Displacement studies indicated the site I as the main binding site for FLX on HSA. The effect of common ions on the binding of FLX to HSA was also investigated.

Response of Fusarium thapsinum to sorghum brown midrib lines and to phenolic metabolites

USDA-ARS?s Scientific Manuscript database

Presentation type: poster Presentation title: Response of Fusarium thapsinum to sorghum brown midrib lines and to phenolic metabolites Sorghum lines were bred for reduced lignin for cellulosic bioenergy uses, through the incorporation of brown midrib (bmr) bmr6 and/or 12 into two gen...

Response of fusarium thapsinum to sorghum brown midrib lines and to phenolic metabolites

USDA-ARS?s Scientific Manuscript database

Sorghum lines were bred for reduced lignin for cellulosic bioenergy uses, through the incorporation of brown midrib (bmr) bmr6 and/or 12 into two genetic backgrounds, either as single or double mutant lines. These lines were assessed for resistance to Fusarium thapsinum stalk rot, a cause of lodging...

Helicoverpa zea (Lepidoptera: Noctuidae) and Spodoptera frugiperda (Lepidoptera: Noctuidae) Responses to Sorghum bicolor (Poales: Poaceae) Tissues From Lowered Lignin Lines

PubMed Central

Dowd, Patrick F.; Sattler, Scott E.

2015-01-01

The presence of lignin within biomass impedes the production of liquid fuels. Plants with altered lignin content and composition are more amenable to lignocellulosic conversion to ethanol and other biofuels but may be more susceptible to insect damage where lignin is an important resistance factor. However, reduced lignin lines of switchgrasses still retained insect resistance in prior studies. Therefore, we hypothesized that sorghum lines with lowered lignin content will also retain insect resistance. Sorghum excised leaves and stalk pith Sorghum bicolor (L.) Moench (Poales: Poaceae) from near isogenic brown midrib (bmr) 6 and 12 mutants lines, which have lowered lignin content and increased lignocellulosic ethanol conversion efficiency, were examined for insect resistance relative to wild-type (normal BTx623). Greenhouse and growth chamber grown plant tissues were fed to first-instar larvae of corn earworms, Helicoverpa zea (Boddie) and fall armyworms Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), two sorghum major pests. Younger bmr leaves had significantly greater feeding damage in some assays than wild-type leaves, but older bmr6 leaves generally had significantly less damage than wild-type leaves. Caterpillars feeding on the bmr6 leaves often weighed significantly less than those feeding on wild-type leaves, especially in the S. frugiperda assays. Larvae fed the pith from bmr stalks had significantly higher mortality compared with those larvae fed on wild-type pith, which suggested that bmr pith was more toxic. Thus, reducing lignin content or changing subunit composition of bioenergy grasses does not necessarily increase their susceptibility to insects and may result in increased resistance, which would contribute to sustainable production. PMID:25601946

Scaling of basal metabolic rate with body mass and temperature in mammals.

PubMed

Clarke, Andrew; Rothery, Peter; Isaac, Nick J B

2010-05-01

1. We present a statistical analysis of the scaling of resting (basal) metabolic rate, BMR, with body mass, B(m) and body temperature, T(b), in mammals. 2. Whilst the majority of the variance in ln BMR is explained by ln B(m), the T(b) term is statistically significant. The best fit model was quadratic, indicating that the scaling of ln BMR with ln B(m) varies with body size; the value of any scaling exponent estimated for a sample of mammals will therefore depend on the size distribution of species in the study. This effect can account for much of the variation in scaling exponents reported in the literature for mammals. 3. In all models, inclusion of T(b) reduced the strength of scaling with ln B(m). The model including T(b) suggests that birds and mammals have a similar underlying thermal dependence of BMR, equivalent to a Q(10) of 2.9 across the range of T(b) values 32-42 degrees C. 4. There was significant heterogeneity in both the mass scaling exponent and mean BMR across mammalian orders, with a tendency for orders dominated by larger taxa to have steeper scaling exponents. This heterogeneity was particularly marked across orders with smaller mean B(m) and the taxonomic composition of the sample will thus also affect the observed scaling exponent. After correcting for the effects of ln B(m) and T(b), Soricomorpha, Didelphimorphia and Artiodactyla had the highest BMR of those orders represented by more than 10 species in the data set. 5. Inclusion of T(b) in the model removed the effect of diet category evident from a model in ln B(m) alone and widely reported in the literature; this was caused by a strong interaction between diet category and T(b) in mammals. 6. Inclusion of mean ambient temperature, T(a), in the model indicated a significant inverse relationship between ln BMR and T(a), complicated by an interaction between T(a) and T(b). All other things being equal, a polar mammal living at -10 degrees C has a body temperature approximately 2.7 degrees C

Response of fusarium thapsinum to sorghum brown midrib lines and to phenolic metabolites

USDA-ARS?s Scientific Manuscript database

Sorghum lines were bred for reduced lignin for cellulosic bioenergy uses, through the incorporation of brown midrib (bmr) bmr6 and/or 12 into two genetic backgrounds, either as single or double mutant lines. When these lines were assessed for resistance to F. thapsinum stalk rot, a cause of lodging,...

Differences in fusarium species in brown midrib sorghum and in air populations in production fields

USDA-ARS?s Scientific Manuscript database

Several Fusarium spp. cause sorghum (Sorghum bicolor) grain mold, resulting in deterioration and mycotoxin production in the field and during storage. Fungal isolates from the air (2005 to 2006) and from leaves and grain from wild-type and brown midrib (bmr)-6 and bmr12 plants (2002 to 2003) were co...

Genetic dissection of bioenerrgy traits in sorghum

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

Vermerris, Wilfred; Kresovich, Stephen; Murray, Seth

Specific Objectives: 1. To identify the gene(s) underlying a major QTL for stem sugar concentration located on chromosome 3. 2. To identify QTL for stem juice volume and stalk sugar concentration and to identify the underlying genes. 3. To classify 60 novel sorghum bmr mutants from the USDA TILLING population in allelic groups based on cell wall chemistry and allelism tests. 4. To select representative bmr mutants from each allelic group and selected NIR spectral mutants for their potential value as feedstock for ethanol production. 5. To clone and characterize those Bmr genes that represent loci other than Bmr12 andmore » Bmr6 using a mapping and a candidate gene approach. Objective 1 The experiments for this objective are largely complete and the data have been analyzed. Data interpretation and follow-up experiments are still in progress. A manuscript is in preparation (Vermerris et al.; see publication list for full details). The main results are: 1) 16 cDNA libraries were prepared and sequenced at Cornell University. The libraries represent internode tissue and flag leaf tissue at booting, internode tissue and peduncle at soft-dough stage, from two plants per sampling time with the Rio allele for the QTL on chromosome 3, and two plants with the BTx623 allele on chromosome 3 (4 tissues x 2 genotypes x 2 replicates) 2) 480 million 86-nucleotide reads were generated from four lanes of Illuminia HiSeqII 3) 74% of the reads could be mapped to the sorghum transcriptome, indicative of good sequence quality 4) Of the 216 genes within the QTL, 17 genes were differentially expressed among plants with and without the Rio QTL. None of these 17 genes had obvious roles in sucrose metabolism 5) Clustering algorithms identified a group of 721 co-expressed genes. One of these genes is a sucrose synthase gene. This cluster also contains 10 genes from the QTL. 6) Among these co-expressed genes are regulatory genes for which knock-out lines in Arabidopsis have been obtained

Cyclophilin B binding to platelets supports calcium-dependent adhesion to collagen.

PubMed

Allain, F; Durieux, S; Denys, A; Carpentier, M; Spik, G

1999-08-01

We have recently reported that cyclophilin B (CyPB), a secreted cyclosporine-binding protein, could bind to T lymphocytes through interactions with two types of binding sites. The first ones, referred to as type I, involve interactions with the conserved domain of CyPB and promote the endocytosis of surface-bound ligand, while the second type of binding sites, termed type II, are represented by glycosaminoglycans (GAG). Here, we further investigated the interactions of CyPB with blood cell populations. In addition to lymphocytes, CyPB was found to interact mainly with platelets. The binding is specific, with a dissociation constant (kd) of 9 +/- 3 nmol/L and the number of sites estimated at 960 +/- 60 per cell. Platelet glycosaminoglycans are not required for the interactions, but the binding is dramatically reduced by active cyclosporine derivatives. We then analyzed the biologic effects of CyPB and found a significant increase in platelet adhesion to collagen. Concurrently, CyPB initiates a transmembranous influx of Ca(2+) and induces the phosphorylation of the P-20 light chains of myosin. Taken together, the present results demonstrate for the first time that extracellular CyPB specifically interacts with platelets through a functional receptor related to the lymphocyte type I binding sites and might act by regulating the activity of a receptor-operated membrane Ca(2+) channel.

The influence of foraging mode and arid adaptation on the basal metabolic rates of burrowing mammals.

PubMed

White, Craig R

2003-01-01

Two competing but nonexclusive hypotheses to explain the reduced basal metabolic rate (BMR) of mammals that live and forage underground (fossorial species) are examined by comparing this group with burrowing mammals that forage on the surface (semifossorial species). These hypotheses suggest that the low BMR of fossorial species either compensates for the enormous energetic demands of subterranean foraging (the cost-of-burrowing hypothesis) or prevents overheating in closed burrow systems (the thermal-stress hypothesis). Because phylogentically informed allometric analysis showed that arid burrowing mammals have a significantly lower BMR than mesic ones, fossorial and semifossorial species were compared within these groups. The BMRs of mesic fossorial and semifossorial mammals could not be reliably distinguished, nor could the BMRs of large (>77 g) arid fossorial and semifossorial mammals. This finding favours the thermal-stress hypothesis, because the groups appear to have similar BMRs despite differences in foraging costs. However, in support of the cost-of-burrowing hypothesis, small (<77 g) arid fossorial mammals were found to have a significantly lower BMR than semifossorial mammals of the similar size. Given the high mass-specific metabolic rates of small animals, they are expected to be under severe energy and water stress in arid environments. Under such conditions, the greatly reduced BMR of small fossorial species may compensate for the enormous energetic demands of subterranean foraging.

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 structure of binding curves and practical identifiability of equilibrium ligand-binding parameters

PubMed Central

Middendorf, Thomas R.

2017-01-01

A critical but often overlooked question in the study of ligands binding to proteins is whether the parameters obtained from analyzing binding data are practically identifiable (PI), i.e., whether the estimates obtained from fitting models to noisy data are accurate and unique. Here we report a general approach to assess and understand binding parameter identifiability, which provides a toolkit to assist experimentalists in the design of binding studies and in the analysis of binding data. The partial fraction (PF) expansion technique is used to decompose binding curves for proteins with n ligand-binding sites exactly and uniquely into n components, each of which has the form of a one-site binding curve. The association constants of the PF component curves, being the roots of an n-th order polynomial, may be real or complex. We demonstrate a fundamental connection between binding parameter identifiability and the nature of these one-site association constants: all binding parameters are identifiable if the constants are all real and distinct; otherwise, at least some of the parameters are not identifiable. The theory is used to construct identifiability maps from which the practical identifiability of binding parameters for any two-, three-, or four-site binding curve can be assessed. Instructions for extending the method to generate identifiability maps for proteins with more than four binding sites are also given. Further analysis of the identifiability maps leads to the simple rule that the maximum number of structurally identifiable binding parameters (shown in the previous paper to be equal to n) will also be PI only if the binding curve line shape contains n resolved components. PMID:27993951

Utilization of extracellular information before ligand-receptor binding reaches equilibrium expands and shifts the input dynamic range

PubMed Central

Ventura, Alejandra C.; Bush, Alan; Vasen, Gustavo; Goldín, Matías A.; Burkinshaw, Brianne; Bhattacharjee, Nirveek; Folch, Albert; Brent, Roger; Chernomoretz, Ariel; Colman-Lerner, Alejandro

2014-01-01

Cell signaling systems sense and respond to ligands that bind cell surface receptors. These systems often respond to changes in the concentration of extracellular ligand more rapidly than the ligand equilibrates with its receptor. We demonstrate, by modeling and experiment, a general “systems level” mechanism cells use to take advantage of the information present in the early signal, before receptor binding reaches a new steady state. This mechanism, pre-equilibrium sensing and signaling (PRESS), operates in signaling systems in which the kinetics of ligand-receptor binding are slower than the downstream signaling steps, and it typically involves transient activation of a downstream step. In the systems where it operates, PRESS expands and shifts the input dynamic range, allowing cells to make different responses to ligand concentrations so high as to be otherwise indistinguishable. Specifically, we show that PRESS applies to the yeast directional polarization in response to pheromone gradients. Consideration of preexisting kinetic data for ligand-receptor interactions suggests that PRESS operates in many cell signaling systems throughout biology. The same mechanism may also operate at other levels in signaling systems in which a slow activation step couples to a faster downstream step. PMID:25172920

Helicoverpa zea (Lepidoptera: Noctuidae) and Spodoptera frugiperda (Lepidoptera: Noctuidae) Responses to Sorghum bicolor (Poales: Poaceae) Tissues From Lowered Lignin Lines.

PubMed

Dowd, Patrick F; Sattler, Scott E

2015-01-01

The presence of lignin within biomass impedes the production of liquid fuels. Plants with altered lignin content and composition are more amenable to lignocellulosic conversion to ethanol and other biofuels but may be more susceptible to insect damage where lignin is an important resistance factor. However, reduced lignin lines of switchgrasses still retained insect resistance in prior studies. Therefore, we hypothesized that sorghum lines with lowered lignin content will also retain insect resistance. Sorghum excised leaves and stalk pith Sorghum bicolor (L.) Moench (Poales: Poaceae) from near isogenic brown midrib (bmr) 6 and 12 mutants lines, which have lowered lignin content and increased lignocellulosic ethanol conversion efficiency, were examined for insect resistance relative to wild-type (normal BTx623). Greenhouse and growth chamber grown plant tissues were fed to first-instar larvae of corn earworms, Helicoverpa zea (Boddie) and fall armyworms Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), two sorghum major pests. Younger bmr leaves had significantly greater feeding damage in some assays than wild-type leaves, but older bmr6 leaves generally had significantly less damage than wild-type leaves. Caterpillars feeding on the bmr6 leaves often weighed significantly less than those feeding on wild-type leaves, especially in the S. frugiperda assays. Larvae fed the pith from bmr stalks had significantly higher mortality compared with those larvae fed on wild-type pith, which suggested that bmr pith was more toxic. Thus, reducing lignin content or changing subunit composition of bioenergy grasses does not necessarily increase their susceptibility to insects and may result in increased resistance, which would contribute to sustainable production. Published by Oxford University Press on behalf of the Entomological Society of America 2015. This work is written by US Government employees and is in the public domain in the US.

Effects of weak/non-complement-binding HLA antibodies on C1q-binding.

PubMed

Hönger, G; Amico, P; Arnold, M-L; Spriewald, B M; Schaub, S

2017-08-01

It is unknown under what conditions and to what extent weak/non-complement (C)-binding IgG subclasses (IgG2/IgG4) can block C1q-binding triggered by C-binding IgG subclasses (IgG1/IgG3). Therefore, we investigated in vitro C1q-binding induced by IgG subclass mixtures targeting the same HLA epitope. Various mixtures of HLA class II specific monoclonal antibodies of different IgG subclasses but identical V-region were incubated with HLA DRB1*07:01 beads and monitored for C1q-binding. The lowest concentration to achieve maximum C1q-binding was measured for IgG3, followed by IgG1, while IgG2 and IgG4 did not show appreciable C1q-binding. C1q-binding occurred only after a critical amount of IgG1/3 has bound and sharply increased thereafter. When both, C-binding and weak/non-C-binding IgG subclasses were mixed, C1q-binding was diminished proportionally to the fraction of IgG2/4. A 2- to 4-fold excess of IgG2/4 inhibited C1q-binding by 50%. Very high levels (10-fold excess) almost completely abrogated C1q-binding even in the presence of significant IgG1/3 levels that would usually lead to strong C1q-binding. In sensitized renal allograft recipients, IgG subclass constellations with ≥ 2-fold excess of IgG2/4 over IgG1/3 were present in 23/66 patients (34.8%) and overall revealed slightly decreased C1q signals. However, spiking of patient sera with IgG2 targeting a different epitope than the patient's IgG1/3 synergistically increased C1q-binding. In conclusion, if targeting the same epitope, an excess of IgG2/4 is repressing the extent of IgG1/3 triggered C1q-binding in vitro. Such IgG subclass constellations are present in about a third of sensitized patients and their net effect on C1q-binding is slightly inhibitory. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales

NASA Astrophysics Data System (ADS)

Qian, Long; Kussell, Edo

2016-10-01

The composition of a genome with respect to all possible short DNA motifs impacts the ability of DNA binding proteins to locate and bind their target sites. Since nonfunctional DNA binding can be detrimental to cellular functions and ultimately to organismal fitness, organisms could benefit from reducing the number of nonfunctional DNA binding sites genome wide. Using in vitro measurements of binding affinities for a large collection of DNA binding proteins, in multiple species, we detect a significant global avoidance of weak binding sites in genomes. We demonstrate that the underlying evolutionary process leaves a distinct genomic hallmark in that similar words have correlated frequencies, a signal that we detect in all species across domains of life. We consider the possibility that natural selection against weak binding sites contributes to this process, and using an evolutionary model we show that the strength of selection needed to maintain global word compositions is on the order of point mutation rates. Likewise, we show that evolutionary mechanisms based on interference of protein-DNA binding with replication and mutational repair processes could yield similar results and operate with similar rates. On the basis of these modeling and bioinformatic results, we conclude that genome-wide word compositions have been molded by DNA binding proteins acting through tiny evolutionary steps over time scales spanning millions of generations.

Hypertension prevalence and influence of basal metabolic rate on blood pressure among adult students in Bangladesh.

PubMed

Ali, Nurshad; Mahmood, Shakil; Manirujjaman, M; Perveen, Rasheda; Al Nahid, Abdullah; Ahmed, Shamim; Khanum, Farida Adib; Rahman, Mustafizur

2017-07-25

Hypertension is a global health issue and is currently increasing at rapid pace in South Asian countries including Bangladesh. Although, some studies on hypertension have been conducted in Bangladesh, there is a lack of scientific evidence in the adult student population that was missing from the previous and recent national cross-sectional studies. Moreover, the specific risk factors of hypertension in the Bangladeshi adults still need to be investigated. This study was conducted to estimate hypertension prevalence among adult students in Bangladesh and to test the hypothesis of Luke et al. (Hypertension 43:555-560, 2004) that basal metabolic rate (BMR) and blood pressure are positively associated independent of body size. The data was collected on 184 adult university students (118 female and 66 male) in Dhaka, Bangladesh. Anthropometric, BMR details and an average of at least two blood pressure measurements were obtained. Hypertension was defined by a systolic blood pressure (SBP) ≥ 140 mmHg and/or, diastolic blood pressure (DBP) ≥ 90 mmHg. Overall, 6.5% of participants had hypertension with significantly (p < 0.001) higher prevalence in male (12.1%) than in the female (3.4%) students. Age and BMI showed positive and significant correlation with hypertension among the students. When adjusted for body mass index (BMI), as well as other potentially confounding variables such as age, sex, smoking status and degree of urbanization, BMR was positively correlated with SBP and DBP (p < 0.001). Thus, higher BMR is associated with SBP and DBP; this is opposite the well documented inverse relationship between physical activity and blood pressure. If the influence of BMR on blood pressure is confirmed, the systematically elevated BMR might be an important predictor that can explain relatively high blood pressure and hypertension in humans. This study reports the prevalence and associated risk factors of hypertension in the Bangladeshi adult students. The

Two nucleotide binding sites modulate ( sup 3 H) glyburide binding to rat cortex membranes

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

Johnson, D.E.; Gopalakrishnan, M.; Triggle, D.J.

1991-03-11

The effects of nucleotides on the binding of the ATP-dependent K{sup +}-channel antagonist ({sup 3}H)glyburide (GLB) to rat cortex membranes were examined. Nucleotide triphosphates (NTPs) and nucleotide diphosphate (NDPs) inhibited the binding of GLB. This effect was dependent on the presence of dithiothreitol (DTT). Inhibition of binding by NTPs, with the exception of ATP{gamma}S, was dependent on the presence of Mg{sup 2+}. GLB binding showed a biphasic response to ADP: up to 3 mM, ADP inhibited binding, and above this concentration GLB binding increased rapidly, and was restored to normal levels by 10 mM ADP. In the presence of Mg{supmore » 2+}, ADP did not stimulate binding. Saturation analysis in the presence of Mg{sup 2+} and increasing concentrations of ADP showed that ADP results primarily in a change of the B{sub max} for GLB binding. The differential effects of NTPS and NDPs indicate that two nucleotide binding sites regulate GLB binding.« less

Porphyrin-substrate binding to murine ferrochelatase: effect on the thermal stability of the enzyme

PubMed Central

2004-01-01

Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the haem biosynthetic pathway, catalyses the chelation of Fe(II) into the protoporphyrin IX ring. The energetics of the binding between murine ferrochelatase and mesoporphyrin were determined using isothermal titration calorimetry, which revealed a stoichiometry of one molecule of mesoporphyrin bound per protein monomer. The binding is strongly exothermic, with a large intrinsic enthalpy (ΔH=−97.1 kJ · mol−1), and is associated with the uptake of two protons from the buffer. This proton transfer suggests that hydrogen bonding between ferrochelatase and mesoporphyrin is a key factor in the thermodynamics of the binding reaction. Differential scanning calorimetry thermograms indicated a co-operative two-state denaturation process with a single transition temperature of 56 °C for wild-type murine ferrochelatase. An increase in the thermal stability of ferrochelatase is dependent upon mesoporphyrin binding. Similarly, murine ferrochelatase variants, in which the active site Glu-289 was replaced by either glutamine or alanine and, when purified, contained specifically-bound protoporphyrin, exhibited enhanced protein stability when compared with wild-type ferrochelatase. However, in contrast with the wild-type enzyme, the thermal denaturation of ferrochelatase variants was best described as a non-co-operative denaturation process. PMID:15496139

Gold Binding by Native and Chemically Modified Hops Biomasses

DOE PAGES

López, M. Laura; Gardea-Torresdey, J. L.; Peralta-Videa, J. R.; ...

2005-01-01

Heavy metals from mining, smelting operations and other industrial processing facilities pollute wastewaters worldwide. Extraction of metals from industrial effluents has been widely studied due to the economic advantages and the relative ease of technical implementation. Consequently, the search for new and improved methodologies for the recovery of gold has increased. In this particular research, the use of cone hops biomass ( Humulus lupulus ) was investigated as a new option for gold recovery. The results showed that the gold binding to native hops biomass was pH dependent from pH 2 to pH 6, with a maximum percentage binding atmore » pH 3. Time dependency studies demonstrated that Au(III) binding to native and modified cone hops biomasses was found to be time independent at pH 2 while at pH 5, it was time dependent. Capacity experiments demonstrated that at pH 2, esterified hops biomass bound 33.4 mg Au/g of biomass, while native and hydrolyzed hops biomasses bound 28.2 and 12.0 mg Au/g of biomass, respectively. However, at pH 5 the binding capacities were 38.9, 37.8 and 11.4 mg of Au per gram of native, esterified and hydrolyzed hops biomasses, respectively.« less

Gold Binding by Native and Chemically Modified Hops Biomasses

PubMed Central

López, M. Laura; Peralta-Videa, J. R.; de la Rosa, G.; Armendáriz, V.; Herrera, I.; Troiani, H.; Henning, J.

2005-01-01

Heavy metals from mining, smelting operations and other industrial processing facilities pollute wastewaters worldwide. Extraction of metals from industrial effluents has been widely studied due to the economic advantages and the relative ease of technical implementation. Consequently, the search for new and improved methodologies for the recovery of gold has increased. In this particular research, the use of cone hops biomass (Humulus lupulus) was investigated as a new option for gold recovery. The results showed that the gold binding to native hops biomass was pH dependent from pH 2 to pH 6, with a maximum percentage binding at pH 3. Time dependency studies demonstrated that Au(III) binding to native and modified cone hops biomasses was found to be time independent at pH 2 while at pH 5, it was time dependent. Capacity experiments demonstrated that at pH 2, esterified hops biomass bound 33.4 mg Au/g of biomass, while native and hydrolyzed hops biomasses bound 28.2 and 12.0 mg Au/g of biomass, respectively. However, at pH 5 the binding capacities were 38.9, 37.8 and 11.4 mg of Au per gram of native, esterified and hydrolyzed hops biomasses, respectively. PMID:18365087

Binding Procurement

NASA Technical Reports Server (NTRS)

Rao, Gopalakrishna M.; Vaidyanathan, Hari

2007-01-01

This viewgraph presentation reviews the use of the binding procurement process in purchasing Aerospace Flight Battery Systems. NASA Engineering and Safety Center (NESC) requested NASA Aerospace Flight Battery Systems Working Group to develop a set of guideline requirements document for Binding Procurement Contracts.

Variation in energy expenditure among black-legged kittiwakes: Effects of activity-specific metabolic rates and activity budgets

USGS Publications Warehouse

Jodice, P.G.R.; Roby, D.D.; Suryan, R.M.; Irons, D.B.; Kaufman, A.M.; Turco, K.R.; Visser, G. Henk

2003-01-01

We sought to determine the effect of variation in time-activity budgets (TABs) and foraging behavior on energy expenditure rates of parent black-legged kittiwakes (Rissa tridactyla). We quantified TABs using direct observations of radio-tagged adults and simultaneously measured field metabolic rates (FMR) of these same individuals (n = 20) using the doubly labeled water technique. Estimated metabolic rates of kittiwakes attending their brood at the nest or loafing near the colony were similar (ca. 1.3 x basal metabolic rate [BMR]), although loafing during foraging trips was more costly (2.9 x BMR). Metabolic rates during commuting flight (7.3 x BMR) and prey-searching flight (6.2 x BMR) were similar, while metabolic rates during plunge diving were much higher (ca. 47 x BMR). The proportion of the measurement interval spent foraging had a positive effect on FMR (R2 = 0.68), while the combined proportion of time engaged in nest attendance and loafing near the colony had a negative effect on FMR (R2 = 0.72). Thus, more than two-thirds of the variation in kittiwake FMR could be explained by the allocation of time among various activities. The high energetic cost of plunge diving relative to straight flight and searching flight suggests that kittiwakes can optimize their foraging strategy under conditions of low food availability by commuting long distances to feed in areas where gross foraging efficiency is high.

Evolution of basal metabolic rate in bank voles from a multidirectional selection experiment.

PubMed

Sadowska, Edyta T; Stawski, Clare; Rudolf, Agata; Dheyongera, Geoffrey; Chrząścik, Katarzyna M; Baliga-Klimczyk, Katarzyna; Koteja, Paweł

2015-05-07

A major theme in evolutionary and ecological physiology of terrestrial vertebrates encompasses the factors underlying the evolution of endothermy in birds and mammals and interspecific variation of basal metabolic rate (BMR). Here, we applied the experimental evolution approach and compared BMR in lines of a wild rodent, the bank vole (Myodes glareolus), selected for 11 generations for: high swim-induced aerobic metabolism (A), ability to maintain body mass on a low-quality herbivorous diet (H) and intensity of predatory behaviour towards crickets (P). Four replicate lines were maintained for each of the selection directions and an unselected control (C). In comparison to C lines, A lines achieved a 49% higher maximum rate of oxygen consumption during swimming, H lines lost 1.3 g less mass in the test with low-quality diet and P lines attacked crickets five times more frequently. BMR was significantly higher in A lines than in C or H lines (60.8, 56.6 and 54.4 ml O2 h(-1), respectively), and the values were intermediate in P lines (59.0 ml O2 h(-1)). Results of the selection experiment provide support for the hypothesis of a positive association between BMR and aerobic exercise performance, but not for the association of adaptation to herbivorous diet with either a high or low BMR. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

Evaluation of galectin binding by frontal affinity chromatography (FAC).

PubMed

Iwaki, Jun; Hirabayashi, Jun

2015-01-01

Frontal affinity chromatography (FAC) is a simple and versatile procedure enabling quantitative determination of diverse biological interactions in terms of dissociation constants (K d), even though these interactions are relatively weak. The method is best applied to glycans and their binding proteins, with the analytical system operating on the basis of highly reproducible isocratic elution by liquid chromatography. Its application to galectins has been successfully developed to characterize their binding specificities in detail. As a result, their minimal requirements for recognition of disaccharides, i.e., β-galactosides, as well as characteristic features of individual galectins, have been elucidated. In this chapter, we describe standard procedures to determine the K d's for interactions between a series of standard glycans and various galectins.

Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site

PubMed Central

Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J.

2016-01-01

ABSTRACT Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. IMPORTANCE We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. PMID:26764003

Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site.

PubMed

Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J; Hogle, James M

2016-01-13

Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Engineering cofactor and ligand binding in an artificial neuroglobin

NASA Astrophysics Data System (ADS)

Zhang, Lei

HP-7 is one artificial mutated oxygen transport protein, which operates via a mechanism akin to human neuroglobin and cytoglobin. This protein destabilizes one of two heme-ligating histidine residues by coupling histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Replacement of these glutamate residues with alanine, which has a neutral hydrophobicity, slows gaseous ligand binding 22-fold, increases the affinity of the distal histidine ligand by a factor of thirteen, and decreases the binding affinity of carbon monoxide, a nonreactive oxygen analogue, three-fold. Paradoxically, it also decreases heme binding affinity by a factor of three in the reduced state and six in the oxidized state. Application of a two-state binding model, in which an initial pentacoordinate binding event is followed by a protein conformational change to hexacoordinate, provides insight into the mechanism of this seemingly counterintuitive result: the initial pentacoordinate encounter complex is significantly destabilized by the loss of the glutamate side chains, and the increased affinity for the distal histidine only partially compensates. These results point to the importance of considering each oxidation and conformational state in the design of functional artificial proteins. We have also examined the effects these mutations have on function. The K d of the nonnreactive oxygen analogue carbon monoxide (CO) is only decreased three-fold, despite the large increase in distal histidine affinity engendered by the 22-fold decrease in the histidine ligand off-rate. This is a result of the four-fold increase in affinity for CO binding to the pentacoordinate state. Oxygen binds to HP7 with a Kd of 117 µM, while the mutant rapidly oxidizes when exposed to oxygen. EPR analysis of both ferric hemoproteins demonstrates that the mutation increases disorder at the heme binding site. NMR-detected deuterium exchange demonstrates that the mutation causes a

To bind or not to bind? Different temporal binding effects from voluntary pressing and releasing actions.

PubMed

Zhao, Ke; Chen, Yu-Hsin; Yan, Wen-Jing; Fu, Xiaolan

2013-01-01

Binding effect refers to the perceptual attraction between an action and an outcome leading to a subjective compression of time. Most studies investigating binding effects exclusively employ the "pressing" action without exploring other types of actions. The present study addresses this issue by introducing another action, releasing action or the voluntary lifting of the finger/wrist, to investigate the differences between voluntary pressing and releasing actions. Results reveal that releasing actions led to robust yet short-lived temporal binding effects, whereas pressing condition had steady temporal binding effects up to super-seconds. The two actions also differ in sensitivity to changes in temporal contiguity and contingency, which could be attributed to the difference in awareness of action. Extending upon current models of "willed action," our results provide insights from a temporal point of view and support the concept of a dual system consisting of predictive motor control and top-down mechanisms.

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

Nuclear binding of progesterone in hen oviduct. Binding to multiple sites in vitro.

PubMed Central

Pikler, G M; Webster, R A; Spelsberg, T C

1976-01-01

Steroid hormones, including progesterone, are known to bind with high affinity (Kd approximately 1x10(-10)M) to receptor proteins once they enter target cells. This complex (the progesterone-receptor) then undergoes a temperature-and/or salt-dependent activation which allows it to migrate to the cell nucleus and to bind to the deoxyribonucleoproteins. The present studies demonstrate that binding the hormone-receptor complex in vitro to isolated nuclei from the oviducts of laying hens required the same conditions as do other studies of bbinding in vitro reported previously, e.g. the hormone must be complexed to intact and activated receptor. The assay of the nuclear binding by using multiple concentrations of progesterone receptor reveals the presence of more than one class of binding site in the oviduct nuclei. The affinity of each of these classes of binding sites range from Kd approximately 1x10(-9)-1x10(-8)M. Assays using free steroid (not complexed with receptor) show no binding to these sites. The binding to each of the classes of sites, displays a differential stability to increasing ionic concentrations, suggesting primarily an ionic-type interaction for all classes. Only the highest-affinity class of binding site is capable of binding progesterone receptor under physioligical-saline conditions. This class represent 6000-10000 sites per cell nucleus and resembles the sites detected in vivo (Spelsberg, 1976, Biochem. J. 156, 391-398) which cause maximal transcriptional response when saturated with the progesterone receptor. The multiple binding sites for the progesterone receptor either are not present or are found in limited numbers in the nuclei of non-target organs. Differences in extent of binding to the nuclear material between a target tissue (oviduct) and other tissues (spleen or erythrocyte) are markedly dependent on the ionic conditions, and are probably due to binding to different classes of sites in the nuclei. PMID:182147

Lactose-containing starburst dendrimers: influence of dendrimer generation and binding-site orientation of receptors (plant/animal lectins and immunoglobulins) on binding properties.

PubMed

André, S; Ortega, P J; Perez, M A; Roy, R; Gabius, H J

1999-11-01

Starburst glycodendrimers offer the potential to serve as high-affinity ligands for clinically relevant sugar receptors. In order to define areas of application, their binding behavior towards sugar receptors with differential binding-site orientation but identical monosaccharide specificity must be evaluated. Using poly(amidoamine) starburst dendrimers of five generations, which contain the p-isothiocyanato derivative of p-aminophenyl-beta-D-lactoside as ligand group, four different types of galactoside-binding proteins were chosen for this purpose, i.e., the (AB)(2)-toxic agglutinin from mistletoe, a human immunoglobulin G fraction, the homodimeric galectin-1 with its two binding sites at opposite ends of the jelly-roll-motif-harboring protein and monomeric galectin-3. Direct solid-phase assays with surface-immobilized glycodendrimers resulted in obvious affinity enhancements by progressive core branching for the plant agglutinin and less pronounced for the antibody and galectin-1. High density of binding of galectin-3 with modest affinity increases only from the level of the 32-mer onwards points to favorable protein-protein interactions of the monomeric lectin and a spherical display of the end groups without a major share of backfolding. When the inhibitory potency of these probes was evaluated as competitor of receptor binding to an immobilized neoglycoprotein or to asialofetuin, a marked selectivity was detected. The 32- and 64-mers were second to none as inhibitors for the plant agglutinin against both ligand-exposing matrices and for galectin-1 on the matrix with a heterogeneous array of interglycoside distances even on the per-sugar basis. In contrast, a neoglycoprotein with the same end group was superior in the case of the antibody and, less pronounced, monomeric galectin-3. Intimate details of topological binding-site presentation and the ligand display on different generations of core assembly are major operative factors which determine the potential

Comparison and correlation of binding mode of ATP in the kinase domains of Hexokinase family

PubMed Central

Kumar, Yellapu Nanda; Kumar, Pasupuleti Santhosh; Sowjenya, Gopal; Rao, Valasani Koteswara; Yeswanth, Sthanikam; Prasad, Uppu Venkateswara; Pradeepkiran, Jangampalli Adi; Sarma, PVGK; Bhaskar, Matcha

2012-01-01

Hexokinases (HKs) are the enzymes that catalyses the ATP dependent phosphorylation of Hexose sugars to Hexose-6-Phosphate (Hex-6-P). There exist four different forms of HKs namely HK-I, HK-II, HK-III and HK-IV and all of them share a common ATP binding site core surrounded by more variable sequence that determine substrate affinities. Although they share a common binding site but they differ in their kinetic functions, hence the present study is aimed to analyze the binding mode of ATP. The analysis revealed that the four ATP binding domains are showing 13 identical, 7 similar and 6 dissimilar residues with similar structural conformation. Molecular docking of ATP into the kinase domains using Molecular Operating Environment (MOE) soft ware tool clearly showed the variation in the binding mode of ATP with variable docking scores. This probably explains the variable phosphorylation rates among hexokinases family. PMID:22829728

In vitro selection of zinc fingers with altered DNA-binding specificity.

PubMed

Jamieson, A C; Kim, S H; Wells, J A

1994-05-17

We have used random mutagenesis and phage display to alter the DNA-binding specificity of Zif268, a transcription factor that contains three zinc finger domains. Four residues in the helix of finger 1 of Zif268 that potentially mediate DNA binding were identified from an X-ray structure of the Zif268-DNA complex. A library was constructed in which these residues were randomly mutated and the Zif268 variants were fused to a truncated version of the gene III coat protein on the surface of M13 filamentous phage particles. The phage displayed the mutant proteins in a monovalent fashion and were sorted by repeated binding and elution from affinity matrices containing different DNA sequences. When the matrix contained the natural nine base pair operator sequence 5'-GCG-TGG-GCG-3', native-like zinc fingers were isolated. New finger 1 variants were found by sorting with two different operators in which the singly modified triplets, GTG and TCG, replaced the native finger 1 triplet, GCG. Overall, the selected finger 1 variants contained a preponderance of polar residues at the four sites. Interestingly, the net charge of the four residues in any selected finger never derived more that one unit from neutrality despite the fact that about half the variants contained three or four charged residues over the four sites. Measurements of the dissociation constants for two of these purified finger 1 variants by gel-shift assay showed their specificities to vary over a 10-fold range, with the greatest affinity being for the DNA binding site for which they were sorted.(ABSTRACT TRUNCATED AT 250 WORDS)

Predicting protein-binding RNA nucleotides with consideration of binding partners.

PubMed

Tuvshinjargal, Narankhuu; Lee, Wook; Park, Byungkyu; Han, Kyungsook

2015-06-01

In recent years several computational methods have been developed to predict RNA-binding sites in protein. Most of these methods do not consider interacting partners of a protein, so they predict the same RNA-binding sites for a given protein sequence even if the protein binds to different RNAs. Unlike the problem of predicting RNA-binding sites in protein, the problem of predicting protein-binding sites in RNA has received little attention mainly because it is much more difficult and shows a lower accuracy on average. In our previous study, we developed a method that predicts protein-binding nucleotides from an RNA sequence. In an effort to improve the prediction accuracy and usefulness of the previous method, we developed a new method that uses both RNA and protein sequence data. In this study, we identified effective features of RNA and protein molecules and developed a new support vector machine (SVM) model to predict protein-binding nucleotides from RNA and protein sequence data. The new model that used both protein and RNA sequence data achieved a sensitivity of 86.5%, a specificity of 86.2%, a positive predictive value (PPV) of 72.6%, a negative predictive value (NPV) of 93.8% and Matthews correlation coefficient (MCC) of 0.69 in a 10-fold cross validation; it achieved a sensitivity of 58.8%, a specificity of 87.4%, a PPV of 65.1%, a NPV of 84.2% and MCC of 0.48 in independent testing. For comparative purpose, we built another prediction model that used RNA sequence data alone and ran it on the same dataset. In a 10 fold-cross validation it achieved a sensitivity of 85.7%, a specificity of 80.5%, a PPV of 67.7%, a NPV of 92.2% and MCC of 0.63; in independent testing it achieved a sensitivity of 67.7%, a specificity of 78.8%, a PPV of 57.6%, a NPV of 85.2% and MCC of 0.45. In both cross-validations and independent testing, the new model that used both RNA and protein sequences showed a better performance than the model that used RNA sequence data alone in

How aromatic compounds block DNA binding of HcaR catabolite regulator

DOE PAGES

Kim, Youngchang; Joachimiak, Grazyna; Bigelow, Lance; ...

2016-04-25

Bacterial catabolism of aromatic compounds from various sources including phenylpropanoids and flavonoids that are abundant in soil plays an important role in the recycling of carbon in the ecosystem. We have determined the crystal structures of apo-HcaR from Acinetobacter sp. ADP1, a MarR/SlyA transcription factor, in complexes with hydroxycinnamates and a specific DNA operator. The protein regulates the expression of the hca catabolic operon in Acinetobacter and related bacterial strains, allowing utilization of hydroxycinnamates as sole sources of carbon. HcaR binds multiple ligands, and as a result the transcription of genes encoding several catabolic enzymes is increased. The 1.9-2.4 Åmore » resolution structures presented here explain how HcaR recognizes four ligands (ferulate, 3,4-dihydroxybenzoate, p-coumarate, and vanillin) using the same binding site. The ligand promiscuity appears to be an adaptation to match a broad specificity of hydroxycinnamate catabolic enzymes while responding to toxic thioester intermediates. Structures of apo-HcaR and in complex with a specific DNA hca operator when combined with binding studies of hydroxycinnamates show how aromatic ligands render HcaR unproductive in recognizing a specific DNA target. Furthermore, the current study contributes to a better understanding of the hca catabolic operon regulation mechanism by the transcription factor HcaR.« less

Environmental Assessment for Ongoing and Future Operations at U.S. Navy Dabob Bay and Hood Canal Military Operating Areas

DTIC Science & Technology

2002-05-01

well as being cultivated in aquaculture operations in Puget Sound, including Hood Canal and Dabob Bay (Figure 3.4-2). Pacific oysters (Crassostrea...gigas) are widely cultivated in aquaculture operations in Puget Sound. Commercial oyster beds exist in Dabob Bay, mostly at the north end. Dabob Bay... Ecotoxicology of metals in aquatic sediments: binding and release, bioavailability, risk assessment, and remediation. Canadian Journal of

CaMELS: In silico prediction of calmodulin binding proteins and their binding sites.

PubMed

Abbasi, Wajid Arshad; Asif, Amina; Andleeb, Saiqa; Minhas, Fayyaz Ul Amir Afsar

2017-09-01

Due to Ca 2+ -dependent binding and the sequence diversity of Calmodulin (CaM) binding proteins, identifying CaM interactions and binding sites in the wet-lab is tedious and costly. Therefore, computational methods for this purpose are crucial to the design of such wet-lab experiments. We present an algorithm suite called CaMELS (CalModulin intEraction Learning System) for predicting proteins that interact with CaM as well as their binding sites using sequence information alone. CaMELS offers state of the art accuracy for both CaM interaction and binding site prediction and can aid biologists in studying CaM binding proteins. For CaM interaction prediction, CaMELS uses protein sequence features coupled with a large-margin classifier. CaMELS models the binding site prediction problem using multiple instance machine learning with a custom optimization algorithm which allows more effective learning over imprecisely annotated CaM-binding sites during training. CaMELS has been extensively benchmarked using a variety of data sets, mutagenic studies, proteome-wide Gene Ontology enrichment analyses and protein structures. Our experiments indicate that CaMELS outperforms simple motif-based search and other existing methods for interaction and binding site prediction. We have also found that the whole sequence of a protein, rather than just its binding site, is important for predicting its interaction with CaM. Using the machine learning model in CaMELS, we have identified important features of protein sequences for CaM interaction prediction as well as characteristic amino acid sub-sequences and their relative position for identifying CaM binding sites. Python code for training and evaluating CaMELS together with a webserver implementation is available at the URL: http://faculty.pieas.edu.pk/fayyaz/software.html#camels. © 2017 Wiley Periodicals, Inc.

Constitutive innate immunity is a component of the pace-of-life syndrome in tropical birds.

PubMed

Irene Tieleman, B; Williams, Joseph B; Ricklefs, Robert E; Klasing, Kirk C

2005-08-22

We studied the relationship between one component of immune function and basal metabolic rate (BMR), an indicator of the 'pace-of-life syndrome', among 12 tropical bird species and among individuals of the tropical house wren (Troglodytes aedon), to gain insights into functional connections between life history and physiology. To assess constitutive innate immunity we introduced a new technique in the field of ecological and evolutionary immunology that quantifies the bactericidal activity of whole blood. This in vitro assay utilises a single blood sample to provide a functional, integrated measure of constitutive innate immunity. We found that the bactericidal activity of whole blood varied considerably among species and among individuals within a species. This variation was not correlated with body mass or whole-organism BMR. However, among species, bacteria killing activity was negatively correlated with mass-adjusted BMR, suggesting that species with a slower pace-of-life have evolved a more robust constitutive innate immune capability. Among individuals of a single species, the house wren, bacteria killing activity was positively correlated with mass-adjusted BMR, pointing to physiological differences in individual quality on which natural selection potentially could act.

The Receptor Binding Domain of Botulinum Neurotoxin Stereotype C Binds Phosphoinositides

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

Zhang, Yanfeng; Varnum, Susan M.

2012-03-01

Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD50 of {approx} 1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a 'dual receptor' mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Here, using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro. BoNT/C-HCR was foundmore » to bind negatively charged phospholipids, preferentially phosphoinositides. Additional interactions to phosphoinositides may help BoNT/C bind membrane more tightly and transduct signals for subsequent steps of intoxication. Our results provide new insights into the mechanisms of host cell membrane recognition by BoNTs.« less

Neural Architecture for Feature Binding in Visual Working Memory.

PubMed

Schneegans, Sebastian; Bays, Paul M

2017-04-05

Binding refers to the operation that groups different features together into objects. We propose a neural architecture for feature binding in visual working memory that employs populations of neurons with conjunction responses. We tested this model using cued recall tasks, in which subjects had to memorize object arrays composed of simple visual features (color, orientation, and location). After a brief delay, one feature of one item was given as a cue, and the observer had to report, on a continuous scale, one or two other features of the cued item. Binding failure in this task is associated with swap errors, in which observers report an item other than the one indicated by the cue. We observed that the probability of swapping two items strongly correlated with the items' similarity in the cue feature dimension, and found a strong correlation between swap errors occurring in spatial and nonspatial report. The neural model explains both swap errors and response variability as results of decoding noisy neural activity, and can account for the behavioral results in quantitative detail. We then used the model to compare alternative mechanisms for binding nonspatial features. We found the behavioral results fully consistent with a model in which nonspatial features are bound exclusively via their shared location, with no indication of direct binding between color and orientation. These results provide evidence for a special role of location in feature binding, and the model explains how this special role could be realized in the neural system. SIGNIFICANCE STATEMENT The problem of feature binding is of central importance in understanding the mechanisms of working memory. How do we remember not only that we saw a red and a round object, but that these features belong together to a single object rather than to different objects in our environment? Here we present evidence for a neural mechanism for feature binding in working memory, based on encoding of visual

New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein

PubMed Central

Hoffmann, Andreas; Kovermann, Michael; Lilie, Hauke; Fiedler, Markus; Balbach, Jochen; Rudolph, Rainer; Pfeifer, Sven

2012-01-01

A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF)-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNF-alpha trimer. Using NMR spectroscopy and other analytical methods the 1∶3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins – designed ankyrin repeat proteins – without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNF-alpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies. PMID:22363609

Hypophysectomy eliminates and growth hormone (GH) maintains the midpregnancy elevation in GH receptor and serum binding protein in the mouse

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

Sanchez-Jimenez, F.; Fielder, P.J.; Martinez, R.R.

1990-02-01

({sup 125}I)Iodomouse GH (({sup 125}I)iodo-mGH) binding to samples of serum and hepatic microsomal membranes was measured in hypophysectomized pregnant, sham-operated pregnant, intact pregnant, and intact adult virgin mice. Surgeries were carried out on day 11 of pregnancy, and the animals were killed on day 14. The binding of mGH to both serum and hepatic microsomal membranes of intact virgin mice was much lower than to those of intact pregnant mice. In hypophysectomized mice, the mGH-binding capacity of both serum and hepatic microsomes decreased to values similar to those of nonpregnant mice. No significant differences were observed between intact and sham-operatedmore » pregnant animals in the maternal serum mGH concentration, the serum GH-binding protein concentration, or the hepatic GH receptor concentration. GH receptor and binding protein-encoding mRNAs were also higher in intact and sham-operated pregnant mice than in virgin and hypophysectomized mice. Hypophysectomized mice were treated with 200 micrograms/day bovine GH, administered by osmotic minipump; after 3 days of treatment, a significant elevation of hepatic GH receptor and serum GH-binding protein levels was observed. These results demonstrate an up-regulation of hepatic GH receptors and serum GH-binding protein by GH during pregnancy in the mouse.« less

Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding

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

Stenmark, Pål; Dong, Min; Dupuy, Jérôme

2011-11-02

Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-{angstrom} X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantlymore » reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.« less

Crystal structure of the botulinum neurotoxin type G binding domain: insight into cell surface binding.

PubMed

Stenmark, Pål; Dong, Min; Dupuy, Jérôme; Chapman, Edwin R; Stevens, Raymond C

2010-04-16

Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-A X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent. Copyright (c) 2010. Published by Elsevier Ltd.

Association of the Pro12Ala polymorphism in peroxisome proliferator-activated receptor gamma2 with decreased basic metabolic rate in women with polycystic ovary syndrome.

PubMed

Koika, Vasiliki; Marioli, Dimitra J; Saltamavros, Alexandros D; Vervita, Vasiliki; Koufogiannis, Kleanthis D; Adonakis, George; Decavalas, George; Georgopoulos, Neoklis A

2009-08-01

The peroxisome proliferator-activated receptor (PPAR)gamma is a transcription factor involved in glucose homeostasis and energy metabolism. A missense mutation at codon 12 in the PPARgamma2 has been associated with increased body mass index (BMI) and attenuated insulin resistance (IR) in polycystic ovary syndrome (PCOS). We have recently shown a decreased basic metabolic rate (BMR) in PCOS. The aim of the present study is to determine the prevalence of the Pro12Ala polymorphism of the PPARgamma2 gene and its associations with indices of IR and BMR in lean and slightly overweight PCOS women. Case-control association study involving 156 PCOS women with biochemical hyperandrogenism, chronic anovulation and polycystic ovarian morphology in ultrasound and 56 unrelated healthy controls. Hormonal determinations were performed by electrochemiluminescence quantitation or RIA. BMR was measured by indirect calorimetry. All subjects were genotyped by a PCR-restriction fragment length polymorphism assay. Genotype frequencies of the Pro12Ala polymorphism in PPARgamma2 did not differ among PCOS women and control subjects. The presence of Pro12Ala polymorphism of PPARgamma2 was associated with lower BMR (P=0.04). This finding was valid in our subgroup of lean PCOS (BMI<25 kg/m(2)), in which the Ala variant was also associated with higher total testosterone values. The Pro12Ala polymorphism in the PPARgamma2 gene is associated with decreased BMR in women with PCOS and biochemical hyperandrogenemia. These young women are therefore at risk to increase their body weight and should restrict their energy intake by diet and enhance their energy expenditure by exercise.

Wrestlers' minimal weight: anthropometry, bioimpedance, and hydrostatic weighing compared.

PubMed

Oppliger, R A; Nielsen, D H; Vance, C G

1991-02-01

The need for accurate assessment of minimal wrestling weight among interscholastic wrestlers has been well documented. Previous research has demonstrated the validity of anthropometric methods for this purpose, but little research has examined the validity of bioelectrical impedance (BIA) measurements. Comparisons between BIA systems has received limited attention. With these two objectives, we compared the prediction of minimal weight (MW) among 57 interscholastic wrestlers using three anthropometric methods (skinfolds (SF) and two skeletal dimensions equations) and three BIA systems (Berkeley Medical Research (BMR), RJL, and Valhalla (VAL]. All methods showed high correlations (r values greater than 0.92) with hydrostatic weighting (HW) and between methods (r values greater than 0.90). The standard errors of estimate (SEE) were relatively small for all methods, especially for SF and the three BIA systems (SEE less than 0.70 kg). The total errors of prediction (E) for RJL and VAL (E = 4.4 and 3.9 kg) were significantly larger than observed nonsignificant BMR and SF values (E = 2.3 and 1.8 kg, respectively). Significant mean differences were observed between HW, RJL, VAL, and the two skeletal dimensions equations, but nonsignificant differences were observed between HW, BMR, and SF. BMR differed significantly from the RJL and VAL systems. The results suggest that RJL and VAL have potential application for this subpopulation. Prediction equation refinement with the addition of selected anthropometric measurement or moderating variables may enhance their utility. However, within the scope of our study, SF and BMR BIA appear to be the most valid methods for determining MW in interscholastic wrestlers.

Body weight and basal metabolic rate in childhood narcolepsy: a longitudinal study.

PubMed

Wang, Zongwen; Wu, Huijuan; Stone, William S; Zhuang, Jianhua; Qiu, Linli; Xu, Xing; Wang, Yan; Zhao, Zhengqing; Han, Fang; Zhao, Zhongxin

2016-09-01

The aims of this study were to document the trajectory of weight gain and body mass index (BMI) in children with type 1 narcolepsy, and to analyze basal metabolic rate (BMR). A total of 65 Chinese children with type 1 narcolepsy with a disease duration ≤12 months were included. In addition, 79 healthy age-matched students were enrolled as controls. Height and body weight were measured every six months for up to 36 months to calculate BMI growth. BMR was measured using COSMED K4b2 indirect calorimetry in 34 patients and 30 healthy controls at six months. At the end of 36 months, the BMR was compared among 18 patients and 16 healthy controls. The children with type 1 narcolepsy showed higher BMIs at follow-up assessments. At the end of the study, 38.46% of the patients were obese and an additional 26.15% were overweight. The patients' BMI growth at six, 12, 18, 24 and 30 months of follow-up was significantly higher, but not at month 36. The patients' basal energy expenditure was significantly lower than that of the controls at six months but not at 36 months. BMI increased rapidly in children with type 1 narcolepsy after disease onset, but BMI growth decreased gradually with prolonged disease. Decreased BMR is an important cause underlying rapid weight gain. The gradual restoration of BMI growth and BMR in narcolepsy emphasizes the importance of compensatory metabolic mechanisms in this disease. Copyright © 2016 Elsevier B.V. All rights reserved.

Human sex hormone-binding globulin binding affinities of 125 structurally diverse chemicals and comparison with their binding to androgen receptor, estrogen receptor, and α-fetoprotein.

PubMed

Hong, Huixiao; Branham, William S; Ng, Hui Wen; Moland, Carrie L; Dial, Stacey L; Fang, Hong; Perkins, Roger; Sheehan, Daniel; Tong, Weida

2015-02-01

One endocrine disruption mechanism is through binding to nuclear receptors such as the androgen receptor (AR) and estrogen receptor (ER) in target cells. The concentration of a chemical in serum is important for its entry into the target cells to bind the receptors, which is regulated by the serum proteins. Human sex hormone-binding globulin (SHBG) is the major transport protein in serum that can bind androgens and estrogens and thus change a chemical's availability to enter the target cells. Sequestration of an androgen or estrogen in the serum can alter the chemical elicited AR- and ER-mediated responses. To better understand the chemical-induced endocrine activity, we developed a competitive binding assay using human pregnancy plasma and measured the binding to the human SHBG for 125 structurally diverse chemicals, most of which were known to bind AR and ER. Eighty seven chemicals were able to bind the human SHBG in the assay, whereas 38 chemicals were nonbinders. Binding data for human SHBG are compared with that for rat α-fetoprotein, ER and AR. Knowing the binding profiles between serum and nuclear receptors will improve assessment of a chemical's potential for endocrine disruption. The SHBG binding data reported here represent the largest data set of structurally diverse chemicals tested for human SHBG binding. Utilization of the SHBG binding data with AR and ER binding data could enable better evaluation of endocrine disrupting potential of chemicals through AR- and ER-mediated responses since sequestration in serum could be considered. Published by Oxford University Press on behalf of the Society of Toxicology 2014. This work is written by US Government employees and is in the public domain in the US.

Structural changes in calcium-binding allergens: use of circular dichroism to study binding characteristics.

PubMed

Hebenstreit, D; Ferreira, F

2005-09-01

Several studies showed that calcium-binding proteins have a fixed place in the spectrum of allergenic substances. Often the binding of a calcium ion induces conformational changes and affects immunoglobulin E-binding to the allergen. Hence, the quantitative characterization of the binding to calcium is of importance to understand both the biologic and allergenic activity of these proteins. In the present study we describe a procedure for determining the stoichiometry and dissociation constant (K(D)) of calcium-binding allergens using circular dichroism (CD) techniques. For the experiments, we used recombinant Bet v 4, a two EF-hand allergen from birch pollen. Solutions of Bet v 4 were titrated with calcium and the change in molar ellipticity at 222 nm was monitored with a CD spectropolarimeter. The determination of the binding stoichiometry as well as of the K(D) for one EF-hand (4 microM) demonstrated the applicability of the method. CD-monitored calcium-titration of protein solutions represents a fast and easy method for determining the binding characteristics of calcium-binding allergens.

A phylogenetic analysis of basal metabolism, total evaporative water loss, and life-history among foxes from desert and mesic regions.

PubMed

Williams, J B; Muñoz-Garcia, A; Ostrowski, S; Tieleman, B I

2004-01-01

We measured basal metabolic rate (BMR) and total evaporative water loss (TEWL) of species of foxes that exist on the Arabian Peninsula, Blanford's fox (Vulpes cana) and two subspecies of Red fox (Vulpes vulpes). Combining these data with that on other canids from the literature, we searched for specialization of physiological traits among desert foxes using both conventional least squares regression and regressions based on phylogenetic independent contrasts. Further, we explored the consequences of reduced body size of foxes on life history parameters such as litter size and neonate mass. For Blanford's foxes, Red foxes from the central desert of Arabia, and Red foxes from the more mesic Asir mountains, body mass averaged 1,285 +/- 52 g, 1,967 +/- 289 g, and 3,060 +/- 482 g, respectively, whereas mean BMR, during summer, was 304.5 +/- 32.3 kJ/day, 418.0 +/- 32.4 kJ/day, and 724.1 +/- 120.2 kJ/day (+/- SD). An analysis of covariance with body mass as a covariate showed no statistical differences in BMR among foxes. Analysis of covariance indicated that Red fox from the Asir mountains had a higher TEWL than Red foxes from central Arabia or than Blanford's foxes also from the mountains. Comparisons of all species of desert and mesic foxes showed no significant differences in BMR, nor did desert foxes have a significantly lower BMR than other carnivores. TEWL of desert foxes was lower than other more mesic carnivores; deviations in TEWL ranged from -17.7% for the Fennec fox (Fennecus zerda) to -57.4% for the Kit fox (Vulpes velox). Although desert foxes have a BMR comparable to other more mesic species, it appears that desert foxes do have a smaller body mass, lowering overall energy requirements. We attribute this reduction in body size to the "resource limitation hypothesis" whereby natural selection favors smaller individuals in a resource-limited environment, especially during periods of severe food shortage. However, until common garden experiments are performed

Recent improvements to Binding MOAD: a resource for protein–ligand binding affinities and structures

PubMed Central

Ahmed, Aqeel; Smith, Richard D.; Clark, Jordan J.; Dunbar, James B.; Carlson, Heather A.

2015-01-01

For over 10 years, Binding MOAD (Mother of All Databases; http://www.BindingMOAD.org) has been one of the largest resources for high-quality protein–ligand complexes and associated binding affinity data. Binding MOAD has grown at the rate of 1994 complexes per year, on average. Currently, it contains 23 269 complexes and 8156 binding affinities. Our annual updates curate the data using a semi-automated literature search of the references cited within the PDB file, and we have recently upgraded our website and added new features and functionalities to better serve Binding MOAD users. In order to eliminate the legacy application server of the old platform and to accommodate new changes, the website has been completely rewritten in the LAMP (Linux, Apache, MySQL and PHP) environment. The improved user interface incorporates current third-party plugins for better visualization of protein and ligand molecules, and it provides features like sorting, filtering and filtered downloads. In addition to the field-based searching, Binding MOAD now can be searched by structural queries based on the ligand. In order to remove redundancy, Binding MOAD records are clustered in different families based on 90% sequence identity. The new Binding MOAD, with the upgraded platform, features and functionalities, is now equipped to better serve its users. PMID:25378330

Regulation of nif expression in Methanococcus maripaludis: roles of the euryarchaeal repressor NrpR, 2-oxoglutarate, and two operators.

PubMed

Lie, Thomas J; Wood, Gwendolyn E; Leigh, John A

2005-02-18

The methanogenic archaean Methanococcus maripaludis can use ammonia, alanine, or dinitrogen as a nitrogen source for growth. The euryarchaeal nitrogen repressor NrpR controls the expression of the nif (nitrogen fixation) operon, resulting in full repression with ammonia, intermediate repression with alanine, and derepression with dinitrogen. NrpR binds to two tandem operators in the nif promoter region, nifOR(1) and nifOR(2). Here we have undertaken both in vivo and in vitro approaches to study the way in which NrpR, nifOR(1), nifOR(2), and the effector 2-oxoglutarate (2OG) combine to regulate nif expression, leading to a comprehensive understanding of this archaeal regulatory system. We show that NrpR binds as a dimer to nifOR(1) and cooperatively as two dimers to both operators. Cooperative binding occurs only with both operators present. nifOR(1) has stronger binding and by itself can mediate the repression of nif transcription during growth on ammonia, unlike the weakly binding nifOR(2). However, nifOR(2) in combination with nifOR(1) is critical for intermediate repression during growth on alanine. Accordingly, NrpR binds to both operators together with higher affinity than to nifOR(1) alone. NrpR responds directly to 2OG, which weakens its binding to the operators. Hence, 2OG is an intracellular indicator of nitrogen deficiency and acts as an inducer of nif transcription via NrpR. This model is upheld by the recent finding (J. A. Dodsworth and J. A. Leigh, submitted for publication) in our laboratory that 2OG levels in M. maripaludis vary with growth on different nitrogen sources.

Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease-associated ATP Binding Cassette (ABC) Transporter, ABCA4*

PubMed Central

Biswas-Fiss, Esther E.; Affet, Stephanie; Ha, Malissa; Biswas, Subhasis B.

2012-01-01

The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport. PMID:23144455

Energetic cost of standard activities in Gurkha and British soldiers.

PubMed

Strickland, S S; Ulijaszek, S J

1990-01-01

Measurements of basal metabolic rate and energy expenditure at lying, sitting, standing, and performing a step test at four levels of exercise, were made on Gurkha soldiers stationed in Britain and on British controls matched by body weight and occupational background. There was no significant difference in basal metabolic rate (BMR), nor in the energy cost of lying, sitting and standing between the two groups. Gurhas showed significantly lower gross and net energy expenditure, and so significantly greater net mechanical efficiency, at the lower levels of step exercise. The ratio of gross energy expenditure to BMR was lower in Gurkhas at the lowest rates of stepping compared with the British controls. These results suggest that the energy cost of some physical activities expressed as multiples of BMR may not be constant across populations.

Prediction of Nucleotide Binding Peptides Using Star Graph Topological Indices.

PubMed

Liu, Yong; Munteanu, Cristian R; Fernández Blanco, Enrique; Tan, Zhiliang; Santos Del Riego, Antonino; Pazos, Alejandro

2015-11-01

The nucleotide binding proteins are involved in many important cellular processes, such as transmission of genetic information or energy transfer and storage. Therefore, the screening of new peptides for this biological function is an important research topic. The current study proposes a mixed methodology to obtain the first classification model that is able to predict new nucleotide binding peptides, using only the amino acid sequence. Thus, the methodology uses a Star graph molecular descriptor of the peptide sequences and the Machine Learning technique for the best classifier. The best model represents a Random Forest classifier based on two features of the embedded and non-embedded graphs. The performance of the model is excellent, considering similar models in the field, with an Area Under the Receiver Operating Characteristic Curve (AUROC) value of 0.938 and true positive rate (TPR) of 0.886 (test subset). The prediction of new nucleotide binding peptides with this model could be useful for drug target studies in drug development. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

RNA binding protein and binding site useful for expression of recombinant molecules

DOEpatents

Mayfield, Stephen P.

2006-10-17

The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

RNA binding protein and binding site useful for expression of recombinant molecules

DOEpatents

Mayfield, Stephen

2000-01-01

The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

Recent improvements to Binding MOAD: a resource for protein-ligand binding affinities and structures.

PubMed

Ahmed, Aqeel; Smith, Richard D; Clark, Jordan J; Dunbar, James B; Carlson, Heather A

2015-01-01

For over 10 years, Binding MOAD (Mother of All Databases; http://www.BindingMOAD.org) has been one of the largest resources for high-quality protein-ligand complexes and associated binding affinity data. Binding MOAD has grown at the rate of 1994 complexes per year, on average. Currently, it contains 23,269 complexes and 8156 binding affinities. Our annual updates curate the data using a semi-automated literature search of the references cited within the PDB file, and we have recently upgraded our website and added new features and functionalities to better serve Binding MOAD users. In order to eliminate the legacy application server of the old platform and to accommodate new changes, the website has been completely rewritten in the LAMP (Linux, Apache, MySQL and PHP) environment. The improved user interface incorporates current third-party plugins for better visualization of protein and ligand molecules, and it provides features like sorting, filtering and filtered downloads. In addition to the field-based searching, Binding MOAD now can be searched by structural queries based on the ligand. In order to remove redundancy, Binding MOAD records are clustered in different families based on 90% sequence identity. The new Binding MOAD, with the upgraded platform, features and functionalities, is now equipped to better serve its users. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Covalent attachment of Arc repressor subunits by a peptide linker enhances affinity for operator DNA.

PubMed

Robinson, C R; Sauer, R T

1996-01-09

By designing a recombinant gene containing tandem copies of the arc coding sequence with intervening DNA encoding the linker sequence GGGSGGGTGGGSGGG, the two subunits of the P22 Are repressor dimer have been covalently linked to form a single-chain protein called Arc-L1-Arc. The 15-residue linker joins the C-terminus of one monomer to the N-terminus of the second, a distance of approximately 45 A in the Arc-operator cocrystal structure. Arc-L1-Arc is expressed at high levels in Escherichia coli, with no evidence of degradation or proteolytic clipping of the linker, and is more active than wild-type Arc in repression assays. The purified Arc-L1-Arc protein has the molecular weight expected for the designed protein and unfolds cooperatively, reversibly, and with no concentration dependence in thermal-denaturation studies. Arc-L1-Arc protects operator DNA in a manner indistinguishable from that of wild-type Arc in DNase I and copper-phenanthroline footprinting studies, but the covalent attachment of the two monomers results in enhanced affinity for operator DNA. Arc-L1-Arc binds operator DNA half-maximally at a concentration of 1.7 pM, compared with the wild-type value of 185 pM, and also binds DNA fragments containing the left or right operator half-sites more tightly than wild type. Because wild-type Arc is monomeric at sub-nanomolar concentrations and must dimerize before binding to the operator, it was anticipated that Arc-L1-Arc would exhibit a lower half-maximal binding concentration. However, even when the change from a monomeric to a dimeric species is taken into account, the affinity of Arc-L1-Arc for operator and half-operator DNA is greater than the wild-type affinity. This tighter binding appears to result from slower dissociation, as Arc-L1-Arc DNA complexes with full or half-site operators dissociate at rates 5-10 times slower than the corresponding Arc--DNA complexes. Hence, the activity of the designed Arc-L1-Arc protein is substantially increased

Data to hardware binding with physical unclonable functions

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

Hamlet, Jason

The various technologies presented herein relate to binding data (e.g., software) to hardware, wherein the hardware is to utilize the data. The generated binding can be utilized to detect whether at least one of the hardware or the data has been modified between an initial moment (enrollment) and a later moment (authentication). During enrollment, an enrollment value is generated that includes a signature of the data, a first response from a PUF located on the hardware, and a code word. During authentication, a second response from the PUF is utilized to authenticate any of the content in the enrollment value,more » and based upon the authentication, a determination can be made regarding whether the hardware and/or the data have been modified. If modification is detected then a mitigating operation can be performed, e.g., the hardware is prevented from utilizing the data. If no modification is detected, the data can be utilized.« less

Non-equilibrium repressor binding kinetics link DNA damage dose to transcriptional timing within the SOS gene network.

PubMed

Culyba, Matthew J; Kubiak, Jeffrey M; Mo, Charlie Y; Goulian, Mark; Kohli, Rahul M

2018-06-01

Biochemical pathways are often genetically encoded as simple transcription regulation networks, where one transcription factor regulates the expression of multiple genes in a pathway. The relative timing of each promoter's activation and shut-off within the network can impact physiology. In the DNA damage repair pathway (known as the SOS response) of Escherichia coli, approximately 40 genes are regulated by the LexA repressor. After a DNA damaging event, LexA degradation triggers SOS gene transcription, which is temporally separated into subsets of 'early', 'middle', and 'late' genes. Although this feature plays an important role in regulating the SOS response, both the range of this separation and its underlying mechanism are not experimentally defined. Here we show that, at low doses of DNA damage, the timing of promoter activities is not separated. Instead, timing differences only emerge at higher levels of DNA damage and increase as a function of DNA damage dose. To understand mechanism, we derived a series of synthetic SOS gene promoters which vary in LexA-operator binding kinetics, but are otherwise identical, and then studied their activity over a large dose-range of DNA damage. In distinction to established models based on rapid equilibrium assumptions, the data best fit a kinetic model of repressor occupancy at promoters, where the drop in cellular LexA levels associated with higher doses of DNA damage leads to non-equilibrium binding kinetics of LexA at operators. Operators with slow LexA binding kinetics achieve their minimal occupancy state at later times than operators with fast binding kinetics, resulting in a time separation of peak promoter activity between genes. These data provide insight into this remarkable feature of the SOS pathway by demonstrating how a single transcription factor can be employed to control the relative timing of each gene's transcription as a function of stimulus dose.

Exploring the sequence-function relationship in transcriptional regulation by the lac O1 operator.

PubMed

Maity, Tuhin S; Jha, Ramesh K; Strauss, Charlie E M; Dunbar, John

2012-07-01

Understanding how binding of a transcription factor to an operator is influenced by the operator sequence is an ongoing quest. It facilitates discovery of alternative binding sites as well as tuning of transcriptional regulation. We investigated the behavior of the Escherichia coli Lac repressor (LacI) protein with a large set of lac O(1) operator variants. The 114 variants examined contained a mean of 2.9 (range 0-4) mutations at positions -4, -2, +2 and +4 in the minimally required 17 bp operator. The relative affinity of LacI for the operators was examined by quantifying expression of a GFP reporter gene and Rosetta structural modeling. The combinations of mutations in the operator sequence created a wide range of regulatory behaviors. We observed variations in the GFP fluorescent signal among the operator variants of more than an order of magnitude under both uninduced and induced conditions. We found that a single nucleotide change may result in changes of up to six- and 12-fold in uninduced and induced GFP signals, respectively. Among the four positions mutated, we found that nucleotide G at position -4 is strongly correlated with strong repression. By Rosetta modeling, we found a significant correlation between the calculated binding energy and the experimentally observed transcriptional repression strength for many operators. However, exceptions were also observed, underscoring the necessity for further improvement in biophysical models of protein-DNA interactions. © 2012 The Authors Journal compilation © 2012 FEBS.

Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

NASA Technical Reports Server (NTRS)

Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

2003-01-01

Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

Impaired visual search in rats reveals cholinergic contributions to feature binding in visuospatial attention.

PubMed

Botly, Leigh C P; De Rosa, Eve

2012-10-01

The visual search task established the feature integration theory of attention in humans and measures visuospatial attentional contributions to feature binding. We recently demonstrated that the neuromodulator acetylcholine (ACh), from the nucleus basalis magnocellularis (NBM), supports the attentional processes required for feature binding using a rat digging-based task. Additional research has demonstrated cholinergic contributions from the NBM to visuospatial attention in rats. Here, we combined these lines of evidence and employed visual search in rats to examine whether cortical cholinergic input supports visuospatial attention specifically for feature binding. We trained 18 male Long-Evans rats to perform visual search using touch screen-equipped operant chambers. Sessions comprised Feature Search (no feature binding required) and Conjunctive Search (feature binding required) trials using multiple stimulus set sizes. Following acquisition of visual search, 8 rats received bilateral NBM lesions using 192 IgG-saporin to selectively reduce cholinergic afferentation of the neocortex, which we hypothesized would selectively disrupt the visuospatial attentional processes needed for efficient conjunctive visual search. As expected, relative to sham-lesioned rats, ACh-NBM-lesioned rats took significantly longer to locate the target stimulus on Conjunctive Search, but not Feature Search trials, thus demonstrating that cholinergic contributions to visuospatial attention are important for feature binding in rats.

S-adenosylmethionine directly inhibits binding of 30S ribosomal subunits to the SMK box translational riboswitch RNA

PubMed Central

Fuchs, Ryan T.; Grundy, Frank J.; Henkin, Tina M.

2007-01-01

The SMK box is a conserved riboswitch motif found in the 5′ untranslated region of metK genes [encoding S-adenosylmethionine (SAM) synthetase] in lactic acid bacteria, including Enterococcus, Streptococcus, and Lactococcus sp. Previous studies showed that this RNA element binds SAM in vitro, and SAM binding causes a structural rearrangement that sequesters the Shine–Dalgarno (SD) sequence by pairing with an anti-SD (ASD) element. A model was proposed in which SAM binding inhibits metK translation by preventing binding of the ribosome to the SD region of the mRNA. In the current work, the addition of SAM was shown to inhibit binding of 30S ribosomal subunits to SMK box RNA; in contrast, the addition of S-adenosylhomocysteine (SAH) had no effect. A mutant RNA, which has a disrupted SD-ASD pairing, was defective in SAM binding and showed no reduction of ribosome binding in the presence of SAM, whereas a compensatory mutation that restored SD-ASD pairing restored the response to SAM. Primer extension inhibition assays provided further evidence for SD-ASD pairing in the presence of SAM. These results strongly support the model that SMK box translational repression operates through occlusion of the ribosome binding site and that SAM binding requires the SD-ASD pairing. PMID:17360376

Synthetic heparin-binding factor analogs

DOEpatents

Pena, Louis A [Poquott, NY; Zamora, Paul O [Gaithersburg, MD; Lin, Xinhua [Plainview, NY; Glass, John D [Shoreham, NY

2010-04-20

The invention provides synthetic heparin-binding growth factor analogs having at least one peptide chain, and preferably two peptide chains branched from a dipeptide branch moiety composed of two trifunctional amino acid residues, which peptide chain or chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a linker, which may be a hydrophobic linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

Spatial Analysis and Quantification of the Thermodynamic Driving Forces in Protein-Ligand Binding: Binding Site Variability

PubMed Central

Raman, E. Prabhu; MacKerell, Alexander D.

2015-01-01

The thermodynamic driving forces behind small molecule-protein binding are still not well understood, including the variability of those forces associated with different types of ligands in different binding pockets. To better understand these phenomena we calculate spatially resolved thermodynamic contributions of the different molecular degrees of freedom for the binding of propane and methanol to multiple pockets on the proteins Factor Xa and p38 MAP kinase. Binding thermodynamics are computed using a statistical thermodynamics based end-point method applied on a canonical ensemble comprising the protein-ligand complexes and the corresponding free states in an explicit solvent environment. Energetic and entropic contributions of water and ligand degrees of freedom computed from the configurational ensemble provides an unprecedented level of detail into the mechanisms of binding. Direct protein-ligand interaction energies play a significant role in both non-polar and polar binding, which is comparable to water reorganization energy. Loss of interactions with water upon binding strongly compensates these contributions leading to relatively small binding enthalpies. For both solutes, the entropy of water reorganization is found to favor binding in agreement with the classical view of the “hydrophobic effect”. Depending on the specifics of the binding pocket, both energy-entropy compensation and reinforcement mechanisms are observed. Notable is the ability to visualize the spatial distribution of the thermodynamic contributions to binding at atomic resolution showing significant differences in the thermodynamic contributions of water to the binding of propane versus methanol. PMID:25625202

Selected wheat seed defense proteins exhibit competitive binding to model microbial lipid interfaces.

PubMed

Sanders, Michael R; Clifton, Luke A; Neylon, Cameron; Frazier, Richard A; Green, Rebecca J

2013-07-17

Puroindolines (Pins) and purothionins (Pths) are basic, amphiphilic, cysteine-rich wheat proteins that play a role in plant defense against microbial pathogens. This study examined the co-adsorption and sequential addition of Pins (Pin-a, Pin-b, and a mutant form of Pin-b with Trp-44 to Arg-44 substitution) and β-purothionin (β-Pth) model anionic lipid layers using a combination of surface pressure measurements, external reflection FTIR spectroscopy, and neutron reflectometry. Results highlighted differences in the protein binding mechanisms and in the competitive binding and penetration of lipid layers between respective Pins and β-Pth. Pin-a formed a blanket-like layer of protein below the lipid surface that resulted in the reduction or inhibition of β-Pth penetration of the lipid layer. Wild-type Pin-b participated in co-operative binding with β-Pth, whereas the mutant Pin-b did not bind to the lipid layer in the presence of β-Pth. The results provide further insight into the role of hydrophobic and cationic amino acid residues in antimicrobial activity.

Effects of helium/oxygen and temperature on aerobic metabolism in the marsupial sugar glider, Petaurus breviceps.

PubMed

Holloway, J C; Geiser, F

2001-01-01

Helox (79% helium and 21% oxygen) has often been used for thermobiological studies, primarily because helium is thought to be metabolically inert and to produce no adverse effects other than increasing heat loss. However, these assumptions have been questioned. As basal metabolic rate (BMR) represents maintenance energy requirements for vital body functions, potential physiological effects of helox should be reflected in changes of BMR. In this study, sugar gliders were subjected to both air and helox atmospheres over a wide range of T(a)'s, including the thermoneutral zone (TNZ), to determine (1) whether helox has any influence other than on heat loss and (2) the maximum heat production (HP(max)) and thermal limits of this species. Although thermal conductance in the TNZ increased in helox, BMR was similar in air and helox (0.55+/-0.07 and 0.57+/-0.06 mL g(-1) h(-1), respectively). The TNZ in helox, however, was shifted upwards by about 3 degrees C. Below the TNZ, sugar gliders were able to withstand an effective temperature of -24.7+/-7.3 degrees C with an HP(max) of 3.14+/-0.36 mL g(-1) h(-1). The low effective temperature tolerated by sugar gliders shows that they are competent thermoregulators despite their apparent lack of functional brown fat. Similarities of BMRs in air and helox suggest that the effect of helox is restricted to an increase of heat loss, and, consequently, helox represents a useful tool for thermal physiologists. Moreover, the lack of increase of BMR in helox despite an increase in thermal conductance of sugar gliders suggests that BMR is not a function of body surface.

[Effect of two hypocaloric diets and their combination with physical exercise on basal metabolic rate and body composition].

PubMed

Bonfanti, Noelia; Fernández, Juan Marcelo; Gomez-Delgado, Francisco; Pérez-Jiménez, Francisco

2014-03-01

Metabolic syndrome (MetS) is diagnosed by the detection of at least three criteria (hypertriglyceridemia, low HDL-C, hypertension, obesity and altered fasting glucose). Visceral fat excess would be the starting point for its development. Scientific evidence supports hypocaloric diets -mediterranean or low fat diet and rich in complex carbohydrates diet included- as the best treatment to reduce fat mass (FM), maximizing its impact by combining them with physical exercise (PE). However, the effects of these treatments on basal metabolic rate (BMR) of patients with MetS, are unknown. To study the effect of the hypocaloric diet - mediterranean or low fat diet- with or without PE on the BMR and body composition (BC) of adults with MetS. 36 volunteers, MetS, both sexes, > 50 years, meeting the inclusion criteria. They were randomly assigned to a group of intervention (3 months) of hypocaloric diet: mediterranean diet (MED), low fat and rich in complex carbohydrates diet (CHO) and both combined with PE (MEDE and CHOE respectively). Anthropometric data was taken (weight, muscle mass (MM) and FM) and BMR was determined by indirect calorimetry, before and after intervention. The addition of PE to both hypocaloric treatments produced greater FM loss and weight loss than dieting alone, being this loss in CHOE > MEDE (p < 0.05). These groups decreased the BMR after treatment being MEDE > CHOE (p < 0.05). Mediterranean diet with or without PE lost MM (p < 0.05) being MEDE > MED CONCLUSIONS: CHOE induces less reduction of BMR while supporting a better profile of BC than MEDE. Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.

Melanin-binding radiopharmaceuticals

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

Packer, S; Fairchild, R G; Watts, K P

The scope of this paper is limited to an analysis of the factors that are important to the relationship of radiopharmaceuticals to melanin. While the authors do not attempt to deal with differences between melanin-binding vs. melanoma-binding, a notable variance is assumed. (PSB)

Optimization of reverse chemical ecology method: false positive binding of Aenasius bambawalei odorant binding protein 1 caused by uncertain binding mechanism.

PubMed

Li, Q L; Yi, S C; Li, D Z; Nie, X P; Li, S Q; Wang, M-Q; Zhou, A M

2018-06-01

Odorant binding proteins (OBPs) are considered as the core molecular targets in reverse chemical ecology, which is a convenient and efficient method by which to screen potential semiochemicals. Herein, we identified a classic OBP, AbamOBP1 from Aenasius bambawalei, which showed high mRNA expression in male antennae. Fluorescence competitive binding assay (FCBA) results demonstrated that AbamOBP1 has higher binding affinity with ligands at acid pH, suggesting the physiologically inconsistent binding affinity of this protein. Amongst the four compounds with the highest binding affinities at acid pH, 2, 4, 4-trimethyl-2-pentene and 1-octen-3-one were shown to have attractant activity for male adults, whereas (-)-limonene and an analogue of 1-octen-3-ol exhibited nonbehavioural activity. Further homology modelling and fluorescence quenching experiments demonstrated that the stoichiometry of the binding of this protein to these ligands was not 1: 1, suggesting that the results of FCBA were false. In contrast, the apparent association constants (Ka) of fluorescence quenching experiments seemed to be more reliable, because 2, 4, 4-trimethyl-2-pentene and 1-octen-3-one had observably higher Ka than (-)-limonene and 1-octen-3-ol at neutral pH. Based on the characteristics of different OBPs, various approaches should be applied to study their binding affinities with ligands, which could modify and complement the results of FCBA and contribute to the application of reverse chemical ecology. © 2018 The Royal Entomological Society.

Intra-Seasonal Flexibility in Avian Metabolic Performance Highlights the Uncoupling of Basal Metabolic Rate and Thermogenic Capacity

PubMed Central

Petit, Magali; Lewden, Agnès; Vézina, François

2013-01-01

Stochastic winter weather events are predicted to increase in occurrence and amplitude at northern latitudes and organisms are expected to cope through phenotypic flexibility. Small avian species wintering in these environments show acclimatization where basal metabolic rate (BMR) and maximal thermogenic capacity (MSUM) are typically elevated. However, little is known on intra-seasonal variation in metabolic performance and on how population trends truly reflect individual flexibility. Here we report intra-seasonal variation in metabolic parameters measured at the population and individual levels in black-capped chickadees ( Poecile atricapillus ). Results confirmed that population patterns indeed reflect flexibility at the individual level. They showed the expected increase in BMR (6%) and MSUM (34%) in winter relative to summer but also, and most importantly, that these parameters changed differently through time. BMR began its seasonal increase in November, while MSUM had already achieved more than 20% of its inter-seasonal increase by October, and declined to its starting level by March, while MSUM remained high. Although both parameters co-vary on a yearly scale, this mismatch in the timing of variation in winter BMR and MSUM likely reflects different constraints acting on different physiological components and therefore suggests a lack of functional link between these parameters. PMID:23840843

Open-wheel race car driving: energy cost for pilots.

PubMed

Beaune, Bruno; Durand, Sylvain; Mariot, Jean-Pierre

2010-11-01

The aim of this study was to evaluate the energy cost of speedway open-wheel race car driving using actimetry. Eight pilot students participated in a training session consisting of 5 successive bouts of around 30 minutes driving at steady speed on the Bugatti speedway of Le Mans (France). Energy expenditure (EE, kcal) was determined continuously by the actimetric method using the standard equation. Energy cost was estimated through physical activity ratio (PAR = EE/BMR ratio, Mets) calculation after basal metabolism rate (BMR, kcal·min-1) estimation. A 1-met PAR value was attributed to the individual BMR of each volunteer. Bout durations and EE were not significantly different between driving bouts. Mean speed was 139.94 ± 2.96 km·h-1. Physical activity ratio values ranged 4.92 ± 0.50 to 5.43 ± 0.47 Mets, corresponding to a 5.27 ± 0.47-Mets mean PAR values and a 1.21 ± 0.41 kcal·min-1 mean BMR value. These results suggest that actimetry is a simple and efficient method for EE and PAR measurements in motor sports. However, further studies are needed in the future to accurately evaluate relationships between PAR and driving intensity or between PAR and race car type.

Structure of the C-terminal effector-binding domain of AhrC bound to its corepressor l-arginine

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

Garnett, James A.; Baumberg, Simon; Stockley, Peter G.

2007-11-01

The crystal structure of the C-terminal domain hexameric core of AhrC, with bound corepressor (l-arginine), has been solved at 1.95 Å resolution. Binding of l-arginine results in a rotation between the two trimers of the hexamer, leading to the activation of the DNA-binding state. The arginine repressor/activator protein (AhrC) from Bacillus subtilis belongs to a large family of multifunctional transcription factors that are involved in the regulation of bacterial arginine metabolism. AhrC interacts with operator sites in the promoters of arginine biosynthetic and catabolic operons, acting as a transcriptional repressor at biosynthetic sites and an activator of transcription at catabolicmore » sites. AhrC is a hexamer of identical subunits, each having two domains. The C-terminal domains form the core of the protein and are involved in oligomerization and l-arginine binding. The N-terminal domains lie on the outside of the compact core and play a role in binding to 18 bp DNA operators called ARG boxes. The C-terminal domain of AhrC has been expressed, purified and characterized, and also crystallized as a hexamer with the bound corepressor l-arginine. Here, the crystal structure refined to 1.95 Å is presented.« less

SHBG (Sex Hormone Binding Globulin)

MedlinePlus

... Links Patient Resources For Health Professionals Subscribe Search Sex Hormone Binding Globulin (SHBG) Send Us Your Feedback ... As Testosterone-estrogen Binding Globulin TeBG Formal Name Sex Hormone Binding Globulin This article was last reviewed ...

Evaluation of a novel virtual screening strategy using receptor decoy binding sites.

PubMed

Patel, Hershna; Kukol, Andreas

2016-08-23

Virtual screening is used in biomedical research to predict the binding affinity of a large set of small organic molecules to protein receptor targets. This report shows the development and evaluation of a novel yet straightforward attempt to improve this ranking in receptor-based molecular docking using a receptor-decoy strategy. This strategy includes defining a decoy binding site on the receptor and adjusting the ranking of the true binding-site virtual screen based on the decoy-site screen. The results show that by docking against a receptor-decoy site with Autodock Vina, improved Receiver Operator Characteristic Enrichment (ROCE) was achieved for 5 out of fifteen receptor targets investigated, when up to 15 % of a decoy site rank list was considered. No improved enrichment was seen for 7 targets, while for 3 targets the ROCE was reduced. The extent to which this strategy can effectively improve ligand prediction is dependent on the target receptor investigated.

Tighter Ligand Binding Can Compensate for Impaired Stability of an RNA-Binding Protein.

PubMed

Wallis, Christopher P; Richman, Tara R; Filipovska, Aleksandra; Rackham, Oliver

2018-06-15

It has been widely shown that ligand-binding residues, by virtue of their orientation, charge, and solvent exposure, often have a net destabilizing effect on proteins that is offset by stability conferring residues elsewhere in the protein. This structure-function trade-off can constrain possible adaptive evolutionary changes of function and may hamper protein engineering efforts to design proteins with new functions. Here, we present evidence from a large randomized mutant library screen that, in the case of PUF RNA-binding proteins, this structural relationship may be inverted and that active-site mutations that increase protein activity are also able to compensate for impaired stability. We show that certain mutations in RNA-protein binding residues are not necessarily destabilizing and that increased ligand-binding can rescue an insoluble, unstable PUF protein. We hypothesize that these mutations restabilize the protein via thermodynamic coupling of protein folding and RNA binding.

Operator design and mechanism for CarA repressor-mediated down-regulation of the photoinducible carB operon in Myxococcus xanthus.

PubMed

López-Rubio, José Juan; Padmanabhan, S; Lázaro, Jose María; Salas, Margarita; Murillo, Francisco José; Elías-Arnanz, Montserrat

2004-07-09

The carB operon encodes all except one of the enzymes involved in light-induced carotenogenesis in Myxococcus xanthus. Expression of its promoter (P(B)) is repressed in the dark by sequence-specific DNA binding of CarA to a palindrome (pI) located between positions -47 and -64 relative to the transcription start site. This promotes subsequent binding of CarA to additional sites that remain to be defined. CarS, produced in the light, interacts physically with CarA, abrogates CarA-DNA binding, and thereby derepresses P(B). In this study, we delineate the operator design that exists for CarA by precisely mapping out the second operator element. For this, we examined how stepwise deletions and site-directed mutagenesis in the region between the palindrome and the transcription start site affect CarA binding around P(B) in vitro and expression of P(B) in vivo. These revealed the second operator element to be an imperfect interrupted palindrome (pII) spanning positions -26 to -40. In vitro assays using purified M. xanthus RNA polymerase showed that CarA abolishes P(B)-RNA polymerase binding and runoff transcription and that both were restored by CarS, thus rationalizing the observations in vivo. CarA binding to pII (after association with pI) effectively occludes RNA polymerase from P(B) and so provides the operative mechanism for the repression of the carB operon by CarA. The bipartite operator design, whereby transcription is blocked by the low affinity CarA-pII binding and is readily restored by CarS, may have evolved to match the needs for a rapid and an effective response to light.

Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

PubMed Central

2011-01-01

Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of

Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

ERIC Educational Resources Information Center

Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

2010-01-01

The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

Binding mode of cytochalasin B to F-actin is altered by lateral binding of regulatory proteins.

PubMed

Suzuki, N; Mihashi, K

1991-01-01

The binding of cytochalasin B (CB) to F-actin was studied using a trace amount of [3H]-cytochalasin B. F-Actin-bound CB was separated from free CB by ultracentrifugation and the amount of F-actin-bound CB was determined by comparing the radioactivity both in the supernatant and in the precipitate. A filament of pure F-actin possessed one high-affinity binding site for CB (Kd = 5.0 nM) at the B-end. When the filament was bound to native tropomyosin (complex of tropomyosin and troponin), two low-affinity binding sites for CB (Kd = 230 nM) were created, while the high-affinity binding site was reserved (Kd = 3.4 nM). It was concluded that the creation of low-affinity binding sites was primarily due to binding of tropomyosin to F-actin, as judged from the following two observations: (1) a filament of F-actin/tropomyosin complex possessed one high-affinity binding site (Kd = 3.9 nM) plus two low-affinity binding sites (Kd = 550 nM); (2) the Ca2(+)-receptive state of troponin C in F-actin/native tropomyosin complex did not affect CB binding.

Modulation of FadR Binding Capacity for Acyl-CoA Fatty Acids Through Structure-Guided Mutagenesis

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

Bacik, John-Paul; Yeager, Chris M.; Twary, Scott N.

FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is hence of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl- CoA, we predicted amino acid positions within the effector binding pocket thatmore » would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in-vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology.« less

Modulation of FadR Binding Capacity for Acyl-CoA Fatty Acids Through Structure-Guided Mutagenesis

DOE PAGES

Bacik, John-Paul; Yeager, Chris M.; Twary, Scott N.; ...

2015-09-18

FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is hence of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl- CoA, we predicted amino acid positions within the effector binding pocket thatmore » would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in-vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology.« less

Calcium binding to Procambarus clarkii sarcoplasmic calcium binding protein splice variants.

PubMed

Rohrback, Suzanne E; Wheatly, Michele G; Gillen, Christopher M

2015-01-01

Sarcoplasmic calcium binding protein (SCP) is a high-affinity calcium buffering protein expressed in muscle of crayfish and other invertebrates. In previous work, we identified three splice variants of Procambarus clarkii SCP (pcSCP1a, pcSCP1b, and pcSCP1c) that differ in a 37 amino acid region that lies mainly between the 2nd and 3ed EF-hand calcium binding domain. To evaluate the function of the proteins encoded by the pcSCP1 transcripts, we produced recombinant pcSCP1 and used tryptophan fluorescence to characterize calcium binding. Tryptophan fluorescence of pcSCP1a decreased in response to increased calcium, while tryptophan fluorescence of the pcSCP1b and pcSCP1c variants increased. We estimated calcium binding constants and Hill coefficients with two different equations: the standard Hill equation and a modified Hill equation that accounts for contributions from two different tryptophans. The approaches gave similar results. Steady-state calcium binding constants (Kd) ranged from 2.7±0.7×10(-8)M to 5.6±0.1×10(-7)M, consistent with previous work. Variants displayed significantly different apparent calcium affinities, which were decreased in the presence of magnesium. Calcium Kd was lowest for pcSCP1a and highest for pcSCP1c. Site-directed mutagenesis of pcSCP1c residues to the amino acids of pcSCP1b decreased the calcium Kd, identifying residues outside the EF-hand domains that contribute to calcium binding in crayfish SCP. Copyright © 2014 Elsevier Inc. All rights reserved.

Synthetic heparin-binding growth factor analogs

DOEpatents

Pena, Louis A.; Zamora, Paul; Lin, Xinhua; Glass, John D.

2007-01-23

The invention provides synthetic heparin-binding growth factor analogs having at least one peptide chain that binds a heparin-binding growth factor receptor, covalently bound to a hydrophobic linker, which is in turn covalently bound to a non-signaling peptide that includes a heparin-binding domain. The synthetic heparin-binding growth factor analogs are useful as soluble biologics or as surface coatings for medical devices.

Phosphatidic acid binding proteins display differential binding as a function of membrane curvature stress and chemical properties.

PubMed

Putta, Priya; Rankenberg, Johanna; Korver, Ruud A; van Wijk, Ringo; Munnik, Teun; Testerink, Christa; Kooijman, Edgar E

2016-11-01

Phosphatidic acid (PA) is a crucial membrane phospholipid involved in de novo lipid synthesis and numerous intracellular signaling cascades. The signaling function of PA is mediated by peripheral membrane proteins that specifically recognize PA. While numerous PA-binding proteins are known, much less is known about what drives specificity of PA-protein binding. Previously, we have described the ionization properties of PA, summarized in the electrostatic-hydrogen bond switch, as one aspect that drives the specific binding of PA by PA-binding proteins. Here we focus on membrane curvature stress induced by phosphatidylethanolamine and show that many PA-binding proteins display enhanced binding as a function of negative curvature stress. This result is corroborated by the observation that positive curvature stress, induced by lyso phosphatidylcholine, abolishes PA binding of target proteins. We show, for the first time, that a novel plant PA-binding protein, Arabidopsis Epsin-like Clathrin Adaptor 1 (ECA1) displays curvature-dependence in its binding to PA. Other established PA targets examined in this study include, the plant proteins TGD2, and PDK1, the yeast proteins Opi1 and Spo20, and, the mammalian protein Raf-1 kinase and the C2 domain of the mammalian phosphatidylserine binding protein Lact as control. Based on our observations, we propose that liposome binding assays are the preferred method to investigate lipid binding compared to the popular lipid overlay assays where membrane environment is lost. The use of complex lipid mixtures is important to elucidate further aspects of PA binding proteins. Copyright © 2016. Published by Elsevier B.V.

Binding Sites Analyser (BiSA): Software for Genomic Binding Sites Archiving and Overlap Analysis

PubMed Central

Khushi, Matloob; Liddle, Christopher; Clarke, Christine L.; Graham, J. Dinny

2014-01-01

Genome-wide mapping of transcription factor binding and histone modification reveals complex patterns of interactions. Identifying overlaps in binding patterns by different factors is a major objective of genomic studies, but existing methods to archive large numbers of datasets in a personalised database lack sophistication and utility. Therefore we have developed transcription factor DNA binding site analyser software (BiSA), for archiving of binding regions and easy identification of overlap with or proximity to other regions of interest. Analysis results can be restricted by chromosome or base pair overlap between regions or maximum distance between binding peaks. BiSA is capable of reporting overlapping regions that share common base pairs; regions that are nearby; regions that are not overlapping; and average region sizes. BiSA can identify genes located near binding regions of interest, genomic features near a gene or locus of interest and statistical significance of overlapping regions can also be reported. Overlapping results can be visualized as Venn diagrams. A major strength of BiSA is that it is supported by a comprehensive database of publicly available transcription factor binding sites and histone modifications, which can be directly compared to user data. The documentation and source code are available on http://bisa.sourceforge.net PMID:24533055

Specific binding of PCBP1 to heavily oxidized RNA to induce cell death.

PubMed

Ishii, Takashi; Hayakawa, Hiroshi; Igawa, Tatsuhiro; Sekiguchi, Takeshi; Sekiguchi, Mutsuo

2018-06-26

In aerobically growing cells, the guanine base of RNA is oxidized to 8-oxo-7,8-dihydroguanine (8-oxoG), which induces alteration in their gene expression. We previously demonstrated that the human AUF1 protein binds to 8-oxoG in RNA to induce the selective degradation of oxidized messenger RNA. We herein report that the poly(C)-binding protein PCBP1 binds to more severely oxidized RNA to activate apoptosis-related reactions. While AUF1 binds to oligoribonucleotides carrying a single 8-oxoG, PCBP1 does not bind to such oligoribonucleotides but instead binds firmly to oligoribonucleotides in which two 8-oxoG residues are located nearby. PCBP1-deficient cells, constructed from the human HeLa S3 line using the CRISPR-Cas9 system, exhibited higher survival rates than HeLa S3 cells when small doses of hydrogen peroxide were applied. The levels of caspase-3 activation and PARP-1 cleavage in the PCBP1-deficient cells were significantly lower than those in wild-type cells. The structure-function relationship of PCBP1 was established with the use of PCBP1 mutant proteins in which the conserved KH domains were defective. Human cells appear to possess two distinct mechanisms, one controlled by AUF1 and the other by PCBP1, with the former functioning when messenger RNA is moderately oxidized and the latter operating when the RNA is more severely damaged.

Evolving nucleotide binding surfaces

NASA Technical Reports Server (NTRS)

Kieber-Emmons, T.; Rein, R.

1981-01-01

An analysis is presented of the stability and nature of binding of a nucleotide to several known dehydrogenases. The employed approach includes calculation of hydrophobic stabilization of the binding motif and its intermolecular interaction with the ligand. The evolutionary changes of the binding motif are studied by calculating the Euclidean deviation of the respective dehydrogenases. Attention is given to the possible structural elements involved in the origin of nucleotide recognition by non-coded primordial polypeptides.

The RNA-Binding Site of Poliovirus 3C Protein Doubles as a Phosphoinositide-Binding Domain.

PubMed

Shengjuler, Djoshkun; Chan, Yan Mei; Sun, Simou; Moustafa, Ibrahim M; Li, Zhen-Lu; Gohara, David W; Buck, Matthias; Cremer, Paul S; Boehr, David D; Cameron, Craig E

2017-12-05

Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using nuclear magnetic resonance spectroscopy. The PIP-binding site was located on a highly dynamic α helix, which also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses. Copyright © 2017 Elsevier Ltd. All rights reserved.

BindML/BindML+: Detecting Protein-Protein Interaction Interface Propensity from Amino Acid Substitution Patterns.

PubMed

Wei, Qing; La, David; Kihara, Daisuke

2017-01-01

Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .

Binding of Radioactive Benzylpenicillin to Sporulating Bacillus Cultures: Chemistry and Fluctuations in Specific Binding Capacity

PubMed Central

Lawrence, Paul J.; Rogolsky, Marvin; Hanh, Vo Thi

1971-01-01

The chemistry of the binding of 14C-benzylpenicillin to sporulating cultures of Bacillus megaterium and B. subtilis is similar to that in a 4-hr vegetative culture of Staphylococcus aureus. Unlabeled penicillins prevent the binding of 14C-benzylpenicillin, but benzylpenicilloic acid and benzylpenilloic acid do not. Bound antibiotic can be removed from cells with neutral hydroxylamine at 25 C. Sporulating cultures display two intervals of enhanced binding, whereas binding to stationaryphase S. aureus cells remains constant. The first period of increased binding activity occurs during formation of the spore septum or cell wall primordium development, and the second coincides with cortex biosynthesis. PMID:4942758

Protein-Binding RNA Aptamers Affect Molecular Interactions Distantly from Their Binding Sites

PubMed Central

Dupont, Daniel M.; Thuesen, Cathrine K.; Bøtkjær, Kenneth A.; Behrens, Manja A.; Dam, Karen; Sørensen, Hans P.; Pedersen, Jan S.; Ploug, Michael; Jensen, Jan K.; Andreasen, Peter A.

2015-01-01

Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site. PMID:25793507

Neurosteroid binding to the amino terminal and glutamate binding domains of ionotropic glutamate receptors.

PubMed

Cameron, Krasnodara; Bartle, Emily; Roark, Ryan; Fanelli, David; Pham, Melissa; Pollard, Beth; Borkowski, Brian; Rhoads, Sarah; Kim, Joon; Rocha, Monica; Kahlson, Martha; Kangala, Melinda; Gentile, Lisa

2012-06-01

The endogenous neurosteroids, pregnenolone sulfate (PS) and 3α-hydroxy-5β-pregnan-20-one sulfate (PREGAS), have been shown to differentially regulate the ionotropic glutamate receptor (iGluR) family of ligand-gated ion channels. Upon binding to these receptors, PREGAS decreases current flow through the channels. Upon binding to non-NMDA or NMDA receptors containing an GluN2C or GluN2D subunit, PS also decreases current flow through the channels, however, upon binding to NMDA receptors containing an GluN2A or GluN2B subunit, flow through the channels increases. To begin to understand this differential regulation, we have cloned the S1S2 and amino terminal domains (ATD) of the NMDA GluN2B and GluN2D and AMPA GluA2 subunits. Here we present results that show that PS and PREGAS bind to different sites in the ATD of the GluA2 subunit, which when combined with previous results from our lab, now identifies two binding domains for each neurosteroid. We also show both neurosteroids bind only to the ATD of the GluN2D subunit, suggesting that this binding is distinct from that of the AMPA GluA2 subunit, with both leading to iGluR inhibition. Finally, we provide evidence that both PS and PREGAS bind to the S1S2 domain of the NMDA GluN2B subunit. Neurosteroid binding to the S1S2 domain of NMDA subunits responsible for potentiation of iGluRs and to the ATD of NMDA subunits responsible for inhibition of iGluRs, provides an interesting option for therapeutic design. Copyright © 2012 Elsevier Inc. All rights reserved.

Compilation of basal metabolic and blood perfusion rates in various multi-compartment, whole-body thermoregulation models

NASA Astrophysics Data System (ADS)

Shitzer, Avraham; Arens, Edward; Zhang, Hui

2016-07-01

The assignments of basal metabolic rates (BMR), basal cardiac output (BCO), and basal blood perfusion rates (BBPR) were compared in nine multi-compartment, whole-body thermoregulation models. The data are presented at three levels of detail: total body, specific body regions, and regional body tissue layers. Differences in the assignment of these quantities among the compared models increased with the level of detail, in the above order. The ranges of variability in the total body BMR was 6.5 % relative to the lowest value, with a mean of 84.3 ± 2 W, and in the BCO, it was 8 % with a mean of 4.70 ± 0.13 l/min. The least variability among the body regions is seen in the combined torso (shoulders, thorax, and abdomen: ±7.8 % BMR and ±5.9 % BBPR) and in the combined head (head, face, and neck ±9.9 % BMR and ±10.9 % BBPR), determined by the ratio of the standard deviation to the mean. Much more variability is apparent in the extremities with the most showing in the BMR of the feet (±117 %), followed by the BBPR in the arms (±61.3 %). In the tissue layers, most of the bone layers were assigned zero BMR and BBPR, except in the shoulders and in the extremities that were assigned non-zero values in a number of models. The next lowest values were assigned to the fat layers, with occasional zero values. Skin basal values were invariably non-zero but involved very low values in certain models, e.g., BBPR in the feet and the hands. Muscle layers were invariably assigned high values with the highest found in the thorax, abdomen, and legs. The brain, lung, and viscera layers were assigned the highest of all values of both basal quantities with those of the brain layers showing rather tight ranges of variability in both basal quantities. Average basal values of the "time-seasoned" models presented in this study could be useful as a first step in future modeling efforts subject to appropriate adjustment of values to conform to most recently available and reliable data.

Binding Site Turnover Produces Pervasive Quantitative Changes in Transcription Factor Binding between Closely Related Drosophila Species

PubMed Central

Trapnell, Cole; Davidson, Stuart; Pachter, Lior; Chu, Hou Cheng; Tonkin, Leath A.; Biggin, Mark D.; Eisen, Michael B.

2010-01-01

Changes in gene expression play an important role in evolution, yet the molecular mechanisms underlying regulatory evolution are poorly understood. Here we compare genome-wide binding of the six transcription factors that initiate segmentation along the anterior-posterior axis in embryos of two closely related species: Drosophila melanogaster and Drosophila yakuba. Where we observe binding by a factor in one species, we almost always observe binding by that factor to the orthologous sequence in the other species. Levels of binding, however, vary considerably. The magnitude and direction of the interspecies differences in binding levels of all six factors are strongly correlated, suggesting a role for chromatin or other factor-independent forces in mediating the divergence of transcription factor binding. Nonetheless, factor-specific quantitative variation in binding is common, and we show that it is driven to a large extent by the gain and loss of cognate recognition sequences for the given factor. We find only a weak correlation between binding variation and regulatory function. These data provide the first genome-wide picture of how modest levels of sequence divergence between highly morphologically similar species affect a system of coordinately acting transcription factors during animal development, and highlight the dominant role of quantitative variation in transcription factor binding over short evolutionary distances. PMID:20351773

Effect of solid surface charge on the binding behaviour of a metal-binding peptide

PubMed Central

Donatan, Senem; Sarikaya, Mehmet; Tamerler, Candan; Urgen, Mustafa

2012-01-01

Over the last decade, solid-binding peptides have been increasingly used as molecular building blocks coupling bio- and nanotechnology. Despite considerable research being invested in this field, the effects of many surface-related parameters that define the binding of peptide to solids are still unknown. In the quest to control biological molecules at solid interfaces and, thereby, tailoring the binding characteristics of the peptides, the use of surface charge of the solid surface may probably play an important role, which then can be used as a potential tuning parameter of peptide adsorption. Here, we report quantitative investigation on the viscoelastic properties and binding kinetics of an engineered gold-binding peptide, 3RGBP1, adsorbed onto the gold surface at different surface charge densities. The experiments were performed in aqueous solutions using an electrochemical dissipative quartz crystal microbalance system. Hydrodynamic mass, hydration state and surface coverage of the adsorbed peptide films were determined as a function of surface charge density of the gold metal substrate. Under each charged condition, binding of 3rGBP1 displayed quantitative differences in terms of adsorbed peptide amount, surface coverage ratio and hydration state. Based on the intrinsically disordered structure of the peptide, we propose a possible mechanism for binding of the peptide that can be used for tuning surface adsorption in further studies. Controlled alteration of peptide binding on solid surfaces, as shown here, may provide novel methods for surface functionalization used for bioenabled processing and fabrication of future micro- and nanodevices. PMID:22491974

Fungal-type carbohydrate binding modules from the coccolithophore Emiliania huxleyi show binding affinity to cellulose and chitin

PubMed Central

Rooijakkers, Bart J. M.

2018-01-01

Six fungal-type cellulose binding domains were found in the genome of the coccolithophore Emiliania huxleyi and cloned and expressed in Escherichia coli. Sequence comparison indicate high similarity to fungal cellulose binding domains, raising the question of why these domains exist in coccolithophores. The proteins were tested for binding with cellulose and chitin as ligands, which resulted in the identification of two functional carbohydrate binding modules: EHUX2 and EHUX4. Compared to benchmark fungal cellulose binding domain Cel7A-CBM1 from Trichoderma reesei, these proteins showed slightly lower binding to birch and bacterial cellulose, but were more efficient chitin binders. Finally, a set of cellulose binding domains was created based on the shuffling of one well-functioning and one non-functional domain. These were characterized in order to get more information of the binding domain’s sequence–function relationship, indicating characteristic differences between the molecular basis of cellulose versus chitin recognition. As previous reports have showed the presence of cellulose in coccoliths and here we find functional cellulose binding modules, a possible connection is discussed. PMID:29782536

Fungal-type carbohydrate binding modules from the coccolithophore Emiliania huxleyi show binding affinity to cellulose and chitin.

PubMed

Rooijakkers, Bart J M; Ikonen, Martina S; Linder, Markus B

2018-01-01

Six fungal-type cellulose binding domains were found in the genome of the coccolithophore Emiliania huxleyi and cloned and expressed in Escherichia coli. Sequence comparison indicate high similarity to fungal cellulose binding domains, raising the question of why these domains exist in coccolithophores. The proteins were tested for binding with cellulose and chitin as ligands, which resulted in the identification of two functional carbohydrate binding modules: EHUX2 and EHUX4. Compared to benchmark fungal cellulose binding domain Cel7A-CBM1 from Trichoderma reesei, these proteins showed slightly lower binding to birch and bacterial cellulose, but were more efficient chitin binders. Finally, a set of cellulose binding domains was created based on the shuffling of one well-functioning and one non-functional domain. These were characterized in order to get more information of the binding domain's sequence-function relationship, indicating characteristic differences between the molecular basis of cellulose versus chitin recognition. As previous reports have showed the presence of cellulose in coccoliths and here we find functional cellulose binding modules, a possible connection is discussed.

Polymorphisms of the FTO gene are associated with variation in energy intake, but not energy expenditure.

PubMed

Speakman, John R; Rance, Kellie A; Johnstone, Alexandra M

2008-08-01

The FTO gene has significant polymorphic variation associated with obesity, but its function is unknown. We screened a population of 150 whites (103F/47M) resident in NE Scotland, United Kingdom, for variants of the FTO gene and linked these to phenotypic variation in their energy expenditure (basal metabolic rate (BMR) and maximal oxygen consumption VO(2)max) and energy intake. There was no significant association between the FTO genotype and BMR or VO(2)max. The FTO genotype was significantly associated (P = 0.024) with variation in energy intake, with average daily intake being 9.0 MJ for the wild-type TT genotype and 10.2 and 9.5 MJ for the "at risk" AT and AA genotypes, respectively. Adjusting intake for BMR did not remove the significance (P = 0.043). FTO genotype probably affects obesity via effects on food intake rather than energy expenditure.

Energy requirements of lactating women derived from doubly labeled water and milk energy output.

PubMed

Butte, N F; Wong, W W; Hopkinson, J M

2001-01-01

Instead of using an incremental approach to assess the energy requirements of lactation, a more comprehensive approach may be taken by measuring total energy expenditure (TEE), milk energy output and energy mobilization from tissue stores. The latter approach avoids assumptions regarding energetic efficiency and changes in physical activity and adiposity. The purpose of this study was threefold: to assess the energy requirements of lactation; to compare these estimates with energy requirements in the nonpregnant, nonlactating state and to test for energetic adaptations in basal metabolic rate (BMR) and physical activity during the energy-demanding process of lactation. Milk production and composition, body weight and composition, TEE, BMR and physical activity levels were measured in 24 well-nourished women during exclusive breastfeeding at 3 mo postpartum and after the cessation of breastfeeding at 18 or 24 mo postpartum. TEE was measured by the doubly labeled water method, milk production by 3-d test-weighing, milk energy by bomb calorimetry on a 24-h milk sample, body composition by dual-energy x-ray absorptiometry and BMR by room respiration calorimetry. TEE, BMR and physical activity level (physical activity level = TEE/BMR) did not differ between the lactating and nonlactating state (TEE 10.0 +/- 1.5 versus 10.6 +/- 2.1 MJ/d). Mean milk energy output was equivalent to 2.02 +/- 0.33 MJ/d. Total energy requirements were greater during lactation than afterward (12.0 +/- 1.4 versus 10.6 +/- 2.1 MJ/d, P: = 0.002). Energy mobilization from tissue stores (-0.65 +/- 0.97 MJ/d) resulted in net energy requirements during lactation of 11.4 +/- 1.8 MJ/d. Because adaptations in basal metabolism and physical activity were not evident in these well-nourished women, energy requirements during lactation were met primarily from the diet and only partially by mobilization of tissue stores.

Cross-validation of bioelectrical impedance analysis of body composition in children and adolescents.

PubMed

Wu, Y T; Nielsen, D H; Cassady, S L; Cook, J S; Janz, K F; Hansen, J R

1993-05-01

The reliability and validity of measurements obtained with two bioelectrical impedance analyzers (BIAs), an RJL Systems model BIA-103 and a Berkeley Medical Research BMR-2000, were investigated using the manufacturers' prediction equations for the assessment of fat-free mass (FFM) (in kilograms) in children and adolescents. Forty-seven healthy children and adolescents (23 male, 24 female), ranging in age from 8 to 20 years (mean = 12.1, SD = 2.3), participated. In the context of a repeated-measures design, the data were analyzed according to gender and maturation (Tanner staging). Hydrostatic weighing (HYDRO) and Lohman's Siri age-adjusted body density prediction equation served as the criteria for validating the BIA-obtained measurements. High intraclass correlation coefficients (ICC > or = .987) demonstrated good test-retest (between-week) measurement reliability for HYDRO and both BIA methods. Between-method (HYDRO versus BIA) correlation coefficients were high for both boys and girls (r > or = .97). The standard errors of estimate (SEEs) for FFM were slightly larger for boys than for girls and were consistently smaller for the RJL system than for the BMR system (RJL SEE = 1.8 kg for boys, 1.3 kg for girls; BMR SEE = 2.4 kg for boys, 1.9 kg for girls). The coefficients of determination were high for both BIA methods (r2 > or = .929). Total prediction errors (TEs) for FFM showed similar between-method trends (RJL TE = 2.1 kg for boys, 1.5 kg for girls; BMR TE = 4.4 kg for boys, 1.9 kg for girls). This study demonstrated that the RJL BIA with the manufacturer's prediction equations can be used to reliably and accurately assess FFM in 8- to 20-year-old children and adolescents. The prediction of FFM by the BMR system was acceptable for girls, but significant overprediction of FFM for boys was noted.

Fundamental considerations in ski binding analysis.

PubMed

Mote, C D; Hull, M L

1976-01-01

1. The static adjustment of a ski binding by hand or by available machines is only an adjustment and is neither a static nor a dynamic evaluation of the binding design. Bindings of different design with identical static adjustments will perform differently in environments in which the forces are static or dynamic. 2. The concept of binding release force is a useful measure of binding adjustment, but it is inappropriate as a criterion for binding evaluation. First, it does not direct attention toward the injury causing mechanism, strain, or displacement in the leg. Second, it is only part of the evaluation in dynamic problems. 3. The binding release decision in present bindings is displacement controlled. The relative displacement of the boot and ski is the system variable. For any specified relative displacement the binding force can be any of an infinite number of possibilities determined by the loading path. 4. The response of the leg-ski system to external impulses applied to the ski is independent of the boot-ski relative motion as long as the boot recenters quickly in the binding. Response is dependent upon the external impulse plus system inertia, damping and stiffness. 5. When tested under half sinusoidal forces applied to a test ski, all bindings will demonstrate static and impulse loading regions. In the static region the force drives the binding to a relative release displacement. In the impulse region the initial velocity of the ski drives the binding to a release displacement. 6. The transition between the static and impulse loading regions is determined by the binding's capacity to store and dissipate energy along the principal loading path. Increased energy capacity necessitates larger external impulses to produce release. 7. In all bindings examined to date, the transmitted leg displacement or strain at release under static loading exceeds leg strain under dynamic or impact loading. Because static loading is responsible for many injuries, a skier

Multiplexed target detection using DNA-binding dye chemistry in droplet digital PCR.

PubMed

McDermott, Geoffrey P; Do, Duc; Litterst, Claudia M; Maar, Dianna; Hindson, Christopher M; Steenblock, Erin R; Legler, Tina C; Jouvenot, Yann; Marrs, Samuel H; Bemis, Adam; Shah, Pallavi; Wong, Josephine; Wang, Shenglong; Sally, David; Javier, Leanne; Dinio, Theresa; Han, Chunxiao; Brackbill, Timothy P; Hodges, Shawn P; Ling, Yunfeng; Klitgord, Niels; Carman, George J; Berman, Jennifer R; Koehler, Ryan T; Hiddessen, Amy L; Walse, Pramod; Bousse, Luc; Tzonev, Svilen; Hefner, Eli; Hindson, Benjamin J; Cauly, Thomas H; Hamby, Keith; Patel, Viresh P; Regan, John F; Wyatt, Paul W; Karlin-Neumann, George A; Stumbo, David P; Lowe, Adam J

2013-12-03

Two years ago, we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a "second generation" ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries. Herein, we describe the operating characteristics of DNA-binding dye based ddPCR and offer a side-by-side comparison to TaqMan probe detection. By partitioning each sample prior to thermal cycling, we demonstrate that it is now possible to use a DNA-binding dye for the quantification of multiple target species from a single reaction. The increased resolution associated with partitioning also made it possible to visualize and account for signals arising from nonspecific amplification products. We expect that the ability to combine the precision of ddPCR with both DNA-binding dye and TaqMan probe detection chemistries will further enable the research community to answer complex and diverse genetic questions.

Library Binding Manual. Revised Edition.

ERIC Educational Resources Information Center

Lakhanpal, S. K.

This procedural manual is designed to be used in bindery sections in public, university and special libraries. It briefly discusses these general matters: administrative control; selection of a binder; when and what to bind; conventional binding; routines; missing issues; schedule for shipments; temporary binding; rare books, maps and newspapers;…

How to deal with multiple binding poses in alchemical relative protein-ligand binding free energy calculations.

PubMed

Kaus, Joseph W; Harder, Edward; Lin, Teng; Abel, Robert; McCammon, J Andrew; Wang, Lingle

2015-06-09

Recent advances in improved force fields and sampling methods have made it possible for the accurate calculation of protein–ligand binding free energies. Alchemical free energy perturbation (FEP) using an explicit solvent model is one of the most rigorous methods to calculate relative binding free energies. However, for cases where there are high energy barriers separating the relevant conformations that are important for ligand binding, the calculated free energy may depend on the initial conformation used in the simulation due to the lack of complete sampling of all the important regions in phase space. This is particularly true for ligands with multiple possible binding modes separated by high energy barriers, making it difficult to sample all relevant binding modes even with modern enhanced sampling methods. In this paper, we apply a previously developed method that provides a corrected binding free energy for ligands with multiple binding modes by combining the free energy results from multiple alchemical FEP calculations starting from all enumerated poses, and the results are compared with Glide docking and MM-GBSA calculations. From these calculations, the dominant ligand binding mode can also be predicted. We apply this method to a series of ligands that bind to c-Jun N-terminal kinase-1 (JNK1) and obtain improved free energy results. The dominant ligand binding modes predicted by this method agree with the available crystallography, while both Glide docking and MM-GBSA calculations incorrectly predict the binding modes for some ligands. The method also helps separate the force field error from the ligand sampling error, such that deviations in the predicted binding free energy from the experimental values likely indicate possible inaccuracies in the force field. An error in the force field for a subset of the ligands studied was identified using this method, and improved free energy results were obtained by correcting the partial charges assigned to the

How To Deal with Multiple Binding Poses in Alchemical Relative Protein–Ligand Binding Free Energy Calculations

PubMed Central

2016-01-01

Recent advances in improved force fields and sampling methods have made it possible for the accurate calculation of protein–ligand binding free energies. Alchemical free energy perturbation (FEP) using an explicit solvent model is one of the most rigorous methods to calculate relative binding free energies. However, for cases where there are high energy barriers separating the relevant conformations that are important for ligand binding, the calculated free energy may depend on the initial conformation used in the simulation due to the lack of complete sampling of all the important regions in phase space. This is particularly true for ligands with multiple possible binding modes separated by high energy barriers, making it difficult to sample all relevant binding modes even with modern enhanced sampling methods. In this paper, we apply a previously developed method that provides a corrected binding free energy for ligands with multiple binding modes by combining the free energy results from multiple alchemical FEP calculations starting from all enumerated poses, and the results are compared with Glide docking and MM-GBSA calculations. From these calculations, the dominant ligand binding mode can also be predicted. We apply this method to a series of ligands that bind to c-Jun N-terminal kinase-1 (JNK1) and obtain improved free energy results. The dominant ligand binding modes predicted by this method agree with the available crystallography, while both Glide docking and MM-GBSA calculations incorrectly predict the binding modes for some ligands. The method also helps separate the force field error from the ligand sampling error, such that deviations in the predicted binding free energy from the experimental values likely indicate possible inaccuracies in the force field. An error in the force field for a subset of the ligands studied was identified using this method, and improved free energy results were obtained by correcting the partial charges assigned to the

Dynamic Factors Affecting Gaseous Ligand Binding in an Artificial Oxygen Transport Protein‡

PubMed Central

Zhang, Lei; Andersen, Eskil M.E.; Khajo, Abdelahad; Magliozzo, Richard S.; Koder, Ronald L.

2013-01-01

We report the functional analysis of an artificial hexacoordinate oxygen transport protein, HP7, which operates via a mechanism similar to that of human neuroglobin and cytoglobin: the destabilization of one of two heme-ligating histidine residues. In the case of HP7 this is the result of the coupling of histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Here we compare gaseous ligand binding, including rates, affinities and oxyferrous state lifetimes, of both heme binding sites in HP7. We find that despite the identical sequence of helices in both binding sites, there are differences in oxygen affinity and oxyferrous state lifetime which may be the result of differences in the freedom of motion imposed by the candelabra fold on the two sites of the protein. We further examine the effect of mutational removal of the buried glutamates on function. Heme iron in the ferrous state of this mutant is rapidly oxidized when when exposed to oxygen. Compared to HP7, distal histidine affinity is increased by a 22-fold decrease in the histidine ligand off-rate. EPR comparison of these ferric hemoproteins demonstrates that the mutation increases disorder at the heme binding site. NMR-detected deuterium exchange demonstrates that the mutation greatly increases water penetration into the protein core. The inability of the mutant protein to bind oxygen may be due to increased water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors. Together these data underline the importance of the control of protein dynamics in the design of functional artificial proteins. PMID:23249163

On the binding determinants of the glutamate agonist with the glutamate receptor ligand binding domain.

PubMed

Speranskiy, Kirill; Kurnikova, Maria

2005-08-30

Ionotropic glutamate receptors (GluRs) are ligand-gated membrane channel proteins found in the central neural system that mediate a fast excitatory response of neurons. In this paper, we report theoretical analysis of the ligand-protein interactions in the binding pocket of the S1S2 (ligand binding) domain of the GluR2 receptor in the closed conformation. By utilizing several theoretical methods ranging from continuum electrostatics to all-atom molecular dynamics simulations and quantum chemical calculations, we were able to characterize in detail glutamate agonist binding to the wild-type and E705D mutant proteins. A theoretical model of the protein-ligand interactions is validated via direct comparison of theoretical and Fourier transform infrared spectroscopy (FTIR) measured frequency shifts of the ligand's carboxylate group vibrations [Jayaraman et al. (2000) Biochemistry 39, 8693-8697; Cheng et al. (2002) Biochemistry 41, 1602-1608]. A detailed picture of the interactions in the binding site is inferred by analyzing contributions to vibrational frequencies produced by protein residues forming the ligand-binding pocket. The role of mobility and hydrogen-bonding network of water in the ligand-binding pocket and the contribution of protein residues exposed in the binding pocket to the binding and selectivity of the ligand are discussed. It is demonstrated that the molecular surface of the protein in the ligand-free state has mainly positive electrostatic potential attractive to the negatively charged ligand, and the potential produced by the protein in the ligand-binding pocket in the closed state is complementary to the distribution of the electrostatic potential produced by the ligand itself. Such charge complementarity ensures specificity to the unique charge distribution of the ligand.

Basal metabolic rate of endotherms can be modeled using heat-transfer principles and physiological concepts: reply to "can the basal metabolic rate of endotherms be explained by biophysical modeling?".

PubMed

Roberts, Michael F; Lightfoot, Edwin N; Porter, Warren P

2011-01-01

Our recent article (Roberts et al. 2010 ) proposes a mechanistic model for the relation between basal metabolic rate (BMR) and body mass (M) in mammals. The model is based on heat-transfer principles in the form of an equation for distributed heat generation within the body. The model can also be written in the form of the allometric equation BMR = aM(b), in which a is the coefficient of the mass term and b is the allometric exponent. The model generates two interesting results: it predicts that b takes the value 2/3, indicating that BMR is proportional to surface area in endotherms. It also provides an explanation of the physiological components that make up a, that is, respiratory heat loss, core-skin thermal conductance, and core-skin thermal gradient. Some of the ideas in our article have been questioned (Seymour and White 2011 ), and this is our response to those questions. We specifically address the following points: whether a heat-transfer model can explain the level of BMR in mammals, whether our test of the model is inadequate because it uses the same literature data that generated the values of the physiological variables, and whether geometry and empirical values combine to make a "coincidence" that makes the model only appear to conform to real processes.

Dissecting the protein architecture of DNA-binding transcription factors in bacteria and archaea.

PubMed

Rivera-Gómez, Nancy; Martínez-Núñez, Mario Alberto; Pastor, Nina; Rodriguez-Vazquez, Katya; Perez-Rueda, Ernesto

2017-08-01

Gene regulation at the transcriptional level is a central process in all organisms where DNA-binding transcription factors play a fundamental role. This class of proteins binds specifically at DNA sequences, activating or repressing gene expression as a function of the cell's metabolic status, operator context and ligand-binding status, among other factors, through the DNA-binding domain (DBD). In addition, TFs may contain partner domains (PaDos), which are involved in ligand binding and protein-protein interactions. In this work, we systematically evaluated the distribution, abundance and domain organization of DNA-binding TFs in 799 non-redundant bacterial and archaeal genomes. We found that the distributions of the DBDs and their corresponding PaDos correlated with the size of the genome. We also identified specific combinations between the DBDs and their corresponding PaDos. Within each class of DBDs there are differences in the actual angle formed at the dimerization interface, responding to the presence/absence of ligands and/or crystallization conditions, setting the orientation of the resulting helices and wings facing the DNA. Our results highlight the importance of PaDos as central elements that enhance the diversity of regulatory functions in all bacterial and archaeal organisms, and our results also demonstrate the role of PaDos in sensing diverse signal compounds. The highly specific interactions between DBDs and PaDos observed in this work, together with our structural analysis highlighting the difficulty in predicting both inter-domain geometry and quaternary structure, suggest that these systems appeared once and evolved with diverse duplication events in all the analysed organisms.

Human mRNA polyadenylate binding protein: evolutionary conservation of a nucleic acid binding motif.

PubMed Central

Grange, T; de Sa, C M; Oddos, J; Pictet, R

1987-01-01

We have isolated a full length cDNA (cDNA) coding for the human poly(A) binding protein. The cDNA derived 73 kd basic translation product has the same Mr, isoelectric point and peptidic map as the poly(A) binding protein. DNA sequence analysis reveals a 70,244 dalton protein. The N terminal part, highly homologous to the yeast poly(A) binding protein, is sufficient for poly(A) binding activity. This domain consists of a four-fold repeated unit of approximately 80 amino acids present in other nucleic acid binding proteins. In the C terminal part there is, as in the yeast protein, a sequence of approximately 150 amino acids, rich in proline, alanine and glutamine which together account for 48% of the residues. A 2,9 kb mRNA corresponding to this cDNA has been detected in several vertebrate cell types and in Drosophila melanogaster at every developmental stage including oogenesis. Images PMID:2885805

Bak Conformational Changes Induced by Ligand Binding: Insight into BH3 Domain Binding and Bak Homo-Oligomerization

PubMed Central

Pang, Yuan-Ping; Dai, Haiming; Smith, Alyson; Meng, X. Wei; Schneider, Paula A.; Kaufmann, Scott H.

2012-01-01

Recently we reported that the BH3-only proteins Bim and Noxa bind tightly but transiently to the BH3-binding groove of Bak to initiate Bak homo-oligomerization. However, it is unclear how such tight binding can induce Bak homo-oligomerization. Here we report the ligand-induced Bak conformational changes observed in 3D models of Noxa·Bak and Bim·Bak refined by molecular dynamics simulations. In particular, upon binding to the BH3-binding groove, Bim and Noxa induce a large conformational change of the loop between helices 1 and 2 and in turn partially expose a remote groove between helices 1 and 6 in Bak. These observations, coupled with the reported experimental data, suggest formation of a pore-forming Bak octamer, in which the BH3-binding groove is at the interface on one side of each monomer and the groove between helices 1 and 6 is at the interface on the opposite side, initiated by ligand binding to the BH3-binding groove. PMID:22355769

Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition.

PubMed

Neth, O; Jack, D L; Dodds, A W; Holzel, H; Klein, N J; Turner, M W

2000-02-01

Mannose-binding lectin (MBL) is a collagenous serum lectin believed to be of importance in innate immunity. Genetically determined low levels of the protein are known to predispose to infections. In this study the binding of purified MBL to pathogens isolated from immunocompromised children was investigated by flow cytometry. Diverse Candida species, Aspergillus fumigatus, Staphylococcus aureus, and beta-hemolytic group A streptococci exhibited strong binding of MBL, whereas Escherichia coli, Klebsiella species, and Haemophilus influenzae type b were characterized by heterogeneous binding patterns. In contrast, beta-hemolytic group B streptococci, Streptococcus pneumoniae, and Staphylococcus epidermidis showed low levels of binding. Bound MBL was able to promote C4 deposition in a concentration-dependent manner. We conclude that MBL may be of importance in first-line immune defense against several important pathogens.

Modeling Conformational Transitions and Energetics of Ligand Binding with the Glutamate Receptor Ligand Binding Domain

NASA Astrophysics Data System (ADS)

Kurnikova, Maria

2009-03-01

Understanding of protein motion and energetics of conformational transitions is crucial to understanding protein function. The glutamate receptor ligand binding domain (GluR2 S1S2) is a two lobe protein, which binds ligand at the interface of two lobes and undergoes conformational transition. The cleft closure conformational transition of S1S2 has been implicated in gating of the ion channel formed by the transmembrane domain of the receptor. In this study we present a composite multi-faceted theoretical analysis of the detailed mechanism of this conformational transition based on rigid cluster decomposition of the protein structure [1] and identifying hydrogen bonds that are responsible for stabilizing the closed conformation [2]. Free energy of the protein reorganization upon ligand binding was calculated using combined Thermodynamic Integration (TI) and Umbrella Sampling (US) simulations [3]. Ligand -- protein interactions in the binding cleft were analyzed using Molecular Dynamics, continuum electrostatics and QM/MM models [4]. All model calculations compare well with corresponding experimental measurements. [4pt] [1] Protein Flexibility using Constraints from Molecular Dynamics Simulations T. Mamonova, B. Hespenheide, R. Straub, M. F. Thorpe, M. G. Kurnikova , Phys. Biol., 2, S137 (2005)[0pt] [2] Theoretical Study of the Glutamate Receptor Ligand Binding Domain Flexibility and Conformational Reorganization T. Mamonova, K. Speranskiy, and M. Kurnikova , Prot.: Struct., Func., Bioinf., 73,656 (2008)[0pt] [3] Energetics of the cleft closing transition and glutamate binding in the Glutamate Receptor ligand Binding Domain T. Mamonova, M. Yonkunas, and M. Kurnikova Biochemistry 47, 11077 (2008)[0pt] [4] On the Binding Determinants of the Glutamate Agonist with the Glutamate Receptor Ligand Binding Domain K. Speranskiy and M. Kurnikova Biochemistry 44, 11208 (2005)

Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor

PubMed Central

Gillard, Nathalie; Goffinont, Stephane; Buré, Corinne; Davidkova, Marie; Maurizot, Jean-Claude; Cadene, Martine; Spotheim-Maurizot, Melanie

2007-01-01

Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with γ-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (OH· radicals) which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece. CD measurements revealed a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements showed a gradual degradation of tyrosine residues. MS was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidized residues. By calculating the relative probabilities of reaction of each amino acid with OH· radicals, we can predict the most probable oxidation targets. By comparing the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hotspots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece. PMID:17263689

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

Caffeine inhibits glucose transport by binding at the GLUT1 nucleotide-binding site

PubMed Central

Sage, Jay M.; Cura, Anthony J.; Lloyd, Kenneth P.

2015-01-01

Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3-O-methylglucose uptake in human erythrocytes [Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3-O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites—the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis. PMID:25715702

Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

PubMed

Oshiro, Satoshi; Honda, Shinya

2014-04-18

Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

Characterization of nicotine binding in mouse brain and comparison with the binding of alpha-bungarotoxin and quinuclidinyl benzilate

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

Marks, M.J.; Collins, A.C.

1982-11-01

The binding of (/sup 3/H)nicotine to mouse brain has been measured and subsequently compared with the binding of (/sup 125/I)alpha-bungarotoxin (alpha-BTX) and L-(/sup 3/H)quinuclidinyl benzilate (QNB). The binding of nicotine was saturable, reversible, and stereospecific. The average KD and Bmax were 59 nM and 88 fmoles/mg of protein, respectively. Although the rates of association and dissociation of nicotine were temperature-dependent, the incubation temperature had no effect on either KD or Bmax. When measured at 20 degrees or 37 degrees, nicotine appeared to bind to a single class of binding sites, but a second, very low-affinity, binding site was observed atmore » 4 degrees. Nicotine binding was unaffected by the addition of NaCl, KCl, CaCl/sub 2/, or MgSO/sub 4/ to the incubation medium. Nicotinic cholinergic agonists were potent inhibitors of nicotine binding; however, nicotinic antagonists were poor inhibitors. The regional distribution of binding was not uniform: midbrain and striatum contained the highest number of receptors, whereas cerebellum had the fewest. Differences in site densities, regional distribution, inhibitor potencies, and thermal denaturation indicated that nicotine binding was not the same as either QNB or alpha-BTX binding, and therefore that receptors for nicotine may represent a unique population of cholinergic receptors.« less

The Baculovirus-Expressed Binding Region of Plasmodium falciparum EBA-140 Ligand and Its Glycophorin C Binding Specificity

PubMed Central

Rydzak, Joanna; Kaczmarek, Radoslaw; Czerwinski, Marcin; Lukasiewicz, Jolanta; Tyborowska, Jolanta; Szewczyk, Boguslaw; Jaskiewicz, Ewa

2015-01-01

The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum DBL family of erythrocyte binding proteins, which are considered as prospective candidates for malaria vaccine development. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They share homology of domain structure, including Region II, which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte receptor interaction during invasion. In this report we describe, for the first time, the glycophorin C specificity of the recombinant, baculovirus-expressed binding region (Region II) of P. falciparum EBA-140 ligand. It was found that the recombinant EBA-140 Region II binds to the endogenous and recombinant glycophorin C, but does not bind to Gerbich-type glycophorin C, neither normal nor recombinant, which lacks amino acid residues 36–63 of its polypeptide chain. Our results emphasize the crucial role of this glycophorin C region in EBA-140 ligand binding. Moreover, the EBA-140 Region II did not bind either to glycophorin D, the truncated form of glycophorin C lacking the N-glycan or to desialylated GPC. These results draw attention to the role of glycophorin C glycans in EBA-140 binding. The full identification of the EBA-140 binding site on glycophorin C molecule, consisting most likely of its glycans and peptide backbone, may help to design therapeutics or vaccines that target the erythrocyte binding merozoite ligands. PMID:25588042

Behind the scenes of vitamin D binding protein: more than vitamin D binding.

PubMed

Delanghe, Joris R; Speeckaert, Reinhart; Speeckaert, Marijn M

2015-10-01

Although being discovered in 1959, the number of published papers in recent years reveals that vitamin D binding protein (DBP), a member of the albuminoid superfamily, is a hot research topic. Besides the three major phenotypes (DBP1F, DBP1S and DBP2), more than 120 unique variants have been described of this polymorphic protein. The presence of DBP has been demonstrated in different body fluids (serum, urine, breast milk, ascitic fluid, cerebrospinal fluid, saliva and seminal fluid) and organs (brain, heart, lungs, kidneys, placenta, spleen, testes and uterus). Although the major function is binding, solubilization and transport of vitamin D and its metabolites, the name of this glycoprotein hides numerous other important biological functions. In this review, we will focus on the analytical aspects of the determination of DBP and discuss in detail the multifunctional capacity [actin scavenging, binding of fatty acids, chemotaxis, binding of endotoxins, influence on T cell response and influence of vitamin D binding protein-macrophage activating factor (DBP-MAF) on bone metabolism and cancer] of this abundant plasma protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

Fine Specificity of Plasmodium vivax Duffy Binding Protein Binding Engagement of the Duffy Antigen on Human Erythrocytes

PubMed Central

Siddiqui, Asim A.; Xainli, Jia; Schloegel, Jesse; Carias, Lenore; Ntumngia, Francis; Shoham, Menachem; Casey, Joanne L.; Foley, Michael; Adams, John H.

2012-01-01

Plasmodium vivax invasion of human erythrocytes requires interaction of the P. vivax Duffy binding protein (PvDBP) with its host receptor, the Duffy antigen (Fy) on the erythrocyte surface. Consequently, PvDBP is a leading vaccine candidate. The binding domain of PvDBP lies in a cysteine-rich portion of the molecule called region II (PvDBPII). PvDBPII contains three distinct subdomains based upon intramolecular disulfide bonding patterns. Subdomain 2 (SD2) is highly polymorphic and is thought to contain many key residues for binding to Fy, while SD1 and SD3 are comparatively conserved and their role in Fy binding is not well understood. To examine the relative contributions of the different subdomains to binding to Fy and their abilities to elicit strain-transcending binding-inhibitory antibodies, we evaluated recombinant proteins from SD1+2, SD2, SD3, and SD3+, which includes 24 residues of SD2. All of the recombinant subdomains, except for SD2, bound variably to human erythrocytes, with constructs containing SD3 showing the best binding. Antisera raised in laboratory animals against SD3, SD3+, and SD2+3 inhibited the binding of full-length PvDBPII, which is strain transcending, whereas antisera generated to SD1+2 and SD2 failed to generate blocking antibodies. All of the murine monoclonal antibodies generated to full-length PvDBPII that had significant binding-inhibitory activity recognized only SD3. Thus, SD3 binds Fy and elicits blocking antibodies, indicating that it contains residues critical to Fy binding that could be the basis of a strain-transcending candidate vaccine against P. vivax. PMID:22615246

Fine specificity of Plasmodium vivax Duffy binding protein binding engagement of the Duffy antigen on human erythrocytes.

PubMed

Siddiqui, Asim A; Xainli, Jia; Schloegel, Jesse; Carias, Lenore; Ntumngia, Francis; Shoham, Menachem; Casey, Joanne L; Foley, Michael; Adams, John H; King, Christopher L

2012-08-01

Plasmodium vivax invasion of human erythrocytes requires interaction of the P. vivax Duffy binding protein (PvDBP) with its host receptor, the Duffy antigen (Fy) on the erythrocyte surface. Consequently, PvDBP is a leading vaccine candidate. The binding domain of PvDBP lies in a cysteine-rich portion of the molecule called region II (PvDBPII). PvDBPII contains three distinct subdomains based upon intramolecular disulfide bonding patterns. Subdomain 2 (SD2) is highly polymorphic and is thought to contain many key residues for binding to Fy, while SD1 and SD3 are comparatively conserved and their role in Fy binding is not well understood. To examine the relative contributions of the different subdomains to binding to Fy and their abilities to elicit strain-transcending binding-inhibitory antibodies, we evaluated recombinant proteins from SD1+2, SD2, SD3, and SD3+, which includes 24 residues of SD2. All of the recombinant subdomains, except for SD2, bound variably to human erythrocytes, with constructs containing SD3 showing the best binding. Antisera raised in laboratory animals against SD3, SD3+, and SD2+3 inhibited the binding of full-length PvDBPII, which is strain transcending, whereas antisera generated to SD1+2 and SD2 failed to generate blocking antibodies. All of the murine monoclonal antibodies generated to full-length PvDBPII that had significant binding-inhibitory activity recognized only SD3. Thus, SD3 binds Fy and elicits blocking antibodies, indicating that it contains residues critical to Fy binding that could be the basis of a strain-transcending candidate vaccine against P. vivax.

Molecular simulations of multimodal ligand-protein binding: elucidation of binding sites and correlation with experiments.

PubMed

Freed, Alexander S; Garde, Shekhar; Cramer, Steven M

2011-11-17

Multimodal chromatography, which employs more than one mode of interaction between ligands and proteins, has been shown to have unique selectivity and high efficacy for protein purification. To test the ability of free solution molecular dynamics (MD) simulations in explicit water to identify binding regions on the protein surface and to shed light on the "pseudo affinity" nature of multimodal interactions, we performed MD simulations of a model protein ubiquitin in aqueous solution of free ligands. Comparisons of MD with NMR spectroscopy of ubiquitin mutants in solutions of free ligands show a good agreement between the two with regard to the preferred binding region on the surface of the protein and several binding sites. MD simulations also identify additional binding sites that were not observed in the NMR experiments. "Bound" ligands were found to be sufficiently flexible and to access a number of favorable conformations, suggesting only a moderate loss of ligand entropy in the "pseudo affinity" binding of these multimodal ligands. Analysis of locations of chemical subunits of the ligand on the protein surface indicated that electrostatic interaction units were located on the periphery of the preferred binding region on the protein. The analysis of the electrostatic potential, the hydrophobicity maps, and the binding of both acetate and benzene probes were used to further study the localization of individual ligand moieties. These results suggest that water-mediated electrostatic interactions help the localization and orientation of the MM ligand to the binding region with additional stability provided by nonspecific hydrophobic interactions.

How Proteins Bind Macrocycles

PubMed Central

Villar, Elizabeth A.; Beglov, Dmitri; Chennamadhavuni, Spandan; Porco, John A.; Kozakov, Dima; Vajda, Sandor; Whitty, Adrian

2014-01-01

The potential utility of synthetic macrocycles as drugs, particularly against low druggability targets such as protein-protein interactions, has been widely discussed. There is little information, however, to guide the design of macrocycles for good target protein-binding activity or bioavailability. To address this knowledge gap we analyze the binding modes of a representative set of macrocycle-protein complexes. The results, combined with consideration of the physicochemical properties of approved macrocyclic drugs, allow us to propose specific guidelines for the design of synthetic macrocycles libraries possessing structural and physicochemical features likely to favor strong binding to protein targets and also good bioavailability. We additionally provide evidence that large, natural product derived macrocycles can bind to targets that are not druggable by conventional, drug-like compounds, supporting the notion that natural product inspired synthetic macrocycles can expand the number of proteins that are druggable by synthetic small molecules. PMID:25038790

A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants

NASA Technical Reports Server (NTRS)

Yang, Tianbao; Poovaiah, B. W.

2002-01-01

We reported earlier that the tobacco early ethylene-responsive gene NtER1 encodes a calmodulin-binding protein (Yang, T., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 38467-38473). Here we demonstrate that there is one NtER1 homolog as well as five related genes in Arabidopsis. These six genes are rapidly and differentially induced by environmental signals such as temperature extremes, UVB, salt, and wounding; hormones such as ethylene and abscisic acid; and signal molecules such as methyl jasmonate, H(2)O(2), and salicylic acid. Hence, they were designated as AtSR1-6 (Arabidopsis thaliana signal-responsive genes). Ca(2+)/calmodulin binds to all AtSRs, and their calmodulin-binding regions are located on a conserved basic amphiphilic alpha-helical motif in the C terminus. AtSR1 targets the nucleus and specifically recognizes a novel 6-bp CGCG box (A/C/G)CGCG(G/T/C). The multiple CGCG cis-elements are found in promoters of genes such as those involved in ethylene signaling, abscisic acid signaling, and light signal perception. The DNA-binding domain in AtSR1 is located on the N-terminal 146 bp where all AtSR1-related proteins share high similarity but have no similarity to other known DNA-binding proteins. The calmodulin-binding nuclear proteins isolated from wounded leaves exhibit specific CGCG box DNA binding activities. These results suggest that the AtSR gene family encodes a family of calmodulin-binding/DNA-binding proteins involved in multiple signal transduction pathways in plants.

Thyroid function and cold acclimation in the hamster, Mesocricetus auratus

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

Tomasi, T.E.; Horwitz, B.A.

1987-02-01

Basal metabolic rate (BMR), thyroxine utilization rate (T4U), and triiodothyronine utilization rate (T3U) were measured in cold-acclimated (CA) and room temperature-acclimated (RA) male golden hamsters, Mesocricetus auratus. Hormone utilization rates were calculated via the plasma disappearance technique using SVI-labeled hormones and measuring serum hormone levels via radioimmunoassay. BMR showed a significant 28% increase with cold acclimation. The same cold exposure also produced a 32% increase in T4U, and a 204% increase in T3U. The much greater increase in T3U implies that previous assessments of the relationship between cold acclimation and thyroid function may have been underestimated and that cold exposuremore » induces both quantitative and qualitative changes in thyroid function. It is concluded that in the cold-acclimated state, T3U more accurately reflects thyroid function than does T4U. A mechanism for the cold-induced change in BMR is proposed.« less

Energy balance in lactating undernourished Indian women.

PubMed

Madhavapeddi, R; Rao, B S

1992-05-01

An energy balance study was conducted in eight lactating poor-income Indian women from delivery to 6 months. Energy intake and expenditure were assessed for 7 days every month (30-37 days). Every month, basal metabolic rate (BMR) and milk ingested by infants was measured. An energy balance was computed. As a group these women were in energy balance, indicated by small body weight changes with respect to time. However, only two of these women were in a positive energy balance. Women with higher body weight lost more weight. Estimated mean energy intake was higher than energy expenditure. BMR showed a slight but not significant fall during the second month of lactation and was not different from the BMR seen in 13 non-pregnant, non-lactating women matched for body weight from the staff of the Institute. The energy cost of lactation was 2.3 MJ (549 kcal), a figure that justifies the Recommended Dietary Allowance for energy recommended by FAO/WHO/UNU (1985) and ICMR (1989).

Analysis of Ethylene Receptors: Ethylene-Binding Assays.

PubMed

Binder, Brad M; Schaller, G Eric

2017-01-01

Plant ethylene receptors bind ethylene with high affinity. Most of the characterization of ethylene binding to the receptors has been carried out using a radioligand-binding assay on functional receptors expressed in yeast. In this chapter, we describe methods for expressing ethylene receptors in yeast and conducting ethylene-binding assays on intact yeast and yeast membranes. The ethylene-binding assays can be modified to analyze ethylene binding to intact plants and other organisms as well as membranes isolated from any biological source.

Accurate pan-specific prediction of peptide-MHC class II binding affinity with improved binding core identification.

PubMed

Andreatta, Massimo; Karosiene, Edita; Rasmussen, Michael; Stryhn, Anette; Buus, Søren; Nielsen, Morten

2015-11-01

A key event in the generation of a cellular response against malicious organisms through the endocytic pathway is binding of peptidic antigens by major histocompatibility complex class II (MHC class II) molecules. The bound peptide is then presented on the cell surface where it can be recognized by T helper lymphocytes. NetMHCIIpan is a state-of-the-art method for the quantitative prediction of peptide binding to any human or mouse MHC class II molecule of known sequence. In this paper, we describe an updated version of the method with improved peptide binding register identification. Binding register prediction is concerned with determining the minimal core region of nine residues directly in contact with the MHC binding cleft, a crucial piece of information both for the identification and design of CD4(+) T cell antigens. When applied to a set of 51 crystal structures of peptide-MHC complexes with known binding registers, the new method NetMHCIIpan-3.1 significantly outperformed the earlier 3.0 version. We illustrate the impact of accurate binding core identification for the interpretation of T cell cross-reactivity using tetramer double staining with a CMV epitope and its variants mapped to the epitope binding core. NetMHCIIpan is publicly available at http://www.cbs.dtu.dk/services/NetMHCIIpan-3.1 .

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

Enhanced protective antibody to a mutant meningococcal factor H-binding protein with low-factor H binding

PubMed Central

Granoff, Dan M.; Giuntini, Serena; Gowans, Flor A.; Lujan, Eduardo; Sharkey, Kelsey; Beernink, Peter T.

2016-01-01

Meningococcal factor H-binding protein (FHbp) is an antigen in 2 serogroup B meningococcal vaccines. FHbp specifically binds human and some nonhuman primate complement FH. To investigate the effect of binding of FH to FHbp on protective antibody responses, we immunized infant rhesus macaques with either a control recombinant FHbp antigen that bound macaque FH or a mutant antigen with 2 amino acid substitutions and >250-fold lower affinity for FH. The mutant antigen elicited 3-fold higher serum IgG anti-FHbp titers and up to 15-fold higher serum bactericidal titers than the control FHbp vaccine. When comparing sera with similar IgG anti-FHbp titers, the antibodies elicited by the mutant antigen gave greater deposition of complement component C4b on live meningococci (classical complement pathway) and inhibited binding of FH, while the anti-FHbp antibodies elicited by the control vaccine enhanced FH binding. Thus, the mutant FHbp vaccine elicited an anti-FHbp antibody repertoire directed at FHbp epitopes within the FH binding site, which resulted in greater protective activity than the antibodies elicited by the control vaccine, which targeted FHbp epitopes outside of the FH combining site. Binding of a host protein to a vaccine antigen impairs protective antibody responses, which can be overcome with low-binding mutant antigens. PMID:27668287

The Verrucomicrobia LexA-Binding Motif: Insights into the Evolutionary Dynamics of the SOS Response.

PubMed

Erill, Ivan; Campoy, Susana; Kılıç, Sefa; Barbé, Jordi

2016-01-01

The SOS response is the primary bacterial mechanism to address DNA damage, coordinating multiple cellular processes that include DNA repair, cell division, and translesion synthesis. In contrast to other regulatory systems, the composition of the SOS genetic network and the binding motif of its transcriptional repressor, LexA, have been shown to vary greatly across bacterial clades, making it an ideal system to study the co-evolution of transcription factors and their regulons. Leveraging comparative genomics approaches and prior knowledge on the core SOS regulon, here we define the binding motif of the Verrucomicrobia, a recently described phylum of emerging interest due to its association with eukaryotic hosts. Site directed mutagenesis of the Verrucomicrobium spinosum recA promoter confirms that LexA binds a 14 bp palindromic motif with consensus sequence TGTTC-N4-GAACA. Computational analyses suggest that recognition of this novel motif is determined primarily by changes in base-contacting residues of the third alpha helix of the LexA helix-turn-helix DNA binding motif. In conjunction with comparative genomics analysis of the LexA regulon in the Verrucomicrobia phylum, electrophoretic shift assays reveal that LexA binds to operators in the promoter region of DNA repair genes and a mutagenesis cassette in this organism, and identify previously unreported components of the SOS response. The identification of tandem LexA-binding sites generating instances of other LexA-binding motifs in the lexA gene promoter of Verrucomicrobia species leads us to postulate a novel mechanism for LexA-binding motif evolution. This model, based on gene duplication, successfully addresses outstanding questions in the intricate co-evolution of the LexA protein, its binding motif and the regulatory network it controls.

Structural analysis of ibuprofen binding to human adipocyte fatty-acid binding protein (FABP4).

PubMed

González, Javier M; Fisher, S Zoë

2015-02-01

Inhibition of human adipocyte fatty-acid binding protein (FABP4) has been proposed as a treatment for type 2 diabetes, fatty liver disease and atherosclerosis. However, FABP4 displays a naturally low selectivity towards hydrophobic ligands, leading to the possibility of side effects arising from cross-inhibition of other FABP isoforms. In a search for structural determinants of ligand-binding selectivity, the binding of FABP4 towards a group of small molecules structurally related to the nonsteroidal anti-inflammatory drug ibuprofen was analyzed through X-ray crystallography. Several specific hydrophobic interactions are shown to enhance the binding affinities of these compounds, whereas an aromatic edge-to-face interaction is proposed to determine the conformation of bound ligands, highlighting the importance of aromatic interactions in hydrophobic environments.

Cd-binding to model membranes

NASA Astrophysics Data System (ADS)

Geszner, R.; Saibene, S.; Butz, T.; Lerf, A.

1990-08-01

The binding of Cd2+ to the model membranes Di-myristoyl L-α-phosphatidic acid (DMPA) and Di-myristoyl L-α-phosphatidylcholine (DMPC) was studied by time differential perturbed angular correlation (TDPAC) on111mCd, via its nuclear quadrupole interaction. Whereas Cd2+ does not bind to the neutral DMPC, it binds to charged DMPA up to a 0.8∶1 Cd/lipid ratio.

Dynamic factors affecting gaseous ligand binding in an artificial oxygen transport protein.

PubMed

Zhang, Lei; Andersen, Eskil M E; Khajo, Abdelahad; Magliozzo, Richard S; Koder, Ronald L

2013-01-22

We report the functional analysis of an artificial hexacoordinate oxygen transport protein, HP7, which operates via a mechanism similar to that of human neuroglobin and cytoglobin: the destabilization of one of two heme-ligating histidine residues. In the case of HP7, this is the result of the coupling of histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Here we compare gaseous ligand binding, including rates, affinities, and oxyferrous state lifetimes, of both heme binding sites in HP7. We find that despite the identical sequence of helices in both binding sites, there are differences in oxygen affinity and oxyferrous state lifetime that may be the result of differences in the freedom of motion imposed by the candelabra fold on the two sites of the protein. We further examine the effect of mutational removal of the buried glutamates on function. Heme iron in the ferrous state of this mutant is rapidly oxidized when exposed to oxygen. Compared to that of HP7, the distal histidine affinity is increased by a 22-fold decrease in the histidine ligand off rate. Electron paramagnetic resonance comparison of these ferric hemoproteins demonstrates that the mutation increases the level of disorder at the heme binding site. Nuclear magnetic resonance-detected deuterium exchange demonstrates that the mutation greatly increases the degree of penetration of water into the protein core. The inability of the mutant protein to bind oxygen may be due to an increased level of water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors. Together, these data underline the importance of the control of protein dynamics in the design of functional artificial proteins.

Binding Properties of General Odorant Binding Proteins from the Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae)

PubMed Central

Li, Guangwei; Chen, Xiulin; Li, Boliao; Zhang, Guohui; Li, Yiping; Wu, Junxiang

2016-01-01

Background The oriental fruit moth Grapholita molesta is a host-switching pest species. The adults highly depend on olfactory cues in locating optimal host plants and oviposition sites. Odorant binding proteins (OBPs) are thought to be responsible for recognizing and transporting hydrophobic odorants across the aqueous sensillum lymph to stimulate the odorant receptors (ORs) within the antennal sensilla and activate the olfactory signal transduction pathway. Exploring the physiological function of these OBPs could facilitate understanding insect chemical communications. Methodology/Principal Finding Two antennae-specific general OBPs (GOBPs) of G. molesta were expressed and purified in vitro. The binding affinities of G. molesta GOBP1 and 2 (GmolGOBP1 and 2) for sex pheromone components and host plant volatiles were measured by fluorescence ligand-binding assays. The distribution of GmolGOBP1 and 2 in the antennal sensillum were defined by whole mount fluorescence immunohistochemistry (WM-FIHC) experiments. The binding sites of GmolGOBP2 were predicted using homology modeling, molecular docking and site-directed mutagenesis. Both GmolGOBP1 and 2 are housing in sensilla basiconica and with no differences in male and female antennae. Recombinant GmolGOBP1 (rGmolGOBP1) exhibited broad binding properties towards host plant volatiles and sex pheromone components; rGmolGOBP2 could not effectively bind host plant volatiles but showed specific binding affinity with a minor sex pheromone component dodecanol. We chose GmolGOBP2 and dodecanol for further homology modeling, molecular docking, and site-directed mutagenesis. Binding affinities of mutants demonstrated that Thr9 was the key binding site and confirmed dodecanol bonding to protein involves a hydrogen bond. Combined with the pH effect on binding affinities of rGmolGOBP2, ligand binding and release of GmolGOBP2 were related to a pH-dependent conformational transition. Conclusion Two rGmolGOBPs exhibit different

The binding cavity of mouse major urinary protein is optimised for a variety of ligand binding modes

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

Pertinhez, Thelma A.; Ferrari, Elena; Casali, Emanuela

2009-12-25

{sup 15}N and {sup 1}HN chemical shift data and {sup 15}N relaxation studies have been used to characterise the binding of N-phenyl-naphthylamine (NPN) to mouse major urinary protein (MUP). NPN binds in the {beta}-barrel cavity of MUP, hydrogen bonding to Tyr120 and making extensive non-bonded contacts with hydrophobic side chains. In contrast to the natural pheromone 2-sec-butyl-4,5-dihydrothiazole, NPN binding gives no change to the overall mobility of the protein backbone of MUP. Comparison with 11 different ligands that bind to MUP shows a range of binding modes involving 16 different residues in the {beta}-barrel cavity. These finding justify why MUPmore » is able to adapt to allow for many successful binding partners.« less

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.

Sugar-Binding Profiles of Chitin-Binding Lectins from the Hevein Family: A Comprehensive Study

PubMed Central

Itakura, Yoko; Nakamura-Tsuruta, Sachiko; Kominami, Junko; Tateno, Hiroaki; Hirabayashi, Jun

2017-01-01

Chitin-binding lectins form the hevein family in plants, which are defined by the presence of single or multiple structurally conserved GlcNAc (N-acetylglucosamine)-binding domains. Although they have been used as probes for chito-oligosaccharides, their detailed specificities remain to be investigated. In this study, we analyzed six chitin-binding lectins, DSA, LEL, PWM, STL, UDA, and WGA, by quantitative frontal affinity chromatography. Some novel features were evident: WGA showed almost comparable affinity for pyridylaminated chitotriose and chitotetraose, while LEL and UDA showed much weaker affinity, and DSA, PWM, and STL had no substantial affinity for the former. WGA showed selective affinity for hybrid-type N-glycans harboring a bisecting GlcNAc residue. UDA showed extensive binding to high-mannose type N-glycans, with affinity increasing with the number of Man residues. DSA showed the highest affinity for highly branched N-glycans consisting of type II LacNAc (N-acetyllactosamine). Further, multivalent features of these lectins were investigated by using glycoconjugate and lectin microarrays. The lectins showed substantial binding to immobilized LacNAc as well as chito-oligosaccharides, although the extents to which they bound varied among them. WGA showed strong binding to heavily sialylated glycoproteins. The above observations will help interpret lectin-glycoprotein interactions in histochemical studies and glyco-biomarker investigations. PMID:28556796

DNABP: Identification of DNA-Binding Proteins Based on Feature Selection Using a Random Forest and Predicting Binding Residues.

PubMed

Ma, Xin; Guo, Jing; Sun, Xiao

2016-01-01

DNA-binding proteins are fundamentally important in cellular processes. Several computational-based methods have been developed to improve the prediction of DNA-binding proteins in previous years. However, insufficient work has been done on the prediction of DNA-binding proteins from protein sequence information. In this paper, a novel predictor, DNABP (DNA-binding proteins), was designed to predict DNA-binding proteins using the random forest (RF) classifier with a hybrid feature. The hybrid feature contains two types of novel sequence features, which reflect information about the conservation of physicochemical properties of the amino acids, and the binding propensity of DNA-binding residues and non-binding propensities of non-binding residues. The comparisons with each feature demonstrated that these two novel features contributed most to the improvement in predictive ability. Furthermore, to improve the prediction performance of the DNABP model, feature selection using the minimum redundancy maximum relevance (mRMR) method combined with incremental feature selection (IFS) was carried out during the model construction. The results showed that the DNABP model could achieve 86.90% accuracy, 83.76% sensitivity, 90.03% specificity and a Matthews correlation coefficient of 0.727. High prediction accuracy and performance comparisons with previous research suggested that DNABP could be a useful approach to identify DNA-binding proteins from sequence information. The DNABP web server system is freely available at http://www.cbi.seu.edu.cn/DNABP/.

Modulation of ouabain binding and potassium pump fluxes by cellular sodium and potassium in human and sheep erythrocytes.

PubMed Central

Joiner, C H; Lauf, P K

1978-01-01

1. Erythrocytes were treated with nystatin to alter internal Na (Nai) and K (Ki) composition. Although the rates of K pumping and [3H]ouabain binding were altered dramatically, the relationship between glycoside binding and K pump inhibition was unaffected. 2. Human cells with high Nai and low Ki exhibited an increased rate of ouabain binding as compared to high Ki, low Nai cells; this paralleled the stimulated K pump activity of high Nai cells. 3. At constant Ki, increasing internal Na stimulated K pump and ouabain binding rates concomitantly. 4. At low Nai, increasing Ki inhibited both K pumping and ouabain binding. However, at high Nai, increasing Ki from 4 to 44 mM stimulated the rate of glycoside binding, parallel to its effect of increasing the rate of active K influx. 5. Anti-L, an isoantibody to low K (LK) sheep red cells, increased the rate of ouabain binding via its stimulation of K pump turnover. Since the latter effect is the result of affinity changes at the internal cation activation site(s) of the pump (Lauf, Rasmusen, Hoffman, Dunham, Cook, Parmelee & Tosteson, 1970), the antibody's effect on ouabain binding reflected the positive correlation between the rates of K pump turnover and glycoside binding. 6. These data provide the first evidence in intact cells for the occurrence of a Nai-induced conformational change in the Na/K pump during its normal operational cycle. PMID:722574

Accurate prediction of RNA-binding protein residues with two discriminative structural descriptors.

PubMed

Sun, Meijian; Wang, Xia; Zou, Chuanxin; He, Zenghui; Liu, Wei; Li, Honglin

2016-06-07

RNA-binding proteins participate in many important biological processes concerning RNA-mediated gene regulation, and several computational methods have been recently developed to predict the protein-RNA interactions of RNA-binding proteins. Newly developed discriminative descriptors will help to improve the prediction accuracy of these prediction methods and provide further meaningful information for researchers. In this work, we designed two structural features (residue electrostatic surface potential and triplet interface propensity) and according to the statistical and structural analysis of protein-RNA complexes, the two features were powerful for identifying RNA-binding protein residues. Using these two features and other excellent structure- and sequence-based features, a random forest classifier was constructed to predict RNA-binding residues. The area under the receiver operating characteristic curve (AUC) of five-fold cross-validation for our method on training set RBP195 was 0.900, and when applied to the test set RBP68, the prediction accuracy (ACC) was 0.868, and the F-score was 0.631. The good prediction performance of our method revealed that the two newly designed descriptors could be discriminative for inferring protein residues interacting with RNAs. To facilitate the use of our method, a web-server called RNAProSite, which implements the proposed method, was constructed and is freely available at http://lilab.ecust.edu.cn/NABind .

Trigger Factor and DnaK possess overlapping substrate pools and binding specificities.

PubMed

Deuerling, Elke; Patzelt, Holger; Vorderwülbecke, Sonja; Rauch, Thomas; Kramer, Günter; Schaffitzel, Elke; Mogk, Axel; Schulze-Specking, Agnes; Langen, Hanno; Bukau, Bernd

2003-03-01

Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.

Binding Modes of Ligands Using Enhanced Sampling (BLUES): Rapid Decorrelation of Ligand Binding Modes via Nonequilibrium Candidate Monte Carlo.

PubMed

Gill, Samuel C; Lim, Nathan M; Grinaway, Patrick B; Rustenburg, Ariën S; Fass, Josh; Ross, Gregory A; Chodera, John D; Mobley, David L

2018-05-31

Accurately predicting protein-ligand binding affinities and binding modes is a major goal in computational chemistry, but even the prediction of ligand binding modes in proteins poses major challenges. Here, we focus on solving the binding mode prediction problem for rigid fragments. That is, we focus on computing the dominant placement, conformation, and orientations of a relatively rigid, fragment-like ligand in a receptor, and the populations of the multiple binding modes which may be relevant. This problem is important in its own right, but is even more timely given the recent success of alchemical free energy calculations. Alchemical calculations are increasingly used to predict binding free energies of ligands to receptors. However, the accuracy of these calculations is dependent on proper sampling of the relevant ligand binding modes. Unfortunately, ligand binding modes may often be uncertain, hard to predict, and/or slow to interconvert on simulation time scales, so proper sampling with current techniques can require prohibitively long simulations. We need new methods which dramatically improve sampling of ligand binding modes. Here, we develop and apply a nonequilibrium candidate Monte Carlo (NCMC) method to improve sampling of ligand binding modes. In this technique, the ligand is rotated and subsequently allowed to relax in its new position through alchemical perturbation before accepting or rejecting the rotation and relaxation as a nonequilibrium Monte Carlo move. When applied to a T4 lysozyme model binding system, this NCMC method shows over 2 orders of magnitude improvement in binding mode sampling efficiency compared to a brute force molecular dynamics simulation. This is a first step toward applying this methodology to pharmaceutically relevant binding of fragments and, eventually, drug-like molecules. We are making this approach available via our new Binding modes of ligands using enhanced sampling (BLUES) package which is freely available on GitHub.

Multiple Ion Binding Equilibria, Reaction Kinetics, and Thermodynamics in Dynamic Models of Biochemical Pathways

PubMed Central

Vinnakota, Kalyan C.; Wu, Fan; Kushmerick, Martin J.; Beard, Daniel A.

2009-01-01

The operation of biochemical systems in vivo and in vitro is strongly influenced by complex interactions between biochemical reactants and ions such as H+, Mg2+, K+, and Ca2+. These are important second messengers in metabolic and signaling pathways that directly influence the kinetics and thermodynamics of biochemical systems. Herein we describe the biophysical theory and computational methods to account for multiple ion binding to biochemical reactants and demonstrate the crucial effects of ion binding on biochemical reaction kinetics and thermodynamics. In simulations of realistic systems, the concentrations of these ions change with time due to dynamic buffering and competitive binding. In turn, the effective thermodynamic properties vary as functions of cation concentrations and important environmental variables such as temperature and overall ionic strength. Physically realistic simulations of biochemical systems require incorporating all of these phenomena into a coherent mathematical description. Several applications to physiological systems are demonstrated based on this coherent simulation framework. PMID:19216922

Characterization of nicotine binding in mouse brain and comparison with the binding of alpha-bungarotoxin and quinuclidinyl benzilate

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

Marks, M.J.; Collins, A.C.

1982-11-01

The binding of (/sup 3/H)nicotine to mouse brain has been measured and subsequently compared with the binding of (/sup 125/I)alpha-bungarotoxin (alpha-BTX) and L-(/sup 3/H)quinuclidinyl benzilate (QNB). The binding of nicotine was saturable, reversible, and stereospecific. Although the rates of association and dissociation of nicotine were temperature-dependent, the incubation temperature had no effect on either KD or Bmax. Nicotine binding was unaffected by the addition of NaCl, KCl, CaCl/sub 2/, or MgSO/sub 4/ to the incubation medium. Nicotinic cholinergic agonists were potent inhibitors of nicotine binding; however, nicotinic antagonists were poor inhibitors. The regional distribution of binding was not uniform: midbrainmore » and striatum contained the highest number of receptors, whereas cerebellum had the fewest. Differences in site densities, regional distribution, inhibitor potencies, and thermal denaturation indicated that nicotine binding was not the same as either QNB or alpha-BTX binding, and therefore that receptors for nicotine may represent a unique population of cholinergic receptors.« less

DNA-aptamers binding aminoglycoside antibiotics.

PubMed

Nikolaus, Nadia; Strehlitz, Beate

2014-02-21

Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminoglycoside antibiotic kanamycin A with the aim of constructing a robust and functional assay that can be used for water analysis. With this work we show that aptamers that were derived from a Capture-SELEX procedure targeting against kanamycin A also display binding to related aminoglycoside antibiotics. The binding patterns differ among all tested aptamers so that there are highly substance specific aptamers and more group specific aptamers binding to a different variety of aminoglycoside antibiotics. Also the region of the aminoglycoside antibiotics responsible for aptamer binding can be estimated. Affinities of the different aptamers for their target substance, kanamycin A, are measured with different approaches and are in the micromolar range. Finally, the proof of principle of an assay for detection of kanamycin A in a real water sample is given.

An RNA motif that binds ATP

NASA Technical Reports Server (NTRS)

Sassanfar, M.; Szostak, J. W.

1993-01-01

RNAs that contain specific high-affinity binding sites for small molecule ligands immobilized on a solid support are present at a frequency of roughly one in 10(10)-10(11) in pools of random sequence RNA molecules. Here we describe a new in vitro selection procedure designed to ensure the isolation of RNAs that bind the ligand of interest in solution as well as on a solid support. We have used this method to isolate a remarkably small RNA motif that binds ATP, a substrate in numerous biological reactions and the universal biological high-energy intermediate. The selected ATP-binding RNAs contain a consensus sequence, embedded in a common secondary structure. The binding properties of ATP analogues and modified RNAs show that the binding interaction is characterized by a large number of close contacts between the ATP and RNA, and by a change in the conformation of the RNA.

Functions of Intracellular Retinoid Binding-Proteins.

PubMed

Napoli, Joseph L

Multiple binding and transport proteins facilitate many aspects of retinoid biology through effects on retinoid transport, cellular uptake, metabolism, and nuclear delivery. These include the serum retinol binding protein sRBP (aka Rbp4), the plasma membrane sRBP receptor Stra6, and the intracellular retinoid binding-proteins such as cellular retinol-binding proteins (CRBP) and cellular retinoic acid binding-proteins (CRABP). sRBP transports the highly lipophilic retinol through an aqueous medium. The major intracellular retinol-binding protein, CRBP1, likely enhances efficient retinoid use by providing a sink to facilitate retinol uptake from sRBP through the plasma membrane or via Stra6, delivering retinol or retinal to select enzymes that generate retinyl esters or retinoic acid, and protecting retinol/retinal from excess catabolism or opportunistic metabolism. Intracellular retinoic acid binding-proteins (CRABP1 and 2, and FABP5) seem to have more diverse functions distinctive to each, such as directing retinoic acid to catabolism, delivering retinoic acid to specific nuclear receptors, and generating non-canonical actions. Gene ablation of intracellular retinoid binding-proteins does not cause embryonic lethality or gross morphological defects. Metabolic and functional defects manifested in knockouts of CRBP1, CRBP2 and CRBP3, however, illustrate their essentiality to health, and in the case of CRBP2, to survival during limited dietary vitamin A. Future studies should continue to address the specific molecular interactions that occur between retinoid binding-proteins and their targets and their precise physiologic contributions to retinoid homeostasis and function.

Plasma sex steroid binding in Chiroptera.

PubMed

Kwiecinski, G G; Damassa, D A; Gustafson, A W; Armao, M E

1987-04-01

Plasma steroid binding was examined in samples obtained from seven species of bats representing four different families. A specific sex steroid-binding protein (SBP) was identified by steady-state polyacrylamide gel electrophoresis in representatives of two families, the phyllostomids and the vespertilionids. In these species, as in primates, SBP not only exhibited high affinity for the androgens testosterone and dihydrotestosterone (DHT), but also for estradiol. A specific SBP was not identified in the tropical American vampire bat or in the two species of pteropodids examined. In all species examined, except for the vampire bat, a specific corticosteroid-binding globulin (CBG) was also identified. In addition to binding glucocorticoids, CBG in these species appeared to bind androgens as well.

The Staphylococcus aureus pSK41 plasmid-encoded ArtA protein is a master regulator of plasmid transmission genes and contains a RHH motif used in alternate DNA-binding modes.

PubMed

Ni, Lisheng; Jensen, Slade O; Ky Tonthat, Nam; Berg, Tracey; Kwong, Stephen M; Guan, Fiona H X; Brown, Melissa H; Skurray, Ronald A; Firth, Neville; Schumacher, Maria A

2009-11-01

Plasmids harbored by Staphylococcus aureus are a major contributor to the spread of bacterial multi-drug resistance. Plasmid conjugation and partition are critical to the dissemination and inheritance of such plasmids. Here, we demonstrate that the ArtA protein encoded by the S. aureus multi-resistance plasmid pSK41 is a global transcriptional regulator of pSK41 genes, including those involved in conjugation and segregation. ArtA shows no sequence homology to any structurally characterized DNA-binding protein. To elucidate the mechanism by which it specifically recognizes its DNA site, we obtained the structure of ArtA bound to its cognate operator, ACATGACATG. The structure reveals that ArtA is representative of a new family of ribbon-helix-helix (RHH) DNA-binding proteins that contain extended, N-terminal basic motifs. Strikingly, unlike most well-studied RHH proteins ArtA binds its cognate operators as a dimer. However, we demonstrate that it is also able to recognize an atypical operator site by binding as a dimer-of-dimers and the extended N-terminal regions of ArtA were shown to be essential for this dimer-of-dimer binding mode. Thus, these data indicate that ArtA is a master regulator of genes critical for both horizontal and vertical transmission of pSK41 and that it can recognize DNA utilizing alternate binding modes.

The Staphylococcus aureus pSK41 plasmid-encoded ArtA protein is a master regulator of plasmid transmission genes and contains a RHH motif used in alternate DNA-binding modes

PubMed Central

Ni, Lisheng; Jensen, Slade O.; Ky Tonthat, Nam; Berg, Tracey; Kwong, Stephen M.; Guan, Fiona H. X.; Brown, Melissa H.; Skurray, Ronald A.; Firth, Neville; Schumacher, Maria A.

2009-01-01

Plasmids harbored by Staphylococcus aureus are a major contributor to the spread of bacterial multi-drug resistance. Plasmid conjugation and partition are critical to the dissemination and inheritance of such plasmids. Here, we demonstrate that the ArtA protein encoded by the S. aureus multi-resistance plasmid pSK41 is a global transcriptional regulator of pSK41 genes, including those involved in conjugation and segregation. ArtA shows no sequence homology to any structurally characterized DNA-binding protein. To elucidate the mechanism by which it specifically recognizes its DNA site, we obtained the structure of ArtA bound to its cognate operator, ACATGACATG. The structure reveals that ArtA is representative of a new family of ribbon–helix–helix (RHH) DNA-binding proteins that contain extended, N-terminal basic motifs. Strikingly, unlike most well-studied RHH proteins ArtA binds its cognate operators as a dimer. However, we demonstrate that it is also able to recognize an atypical operator site by binding as a dimer-of-dimers and the extended N-terminal regions of ArtA were shown to be essential for this dimer-of-dimer binding mode. Thus, these data indicate that ArtA is a master regulator of genes critical for both horizontal and vertical transmission of pSK41 and that it can recognize DNA utilizing alternate binding modes. PMID:19759211

Neuroscience: toward unbinding the binding problem.

PubMed

Whitney, David

2009-03-24

How the brain 'binds' information to create a coherent perceptual experience is an enduring question. Recent research in the psychophysics of perceptual binding and developments in fMRI analysis techniques are bringing us closer to an understanding of how the brain solves the binding problem.

Multiple conformations of the cytidine repressor DNA-binding domain coalesce to one upon recognition of a specific DNA surface.

PubMed

Moody, Colleen L; Tretyachenko-Ladokhina, Vira; Laue, Thomas M; Senear, Donald F; Cocco, Melanie J

2011-08-09

The cytidine repressor (CytR) is a member of the LacR family of bacterial repressors with distinct functional features. The Escherichia coli CytR regulon comprises nine operons whose palindromic operators vary in both sequence and, most significantly, spacing between the recognition half-sites. This suggests a strong likelihood that protein folding would be coupled to DNA binding as a mechanism to accommodate the variety of different operator architectures to which CytR is targeted. Such coupling is a common feature of sequence-specific DNA-binding proteins, including the LacR family repressors; however, there are no significant structural rearrangements upon DNA binding within the three-helix DNA-binding domains (DBDs) studied to date. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the CytR DBD free in solution and to determine the high-resolution structure of a CytR DBD monomer bound specifically to one DNA half-site of the uridine phosphorylase (udp) operator. We find that the free DBD populates multiple distinct conformations distinguished by up to four sets of NMR peaks per residue. This structural heterogeneity is previously unknown in the LacR family. These stable structures coalesce into a single, more stable udp-bound form that features a three-helix bundle containing a canonical helix-turn-helix motif. However, this structure differs from all other LacR family members whose structures are known with regard to the packing of the helices and consequently their relative orientations. Aspects of CytR activity are unique among repressors; we identify here structural properties that are also distinct and that might underlie the different functional properties. © 2011 American Chemical Society

Binding of corroded ions to human saliva.

PubMed

Mueller, H J

1985-05-01

Employing equilibrium dialysis, the binding abilities of Cu, Al, Co and Cr ions from corroded Cu-Al and Co-Cr dental casting alloys towards human saliva and two of its gel chromatographic fractions were determined. Results indicate that both Cu and Co bind to human saliva i.e. 0.045 and 0.027 mg/mg protein, respectively. Besides possessing the largest binding ability, Cu also possessed the largest binding capacity. The saturation of Cu binding was not reached up to the limit of 0.35 mg protein/ml employed in the tests, while Co reached full saturation at about 0.2 mg protein/ml. Chromium showed absolutely no binding to human saliva while Al ions did not pass through the dialysis membranes. Compared to the binding with solutions that were synthetically made up to contain added salivary-type proteins, it is shown that the binding to human saliva is about 1 order of magnitude larger, at least for Cu ions.

Feature binding in visual short-term memory is unaffected by task-irrelevant changes of location, shape, and color.

PubMed

Logie, Robert H; Brockmole, James R; Jaswal, Snehlata

2011-01-01

Three experiments used a change detection paradigm across a range of study-test intervals to address the respective contributions of location, shape, and color to the formation of bindings of features in sensory memory and visual short-term memory (VSTM). In Experiment 1, location was designated task irrelevant and was randomized between study and test displays. The task was to detect changes in the bindings between shape and color. In Experiments 2 and 3, shape and color, respectively, were task irrelevant and randomized, with bindings tested between location and color (Experiment 2) and location and shape (Experiment 3). At shorter study-test intervals, randomizing location was most disruptive, followed by shape and then color. At longer intervals, randomizing any task-irrelevant feature had no impact on change detection for bindings between features, and location had no special role. Results suggest that location is crucial for initial perceptual binding but loses that special status once representations are formed in VSTM, which operates according to different principles, than do visual attention and perception.

The effect of hybrid type and dietary proportions of corn silage on the lactation performance of high-producing dairy cows.

PubMed

Lim, J M; Nestor, K E; Kung, L

2015-02-01

We evaluated the effects of corn silage hybrids [control vs. brown midrib (BMR)] and the proportion of corn silage in rations on the performance of high-producing dairy cows. The chemical composition of the corn silages was similar except for lignin, which was higher in the control hybrid [3.09%, dry matter (DM) basis] compared with the BMR hybrid (2.19%). The 30-h in vitro neutral detergent fiber (NDF) digestibility was also higher (62.8% of NDF) in the BMR hybrid than in the control hybrid (52.2%). Twenty-seven Holstein cows were fed 1 of 3 diets comprising 62% forage and 38% concentrate (DM basis) containing 35% (DM basis) corn silage from the control hybrid (NLO), 35% of the BMR hybrid (BLO), or 50% of the BMR (BHI). Cows were fed the diets in a replicated 3×3 Latin square design with 28-d periods. Intake of DM was similar among treatments but milk production was greater for cows fed BLO (50.1kg/d) and BHI (51.1kg/d) than for NLO (47.9kg/d). Milk fat percentage was lower for cows fed BHI (3.37%) than for those fed BLO (3.55%) and NLO (3.56%) but yield of milk fat was similar among treatments. Yield and percentage of milk protein was higher for cows in BHI compared with NLO. The concentration of milk urea N was lower in cows fed BHI (14.0mg/dL) than in those fed NLO (14.7mg/dL) and intermediate for BLO (14.5mg/dL). The yield of 3.5% fat-corrected milk was higher in cows fed BLO (50.2kg/d) than in NLO (48.2kg/d) and was intermediate for BHI (49.8kg/d). The total-tract digestibility of dietary DM, organic matter, starch, and crude protein was lower for cows in NLO compared with the other treatments. The total-tract digestibility of NDF was highest for BHI (54.4%), intermediate for BLO (50.9%), and lowest for NLO (43.2%). We conclude that BMR corn silage can be included in rations at moderate and high proportions of a total ration, resulting in high levels of milk production. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All

Production response to corn silage produced from normal, brown midrib, or waxy corn hybrids.

PubMed

Barlow, J S; Bernard, J K; Mullis, N A

2012-08-01

The objective was to evaluate the nutrient intake and digestibility and milk production response of lactating dairy cows fed diets based on corn silage produced from 3 different types of corn hybrids. Experimental diets contained 36.4% of the dietary dry matter (DM) from corn silage produced from normal (Agratech 1021, AgraTech Seeds Inc., Atlanta, GA), brown midrib (BMR; Mycogen F2F797, Mycogen Seeds, Indianapolis, IN), or waxy (Master's Choice 590, Master's Choice Hybrids, Ullin, IL) hybrids. Thirty-six multiparous and primiparous Holstein cows (66 ± 22 d in milk, 41 ± 8 kg/d of milk) were used in an 11-wk completely randomized design trial during the fall of 2009. All cows were fed a diet containing normal corn silage during the first 2wk of the trial before being assigned to 1 of 3 treatments for the following 9 wk. Data collected during the first 2 wk were used as a covariate in the statistical analysis. No difference was observed in dry matter intake (DMI) among treatments, which averaged 22.6 kg/d. Milk yield was higher for cows fed BMR (37.6 kg/d) compared with waxy (35.2 kg/d) but was similar to that of cows fed control (36.2 kg/d). Milk fat percentage tended to be lower for cows fed control (3.28%) compared with those fed BMR (3.60%) or waxy (3.55%) corn silage. Milk protein percentage tended to be lower for cows fed control (2.79%) compared with waxy (2.89%) but similar to that of those fed BMR (2.85%). No differences were observed in yield of milk components. Energy-corrected milk (ECM) yield and dairy efficiency (ECM:DMI) did not differ among treatments. Cows fed BMR tended to gain more body weight compared with those fed control and waxy. Results of this trial are consistent with previous reports in which cows fed diets based on corn silage produced from BMR hybrids have higher milk yield compared with those fed other hybrids. Corn silage produced from the waxy hybrid supported a similar yield of ECM because of higher milk components, but milk yield

Integrating water exclusion theory into βcontacts to predict binding free energy changes and binding hot spots

PubMed Central

2014-01-01

Background Binding free energy and binding hot spots at protein-protein interfaces are two important research areas for understanding protein interactions. Computational methods have been developed previously for accurate prediction of binding free energy change upon mutation for interfacial residues. However, a large number of interrupted and unimportant atomic contacts are used in the training phase which caused accuracy loss. Results This work proposes a new method, βACV ASA , to predict the change of binding free energy after alanine mutations. βACV ASA integrates accessible surface area (ASA) and our newly defined β contacts together into an atomic contact vector (ACV). A β contact between two atoms is a direct contact without being interrupted by any other atom between them. A β contact’s potential contribution to protein binding is also supposed to be inversely proportional to its ASA to follow the water exclusion hypothesis of binding hot spots. Tested on a dataset of 396 alanine mutations, our method is found to be superior in classification performance to many other methods, including Robetta, FoldX, HotPOINT, an ACV method of β contacts without ASA integration, and ACV ASA methods (similar to βACV ASA but based on distance-cutoff contacts). Based on our data analysis and results, we can draw conclusions that: (i) our method is powerful in the prediction of binding free energy change after alanine mutation; (ii) β contacts are better than distance-cutoff contacts for modeling the well-organized protein-binding interfaces; (iii) β contacts usually are only a small fraction number of the distance-based contacts; and (iv) water exclusion is a necessary condition for a residue to become a binding hot spot. Conclusions βACV ASA is designed using the advantages of both β contacts and water exclusion. It is an excellent tool to predict binding free energy changes and binding hot spots after alanine mutation. PMID:24568581

A novel substance P binding site in rat brain regions modulates TRH receptor binding.

PubMed

Sharif, N A

1990-10-01

Binding sites for thyrotropin-releasing hormone (TRH) were labelled with [3H](2-Me-His3)TRH ([3H]MeTRH) on membranes from rat brain regions at 0 degrees C for 5 h. Amygdaloid membranes bound [3H]MeTRH with high-affinity (Kd = 3.1 +/- 0.5 nM (n = 4)). Five TRH analogs competed for this binding with the same rank order and with affinities that matched the pharmacological specificity of pituitary TRH receptors. Substance P (SP) and its C-terminal fragments reduced amygdaloid TRH receptor binding in a concentration dependent manner (IC50 for SP = 65 microM). The rank order of potency of SP analogs at inhibiting TRH receptor binding was: SP greater than nonapeptide (3-11) greater than hexapeptide (6-11) greater than heptapeptide (5-11) greater than pentapeptide (7-11). However, other tachykinins were inactive in this system. SP was a potent inhibitor of [3H]MeTRH binding in hippocampus greater than spinal cord greater than retina greater than n. accumbens greater than hypothalamus greater than amygdaloid greater than olfactory bulb greater than or equal to pituitary greater than pons/medulla in parallel assays. In amygdaloid membranes SP (50 microM) reduced the apparent maximum receptor density by 39% (p less than 0.01) without altering the binding affinity, and 100 microM SP induced a biphasic dissociation of [3H]MeTRH with kinetics faster than those induced by both TRH (10 microM) and serotonin (100 microM). In contrast, other neuropeptides such as neurotensin, proctolin, angiotensin II, bombesin and luteinizing hormone releasing hormone did not significantly inhibit [3H]MeTRH binding to amygdaloid membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Mass-action equilibrium and non-specific interactions in protein binding networks

NASA Astrophysics Data System (ADS)

Maslov, Sergei

2009-03-01

Large-scale protein binding networks serve as a paradigm of complex properties of living cells. These networks are naturally weighted with edges characterized by binding strength and protein-nodes -- by their concentrations. However, the state-of-the-art high-throughput experimental techniques generate just a binary (yes or no) information about individual interactions. As a result, most of the previous research concentrated just on topology of these networks. In a series of recent publications [1-4] my collaborators and I went beyond purely topological studies and calculated the mass-action equilibrium of a genome-wide binding network using experimentally determined protein concentrations, localizations, and reliable binding interactions in baker's yeast. We then studied how this equilibrium responds to large perturbations [1-2] and noise [3] in concentrations of proteins. We demonstrated that the change in the equilibrium concentration of a protein exponentially decays (and sign-alternates) with its network distance away from the perturbed node. This explains why, despite a globally connected topology, individual functional modules in such networks are able to operate fairly independently. In a separate study [4] we quantified the interplay between specific and non-specific binding interactions under crowded conditions inside living cells. We show how the need to limit the waste of resources constrains the number of types and concentrations of proteins that are present at the same time and at the same place in yeast cells. [1] S Maslov, I. Ispolatov, PNAS 104:13655 (2007). [2] S. Maslov, K. Sneppen, I. Ispolatov, New J. of Phys. 9: 273 (2007). [3] K-K. Yan, D. Walker, S. Maslov, PRL accepted (2008). [4] J. Zhang, S. Maslov, and E. I. Shakhnovich, Mol Syst Biol 4, 210 (2008).

Thermal conductance and basal metabolic rate are part of a coordinated system for heat transfer regulation

PubMed Central

Naya, Daniel E.; Spangenberg, Lucía; Naya, Hugo; Bozinovic, Francisco

2013-01-01

Thermal conductance measures the ease with which heat leaves or enters  an organism's body. Although the analysis of this physiological variable in relation to climatic and ecological factors can be traced to studies by Scholander and colleagues, only small advances have occurred ever since. Here, we analyse the relationship between minimal thermal conductance estimated during summer (Cmin) and several ecological, climatic and geographical factors for 127 rodent species, in order to identify the exogenous factors that have potentially affected the evolution of thermal conductance. In addition, we evaluate whether there is compensation between Cmin and basal metabolic rate (BMR)—in such a way that a scale-invariant ratio between both variables is equal to one—as could be expected from the Scholander–Irving model of heat transfer. Our major findings are (i) annual mean temperature is the best single predictor of mass-independent Cmin. (ii) After controlling for the effect of body mass, there is a strong positive correlation between log10 (Cmin) and log10 (BMR). Further, the slope of this correlation is close to one, indicating an almost perfect compensation between both physiological variables. (iii) Structural equation modelling indicated that Cmin values are adjusted to BMR values and not the other way around. Thus, our results strongly suggest that BMR and thermal conductance integrate a coordinated system for heat regulation in endothermic animals and that summer conductance values are adjusted (in an evolutionary sense) to track changes in BMRs. PMID:23902915

The centrifugal and centripetal force influence on spatial competition of agricultural land in Bandung Metropolitan Region

NASA Astrophysics Data System (ADS)

Sadewo, E.

2017-06-01

Agricultural activity has suffered a massive land functional shift caused by market mechanism in Bandung metropolitan region (BMR). We argue that the existence of agricultural land in urban spatial structure is the result of interaction between centrifugal and centripetal force on spatial competition. This research aims to explore how several recognized centrifugal and centripetal force influence to the existence of agricultural land in BMR land development. The analysis using multivariate regression indicates that there exists spatial competition between population density and degree of urbanization with agricultural land areas. Its extended spatial regression model suggested that neighboring situation plays an important role to preserve agricultural land areas existences in BMR. Meanwhile, the influence of distance between the location of the city center and employment opportunities is found to be insignificant in the spatial competition. It is opposed to the theory of von Thünen and monocentric model in general. One of the possible explanation of such condition is that the assumption of centrality does not met. In addition, the agricultural land density decay in the southern parts of the area was related to its geographical conditions as protected areas or unfavorable for farming activity. It is suggested that BMR was in the early phase of polycentric development. Hence, better policies that lead redirected development to the southern part of the region is needed as well as population control and regulation of land use.

Thermal conductance and basal metabolic rate are part of a coordinated system for heat transfer regulation.

PubMed

Naya, Daniel E; Spangenberg, Lucía; Naya, Hugo; Bozinovic, Francisco

2013-09-22

Thermal conductance measures the ease with which heat leaves or enters an organism's body. Although the analysis of this physiological variable in relation to climatic and ecological factors can be traced to studies by Scholander and colleagues, only small advances have occurred ever since. Here, we analyse the relationship between minimal thermal conductance estimated during summer (Cmin) and several ecological, climatic and geographical factors for 127 rodent species, in order to identify the exogenous factors that have potentially affected the evolution of thermal conductance. In addition, we evaluate whether there is compensation between Cmin and basal metabolic rate (BMR)-in such a way that a scale-invariant ratio between both variables is equal to one-as could be expected from the Scholander-Irving model of heat transfer. Our major findings are (i) annual mean temperature is the best single predictor of mass-independent Cmin. (ii) After controlling for the effect of body mass, there is a strong positive correlation between log10 (Cmin) and log10 (BMR). Further, the slope of this correlation is close to one, indicating an almost perfect compensation between both physiological variables. (iii) Structural equation modelling indicated that Cmin values are adjusted to BMR values and not the other way around. Thus, our results strongly suggest that BMR and thermal conductance integrate a coordinated system for heat regulation in endothermic animals and that summer conductance values are adjusted (in an evolutionary sense) to track changes in BMRs.

Rate Constants and Mechanisms of Protein–Ligand Binding

PubMed Central

Pang, Xiaodong; Zhou, Huan-Xiang

2017-01-01

Whereas protein–ligand binding affinities have long-established prominence, binding rate constants and binding mechanisms have gained increasing attention in recent years. Both new computational methods and new experimental techniques have been developed to characterize the latter properties. It is now realized that binding mechanisms, like binding rate constants, can and should be quantitatively determined. In this review, we summarize studies and synthesize ideas on several topics in the hope of providing a coherent picture of and physical insight into binding kinetics. The topics include microscopic formulation of the kinetic problem and its reduction to simple rate equations; computation of binding rate constants; quantitative determination of binding mechanisms; and elucidation of physical factors that control binding rate constants and mechanisms. PMID:28375732

Calcium-binding proteins and development

NASA Technical Reports Server (NTRS)

Beckingham, K.; Lu, A. Q.; Andruss, B. F.; McIntire, L. V. (Principal Investigator)

1998-01-01

The known roles for calcium-binding proteins in developmental signaling pathways are reviewed. Current information on the calcium-binding characteristics of three classes of cell-surface developmental signaling proteins (EGF-domain proteins, cadherins and integrins) is presented together with an overview of the intracellular pathways downstream of these surface receptors. The developmental roles delineated to date for the universal intracellular calcium sensor, calmodulin, and its targets, and for calcium-binding regulators of the cytoskeleton are also reviewed.

Feature-Based Binding and Phase Theory

ERIC Educational Resources Information Center

Antonenko, Andrei

2012-01-01

Current theories of binding cannot provide a uniform account for many facts associated with the distribution of anaphors, such as long-distance binding effects and the subject-orientation of monomorphemic anaphors. Further, traditional binding theory is incompatible with minimalist assumptions. In this dissertation I propose an analysis of…

Coupled binding-bending-folding: The complex conformational dynamics of protein-DNA binding studied by atomistic molecular dynamics simulations.

PubMed

van der Vaart, Arjan

2015-05-01

Protein-DNA binding often involves dramatic conformational changes such as protein folding and DNA bending. While thermodynamic aspects of this behavior are understood, and its biological function is often known, the mechanism by which the conformational changes occur is generally unclear. By providing detailed structural and energetic data, molecular dynamics simulations have been helpful in elucidating and rationalizing protein-DNA binding. This review will summarize recent atomistic molecular dynamics simulations of the conformational dynamics of DNA and protein-DNA binding. A brief overview of recent developments in DNA force fields is given as well. Simulations have been crucial in rationalizing the intrinsic flexibility of DNA, and have been instrumental in identifying the sequence of binding events, the triggers for the conformational motion, and the mechanism of binding for a number of important DNA-binding proteins. Molecular dynamics simulations are an important tool for understanding the complex binding behavior of DNA-binding proteins. With recent advances in force fields and rapid increases in simulation time scales, simulations will become even more important for future studies. This article is part of a Special Issue entitled Recent developments of molecular dynamics. Copyright © 2014. Published by Elsevier B.V.

A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone

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

Kim, Il Hwan; Pham, Van; Jablonka, Willy

Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes, Culex, and Anopheles mosquito species. Using isothermal titration calorimetry,more » we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary “long” D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.« less

A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone.

PubMed

Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G; Ribeiro, José M C; Andersen, John F

2017-09-15

Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes , Culex , and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary "long" D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10 R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

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

Erythropoietin binding protein from mammalian serum

DOEpatents

Clemons, Gisela K.

1997-01-01

Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.

Adrenocortical nuclear progesterone-binding protein: Identification by photoaffinity labeling and evidence for deoxyribonucleic acid binding and stimulation by adrenocorticotropin

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

Demura, T.; Driscoll, W.J.; Lee, Y.C.

1991-01-01

Nuclei of the guinea pig adrenal cortex contain a protein that specifically binds progesterone and that, biochemically, is clearly distinct from the classical progesterone receptor. The adrenocortical nuclear progesterone-binding protein has now been purified more than 2000-fold by steroid-affinity chromatography with a 75% yield. The purified protein preparation demonstrated three major bands on sodium dodecyl sulfate-polyacrylamide gel of 79K, 74K, and 50K. To determine which of the three might represent the progesterone-binding protein, steroid photoaffinity labeling was performed which resulted in the specific and exclusive labeling of a 50K band. Thus, the adrenocortical nuclear progesterone-binding protein appears to be distinctmore » from the classical progesterone receptor not only biochemically, but also on the basis of molecular size. To test whether the adrenocortical nuclear progesterone-binding protein can be hormonally stimulated, guinea pigs were treated with ACTH. The chronic administration of ACTH caused a 4- to 6-fold increase in the specific progesterone binding capacity without a change in the binding affinity. There appeared to be no significant difference in nuclear progesterone binding between the zona fasciculata and zona reticularis. This finding suggests a mediating role for the progesterone-binding protein in ACTH action. In addition, the nuclear progesterone-binding protein bound to nonspecific DNA sequences, further suggesting a possible transcriptional regulatory role.« less

Recognition of xyloglucan by the crystalline cellulose-binding site of a family 3a carbohydrate-binding module

PubMed Central

Hernandez-Gomez, Mercedes C.; Rydahl, Maja G.; Rogowski, Artur; Morland, Carl; Cartmell, Alan; Crouch, Lucy; Labourel, Aurore; Fontes, Carlos M. G. A.; Willats, William G. T.; Gilbert, Harry J; Knox, J. Paul

2018-01-01

Type A non-catalytic carbohydrate-binding modules (CBMs), exemplified by CtCBM3acipA, are widely believed to specifically target crystalline cellulose through entropic forces. Here we have tested the hypothesis that type A CBMs can also bind to xyloglucan, a soluble β-1,4-glucan containing α-1,6-xylose side chains. CtCBM3acipA bound to xyloglucan in cell walls and arrayed on solid surfaces. Xyloglucan and cellulose were shown to bind to the same planar surface on CBM3acipA. A range of type A CBMs from different families were shown to bind to xyloglucan in solution with ligand binding driven by enthalpic changes. The nature of CBM-polysaccharide interactions is discussed. PMID:26193423

Erythropoietin binding protein from mammalian serum

DOEpatents

Clemons, G.K.

1997-04-29

Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.

Superresolution microscopy with transient binding.

PubMed

Molle, Julia; Raab, Mario; Holzmeister, Susanne; Schmitt-Monreal, Daniel; Grohmann, Dina; He, Zhike; Tinnefeld, Philip

2016-06-01

For single-molecule localization based superresolution, the concentration of fluorescent labels has to be thinned out. This is commonly achieved by photophysically or photochemically deactivating subsets of molecules. Alternatively, apparent switching of molecules can be achieved by transient binding of fluorescent labels. Here, a diffusing dye yields bright fluorescent spots when binding to the structure of interest. As the binding interaction is weak, the labeling is reversible and the dye ligand construct diffuses back into solution. This approach of achieving superresolution by transient binding (STB) is reviewed in this manuscript. Different realizations of STB are discussed and compared to other localization-based superresolution modalities. We propose the development of labeling strategies that will make STB a highly versatile tool for superresolution microscopy at highest resolution. Copyright © 2015 Elsevier Ltd. All rights reserved.

Lactoferrin-binding proteins in Shigella flexneri.

PubMed Central

Tigyi, Z; Kishore, A R; Maeland, J A; Forsgren, A; Naidu, A S

1992-01-01

The ability of Shigella flexneri to interact with lactoferrin (Lf) was examined with a 125I-labeled protein-binding assay. The percent binding of human lactoferrin (HLf) and bovine lactoferrin (BLf) to 45 S. flexneri strains was 19 +/- 3 and 21 +/- 3 (mean +/- standard error of the mean), respectively. 125I-labeled HLf and BLf binding to strain M90T reached an equilibrium within 2 h. Unlabeled HLf and BLf displaced the 125I-HLf-bacteria interaction in a dose-dependent manner. The Lf-bacterium complex was uncoupled by KSCN or urea, but not by NaCl. The interaction was specific, and approximately 4,800 HLf binding sites (affinity constant [Ka], 690 nM) or approximately 5,700 BLf binding sites (Ka, 104 nM) per cell were estimated in strain M90T by a Scatchard plot analysis. The native cell envelope (CE) and outer membrane (OM) did not reveal Lf-binding components in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, after being boiled, the CE and OM preparations showed three distinct horseradish peroxidase-Lf reactive bands of about 39, 22, and 16 kDa. The 39-kDa component was also reactive to a monoclonal antibody specific for porin (PoI) proteins of members of the family Enterobacteriaceae. The Lf-binding protein pattern was similar with BLf or HLf, for Crb+ and Crb- strains. The protein-Lf complex was dissociable by KSCN or urea and was stable after treatment with NaCl. Variation (loss) in the O chain of lipopolysaccharide (LPS) markedly enhanced the Lf-binding capacity in the isogenic rough strain SFL1070-15 compared with its smooth parent strain, SFL1070. These data establish that Lf binds to specific components in the bacterial OM; the heat-modifiable, anti-PoI-reactive, and LPS-associated properties suggested that the Lf-binding proteins are porins in S. flexneri. Images PMID:1319403

Discover binding pathways using the sliding binding-box docking approach: application to binding pathways of oseltamivir to avian influenza H5N1 neuraminidase

NASA Astrophysics Data System (ADS)

Tran, Diem-Trang T.; Le, Ly T.; Truong, Thanh N.

2013-08-01

Drug binding and unbinding are transient processes which are hardly observed by experiment and difficult to analyze by computational techniques. In this paper, we employed a cost-effective method called "pathway docking" in which molecular docking was used to screen ligand-receptor binding free energy surface to reveal possible paths of ligand approaching protein binding pocket. A case study was applied on oseltamivir, the key drug against influenza a virus. The equilibrium pathways identified by this method are found to be similar to those identified in prior studies using highly expensive computational approaches.

Energy expenditure and intake during puberty in healthy nonobese adolescents: a systematic review.

PubMed

Cheng, Hoi Lun; Amatoury, Mazen; Steinbeck, Katharine

2016-10-01

Puberty is a time of rapid growth and changing energy requirements and is a risk period for obesity. There is little high-quality evidence on the pubertal alterations of energy expenditure and intake, and this has limited our understanding of energy balance during this important life stage. The purpose of this study was to summarize existing evidence on pubertal energy expenditure and intake in healthy nonobese adolescents. Studies were identified through CINAHL, the Cochrane Library, Embase, MEDLINE, and Web of Science databases up to August 2015. Articles presenting objectively measured data for basal or resting metabolic rate (BMR/RMR), total daily energy expenditure (TDEE), and/or energy intake (EI) for ≥2 categories of puberty were included. Relevant data adjusted for fat-free mass (FFM) also were extracted. Data were dichotomized into prepubertal and pubertal groups and compared through the use of standardized mean differences (SMDs). Heterogeneous study methodologies precluded meta-analysis. The search netted 6770 articles, with 12 included for review. From these, 6 of 9 studies supported significantly higher absolute BMR/RMR during puberty (SMD: 1.10-5.93), and all of the studies favored significantly higher absolute TDEE during puberty (SMD: 0.46-9.55). These corresponded to a 12% difference and an 18% difference in absolute BMR/RMR and TDEE, respectively. Results adjusted for FFM were equivocal, with 3 studies favoring higher (1 significantly) and 3 favoring significantly lower adjusted BMR/RMR during puberty. Only 1 study reported EI, showing 41% and 25% greater absolute intakes in pubertal males and females, respectively. These differences were not significant after adjustment for FFM. Reasonably consistent evidence exists to support higher absolute BMR/RMR and TDEE in pubertal than in prepubertal adolescents. Differences are largely accounted for by FFM, among other potential factors such as growth- and puberty-related hormones. This review argues

Simulation of Reversible Protein–Protein Binding and Calculation of Binding Free Energies Using Perturbed Distance Restraints

PubMed Central

2017-01-01

Virtually all biological processes depend on the interaction between proteins at some point. The correct prediction of biomolecular binding free-energies has many interesting applications in both basic and applied pharmaceutical research. While recent advances in the field of molecular dynamics (MD) simulations have proven the feasibility of the calculation of protein–protein binding free energies, the large conformational freedom of proteins and complex free energy landscapes of binding processes make such calculations a difficult task. Moreover, convergence and reversibility of resulting free-energy values remain poorly described. In this work, an easy-to-use, yet robust approach for the calculation of standard-state protein–protein binding free energies using perturbed distance restraints is described. In the binding process the conformations of the proteins were restrained, as suggested earlier. Two approaches to avoid end-state problems upon release of the conformational restraints were compared. The method was evaluated by practical application to a small model complex of ubiquitin and the very flexible ubiquitin-binding domain of human DNA polymerase ι (UBM2). All computed free energy differences were closely monitored for convergence, and the calculated binding free energies had a mean unsigned deviation of only 1.4 or 2.5 kJ·mol–1 from experimental values. Statistical error estimates were in the order of thermal noise. We conclude that the presented method has promising potential for broad applicability to quantitatively describe protein–protein and various other kinds of complex formation. PMID:28898077

BclxL changes conformation upon binding to wild-type but not mutant p53 DNA binding domain.

PubMed

Hagn, Franz; Klein, Christian; Demmer, Oliver; Marchenko, Natasha; Vaseva, Angelina; Moll, Ute M; Kessler, Horst

2010-01-29

p53 can induce apoptosis through mitochondrial membrane permeabilization by interaction of its DNA binding region with the anti-apoptotic proteins BclxL and Bcl2. However, little is known about the action of p53 at the mitochondria in molecular detail. By using NMR spectroscopy and fluorescence polarization we characterized the binding of wild-type and mutant p53 DNA binding domains to BclxL and show that the wild-type p53 DNA binding domain leads to structural changes in the BH3 binding region of BclxL, whereas mutants fail to induce such effects due to reduced affinity. This was probed by induced chemical shift and residual dipolar coupling data. These data imply that p53 partly achieves its pro-apoptotic function at the mitochondria by facilitating interaction between BclxL and BH3-only proteins in an allosteric mode of action. Furthermore, we characterize for the first time the binding behavior of Pifithrin-mu, a specific small molecule inhibitor of the p53-BclxL interaction, and present a structural model of the protein-ligand complex. A rather unusual behavior is revealed whereby Pifithrin-mu binds to both sides of the protein-protein complex. These data should facilitate the rational design of more potent specific BclxL-p53 inhibitors.

Stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1.

PubMed

Schneider, G J; Geiduschek, E P

1990-06-25

The stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1 has been determined. 3H-Labeled TF1 was allowed to bind to a 32P-labeled DNA fragment containing a TF1 binding site. Multiple TF1-DNA complexes were resolved from each other and from unbound DNA by native gel electrophoresis. DNA-protein complexes were cut from polyacrylamide gels, and the amounts of 3H and 32P contained in each slice were measured. A ratio of 1.12 +/- 0.06 TF1 dimer/DNA molecule was calculated for the fastest-migrating TF1-DNA complex. We conclude that TF1 has a DNA-binding unit of one dimer. More slowly migrating complexes are apparently formed by serial addition of single TF1 dimers.

An alternate binding site for PPARγ ligands

PubMed Central

Hughes, Travis S.; Giri, Pankaj Kumar; de Vera, Ian Mitchelle S.; Marciano, David P.; Kuruvilla, Dana S.; Shin, Youseung; Blayo, Anne-Laure; Kamenecka, Theodore M.; Burris, Thomas P.; Griffin, Patrick R.; Kojetin, Douglas J.

2014-01-01

PPARγ is a target for insulin sensitizing drugs such as glitazones, which improve plasma glucose maintenance in patients with diabetes. Synthetic ligands have been designed to mimic endogenous ligand binding to a canonical ligand-binding pocket to hyperactivate PPARγ. Here we reveal that synthetic PPARγ ligands also bind to an alternate site, leading to unique receptor conformational changes that impact coregulator binding, transactivation and target gene expression. Using structure-function studies we show that alternate site binding occurs at pharmacologically relevant ligand concentrations, and is neither blocked by covalently bound synthetic antagonists nor by endogenous ligands indicating non-overlapping binding with the canonical pocket. Alternate site binding likely contributes to PPARγ hyperactivation in vivo, perhaps explaining why PPARγ full and partial or weak agonists display similar adverse effects. These findings expand our understanding of PPARγ activation by ligands and suggest that allosteric modulators could be designed to fine tune PPARγ activity without competing with endogenous ligands. PMID:24705063

Unusual binding of ursodeoxycholic acid to ileal bile acid binding protein: role in activation of FXRα.

PubMed

Fang, Changming; Filipp, Fabian V; Smith, Jeffrey W

2012-04-01

Ursodeoxycholic acid (UDCA, ursodiol) is used to prevent damage to the liver in patients with primary biliary cirrhosis. The drug also prevents the progression of colorectal cancer and the recurrence of high-grade colonic dysplasia. However, the molecular mechanism by which UDCA elicits its beneficial effects is not entirely understood. The aim of this study was to determine whether ileal bile acid binding protein (IBABP) has a role in mediating the effects of UDCA. We find that UDCA binds to a single site on IBABP and increases the affinity for major human bile acids at a second binding site. As UDCA occupies one of the bile acid binding sites on IBABP, it reduces the cooperative binding that is often observed for the major human bile acids. Furthermore, IBABP is necessary for the full activation of farnesoid X receptor α (FXRα) by bile acids, including UDCA. These observations suggest that IBABP may have a role in mediating some of the intestinal effects of UDCA.

Engineered proteins as specific binding reagents.

PubMed

Binz, H Kaspar; Plückthun, Andreas

2005-08-01

Over the past 30 years, monoclonal antibodies have become the standard binding proteins and currently find applications in research, diagnostics and therapy. Yet, monoclonal antibodies now face strong competition from synthetic antibody libraries in combination with powerful library selection technologies. More recently, an increased understanding of other natural binding proteins together with advances in protein engineering, selection and evolution technologies has also triggered the exploration of numerous other protein architectures for the generation of designed binding molecules. Valuable protein-binding scaffolds have been obtained and represent promising alternatives to antibodies for biotechnological and, potentially, clinical applications.

Solar Cycle Variability and Grand Minima Induced by Joy's Law Scatter

NASA Astrophysics Data System (ADS)

Karak, Bidya Binay; Miesch, Mark S.

2017-08-01

The strength of the solar cycle varies from one cycle to another in an irregular manner and the extreme example of this irregularity is the Maunder minimum when Sun produced only a few spots for several years. We explore the cause of these variabilities using a 3D Babcock--Leighton dynamo. In this model, based on the toroidal flux at the base of the convection zone, bipolar magnetic regions (BMRs) are produced with flux, tilt angle, and time of emergence all obtain from their observed distributions. The dynamo growth is limited by a tilt quenching.The randomnesses in the BMR emergences make the poloidal field unequal and eventually cause an unequal solar cycle. When observed fluctuations of BMR tilts around Joy's law, i.e., a standard deviation of 15 degrees, are considered, our model produces a variation in the solar cycle comparable to the observed solar cycle variability. Tilt scatter also causes occasional Maunder-like grand minima, although the observed scatter does not reproduce correct statistics of grand minima. However, when we double the tilt scatter, we find grand minima consistent with observations. Importantly, our dynamo model can operate even during grand minima with only a few BMRs, without requiring any additional alpha effect.

Nonspecific DNA Binding and Bending by HUαβ: Interfaces of the Three Binding Modes Characterized by Salt Dependent Thermodynamics

PubMed Central

Koh, Junseock; Shkel, Irina; Saecker, Ruth M.; Record, M. Thomas

2011-01-01

Previous ITC and FRET studies demonstrated that Escherichia coli HUαβ binds nonspecifically to duplex DNA in three different binding modes: a tighter-binding 34 bp mode which interacts with DNA in large (>34 bp) gaps between bound proteins, reversibly bending it 140° and thereby increasing its flexibility, and two weaker, modestly cooperative small-site-size modes (10 bp, 6 bp) useful for filling gaps between bound proteins shorter than 34 bp. Here we use ITC to determine the thermodynamics of these binding modes as a function of salt concentration, and deduce that DNA in the 34 bp mode is bent around but not wrapped on the body of HU, in contrast to specific binding of IHF. Analyses of binding isotherms (8, 15, 34 bp DNA) and initial binding heats (34, 38, 160 bp DNA) reveal that all three modes have similar log-log salt concentration derivatives of the binding constants (Ski) even though their binding site sizes differ greatly; most probable values of Ski on 34 bp or larger DNA are − 7.5 ± 0.5. From the similarity of Ski values, we conclude that binding interfaces of all three modes involve the same region of the arms and saddle of HU. All modes are entropy-driven, as expected for nonspecific binding driven by the polyelectrolyte effect. The bent-DNA 34 bp mode is most endothermic, presumably because of the cost of HU-induced DNA bending, while the 6 bp mode is modestly exothermic at all salt concentrations examined. Structural models consistent with the observed Ski values are proposed. PMID:21513716

Stereochemical determinants of C-terminal specificity in PDZ peptide-binding domains: a novel contribution of the carboxylate-binding loop.

PubMed

Amacher, Jeanine F; Cushing, Patrick R; Bahl, Christopher D; Beck, Tobias; Madden, Dean R

2013-02-15

PDZ (PSD-95/Dlg/ZO-1) binding domains often serve as cellular traffic engineers, controlling the localization and activity of a wide variety of binding partners. As a result, they play important roles in both physiological and pathological processes. However, PDZ binding specificities overlap, allowing multiple PDZ proteins to mediate distinct effects on shared binding partners. For example, several PDZ domains bind the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), an epithelial ion channel mutated in CF. Among these binding partners, the CFTR-associated ligand (CAL) facilitates post-maturational degradation of the channel and is thus a potential therapeutic target. Using iterative optimization, we previously developed a selective CAL inhibitor peptide (iCAL36). Here, we investigate the stereochemical basis of iCAL36 specificity. The crystal structure of iCAL36 in complex with the CAL PDZ domain reveals stereochemical interactions distributed along the peptide-binding cleft, despite the apparent degeneracy of the CAL binding motif. A critical selectivity determinant that distinguishes CAL from other CFTR-binding PDZ domains is the accommodation of an isoleucine residue at the C-terminal position (P(0)), a characteristic shared with the Tax-interacting protein-1. Comparison of the structures of these two PDZ domains in complex with ligands containing P(0) Leu or Ile residues reveals two distinct modes of accommodation for β-branched C-terminal side chains. Access to each mode is controlled by distinct residues in the carboxylate-binding loop. These studies provide new insights into the primary sequence determinants of binding motifs, which in turn control the scope and evolution of PDZ interactomes.

Sequential memory: Binding dynamics

NASA Astrophysics Data System (ADS)

Afraimovich, Valentin; Gong, Xue; Rabinovich, Mikhail

2015-10-01

Temporal order memories are critical for everyday animal and human functioning. Experiments and our own experience show that the binding or association of various features of an event together and the maintaining of multimodality events in sequential order are the key components of any sequential memories—episodic, semantic, working, etc. We study a robustness of binding sequential dynamics based on our previously introduced model in the form of generalized Lotka-Volterra equations. In the phase space of the model, there exists a multi-dimensional binding heteroclinic network consisting of saddle equilibrium points and heteroclinic trajectories joining them. We prove here the robustness of the binding sequential dynamics, i.e., the feasibility phenomenon for coupled heteroclinic networks: for each collection of successive heteroclinic trajectories inside the unified networks, there is an open set of initial points such that the trajectory going through each of them follows the prescribed collection staying in a small neighborhood of it. We show also that the symbolic complexity function of the system restricted to this neighborhood is a polynomial of degree L - 1, where L is the number of modalities.

Sequential memory: Binding dynamics.

PubMed

Afraimovich, Valentin; Gong, Xue; Rabinovich, Mikhail

2015-10-01

Temporal order memories are critical for everyday animal and human functioning. Experiments and our own experience show that the binding or association of various features of an event together and the maintaining of multimodality events in sequential order are the key components of any sequential memories-episodic, semantic, working, etc. We study a robustness of binding sequential dynamics based on our previously introduced model in the form of generalized Lotka-Volterra equations. In the phase space of the model, there exists a multi-dimensional binding heteroclinic network consisting of saddle equilibrium points and heteroclinic trajectories joining them. We prove here the robustness of the binding sequential dynamics, i.e., the feasibility phenomenon for coupled heteroclinic networks: for each collection of successive heteroclinic trajectories inside the unified networks, there is an open set of initial points such that the trajectory going through each of them follows the prescribed collection staying in a small neighborhood of it. We show also that the symbolic complexity function of the system restricted to this neighborhood is a polynomial of degree L - 1, where L is the number of modalities.

The α-galactomannan Davanat binds galectin-1 at a site different from the conventional galectin carbohydrate binding domain

PubMed Central

Miller, Michelle C; Klyosov, Anatole; Mayo, Kevin H

2009-01-01

Galectins are a sub-family of lectins, defined by their highly conserved β-sandwich structures and ability to bind to β-galactosides, like Gal β1-4 Glc (lactose). Here, we used 15N-1H HSQC and pulse field gradient (PFG) NMR spectroscopy to demonstrate that galectin-1 (gal-1) binds to the relatively large galactomannan Davanat, whose backbone is composed of β1-4-linked d-mannopyranosyl units to which single d-galactopyranosyl residues are periodically attached via α1-6 linkage (weight-average MW of 59 kDa). The Davanat binding domain covers a relatively large area on the surface of gal-1 that runs across the dimer interface primarily on that side of the protein opposite to the lactose binding site. Our data show that gal-1 binds Davanat with an apparent equilibrium dissociation constant (Kd) of 10 × 10−6 M, compared to 260 × 10−6 M for lactose, and a stiochiometry of about 3 to 6 gal-1 molecules per Davanat molecule. Mannan also interacts at the same galactomannan binding domain on gal-1, but with at least 10-fold lower avidity, supporting the role of galactose units in Davanat for relatively strong binding to gal-1. We also found that the β-galactoside binding domain remains accessible in the gal-1/Davanat complex, as lactose can still bind with no apparent loss in affinity. In addition, gal-1 binding to Davanat also modifies the supermolecular structure of the galactomannan and appears to reduce its hydrodynamic radius and disrupt inter-glycan interactions thereby reducing glycan-mediated solution viscosity. Overall, our findings contribute to understanding gal-1–carbohydrate interactions and provide insight into gal-1 function with potentially significant biological consequences. PMID:19541770

An Electrostatic Funnel in the GABA-Binding Pathway

PubMed Central

Lightstone, Felice C.

2016-01-01

The γ-aminobutyric acid type A receptor (GABAA-R) is a major inhibitory neuroreceptor that is activated by the binding of GABA. The structure of the GABAA-R is well characterized, and many of the binding site residues have been identified. However, most of these residues are obscured behind the C-loop that acts as a cover to the binding site. Thus, the mechanism by which the GABA molecule recognizes the binding site, and the pathway it takes to enter the binding site are both unclear. Through the completion and detailed analysis of 100 short, unbiased, independent molecular dynamics simulations, we have investigated this phenomenon of GABA entering the binding site. In each system, GABA was placed quasi-randomly near the binding site of a GABAA-R homology model, and atomistic simulations were carried out to observe the behavior of the GABA molecules. GABA fully entered the binding site in 19 of the 100 simulations. The pathway taken by these molecules was consistent and non-random; the GABA molecules approach the binding site from below, before passing up behind the C-loop and into the binding site. This binding pathway is driven by long-range electrostatic interactions, whereby the electrostatic field acts as a ‘funnel’ that sweeps the GABA molecules towards the binding site, at which point more specific atomic interactions take over. These findings define a nuanced mechanism whereby the GABAA-R uses the general zwitterionic features of the GABA molecule to identify a potential ligand some 2 nm away from the binding site. PMID:27119953

Warfarin and Flavonoids Do Not Share the Same Binding Region in Binding to the IIA Subdomain of Human Serum Albumin.

PubMed

Rimac, Hrvoje; Dufour, Claire; Debeljak, Željko; Zorc, Branka; Bojić, Mirza

2017-07-11

Human serum albumin (HSA) binds a variety of xenobiotics, including flavonoids and warfarin. The binding of another ligand to the IIA binding site on HSA can cause warfarin displacement and potentially the elevation of its free concentration in blood. Studies dealing with flavonoid-induced warfarin displacement from HSA provided controversial results: estimated risk of displacement ranged from none to serious. To resolve these controversies, in vitro study of simultaneous binding of warfarin and eight different flavonoid aglycons and glycosides to HSA was carried out by fluorescence spectroscopy as well as molecular docking. Results show that warfarin and flavonoids do not share the same binding region in binding to HSA. Interactions were only observed at high warfarin concentrations not attainable under recommended dosing regimes. Docking experiments show that flavonoid aglycons and glycosides do not bind at warfarin high affinity sites, but rather to different regions within the IIA HSA subdomain. Thus, the risk of clinically significant warfarin-flavonoid interaction in binding to HSA should be regarded as negligible.

Binding of Diphtheria Toxin to Phospholipids in Liposomes

NASA Astrophysics Data System (ADS)

Alving, Carl R.; Iglewski, Barbara H.; Urban, Katharine A.; Moss, Joel; Richards, Roberta L.; Sadoff, Jerald C.

1980-04-01

Diphtheria toxin bound to the phosphate portion of some, but not all, phospholipids in liposomes. Liposomes consisting of dimyristoyl phosphatidylcholine and cholesterol did not bind toxin. Addition of 20 mol% (compared to dimyristoyl phosphatidylcholine) of dipalmitoyl phosphatidic acid, dicetyl phosphate, phosphatidylinositol phosphate, cardiolipin, or phosphatidylserine in the liposomes resulted in substantial binding of toxin. Inclusion of phosphatidylinositol in dimyristol phosphatidylcholine / cholesterol liposomes did not result in toxin binding. The calcium salt of dipalmitoyl phosphatidic acid was more effective than the sodium salt, and the highest level of binding occurred with liposomes consisting only of dipalmitoyl phosphatidic acid (calcium salt) and cholesterol. Binding of toxin to liposomes was dependent on pH, and the pattern of pH dependence varied with liposomes having different compositions. Incubation of diphtheria toxin with liposomes containing dicetyl phosphate resulted in maximal binding at pH 3.6, whereas binding to liposomes containing phosphatidylinositol phosphate was maximal above pH 7. Toxin did not bind to liposomes containing 20 mol% of a free fatty acid (palmitic acid) or a sulfated lipid (3-sulfogalactosylceramide). Toxin binding to dicetyl phosphate or phosphatidylinositol phosphate was inhibited by UTP, ATP, phosphocholine, or p-nitrophenyl phosphate, but not by uracil. We conclude that (a) diphtheria toxin binds specifically to the phosphate portion of certain phospholipids, (b) binding to phospholipids in liposomes is dependent on pH, but is not due only to electrostatic interaction, and (c) binding may be strongly influenced by the composition of adjacent phospholipids that do not bind toxin. We propose that a minor membrane phospholipid (such as phosphatidylinositol phosphate or phosphatidic acid), or that some other phosphorylated membrane molecule (such as a phosphoprotein) may be important in the initial binding of

Characterization and cross calibration of Agfa D4, D7, and D8 and Kodak SR45 x-ray films against direct exposure film at 4.0-5.5 keV

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

Lanier, N.E.; Cowan, J.S.; Workman, J.

2006-04-15

Kodak direct exposure film (DEF) [B. L. Henke et al., J. Opt. Soc. Am. B 3, 1540 (1986)] has been the standard for moderate energy (1-10 keV) x-ray diagnostic applications among the high-energy-density and inertial confinement fusion research communities. However, market forces have prompted Kodak to discontinue production of DEF, leaving these specialized communities searching for a replacement. We have conducted cross-calibration experiments and film characterizations on five possible substitutes for Kodak DEF. The film types studied were Kodak's Biomax MR (BMR) and SR45 along with Agfa's D8, D7, and D4sc. None of the films tested matched the speed ofmore » DEF. BMR and D8 were closest but D8 exhibited lower noise, with superior resolution and dynamic range. Agfa D7, Agfa D4sc, and Kodak SR45 were significantly less sensitive than BMR and D8, however, the improvements they yielded in resolution and dynamic range warrant their use if experimental constraints allow.« less

Effects of dietary quality on basal metabolic rate and internal morphology of European starlings (Sturnus vulgaris)

USGS Publications Warehouse

Geluso, Keith; Hayes, J.P.

1999-01-01

European starlings (Sturnus vulgaris) were fed either a low- or high-quality diet to test the effects of dietary quality on basal metabolic rate (BMR) and internal morphology. Basal metabolic rate did not differ significantly between the two dietary groups, but internal morphology differed greatly. Starlings fed the low-quality diet had heavier gastrointestinal tracts, gizzards, and livers. Starlings fed the high-quality diet had heavier breast muscles. Starlings on the low-quality diet maintained mass, while starlings on the high-quality diet gained mass. Dry matter digestibility and energy digestibility were lower for starlings fed the low-quality diet, and their food and water intake were greater than starlings on the high-quality diet. The lack of dietary effect on BMR may be the result of increased energy expenditure of digestive organs paralleling a reduction of energy expenditure of organs and tissues not related to digestion (i.e., skeletal muscle). This trade-off in energy allocation among organs suggests a mechanism by which organisms may alter BMR in response to a change in seasonal variation in food availability.

Metabolic rate and evaporative water loss in the silky starling (Sturnus sericeus)

PubMed Central

Huan-Huan, BAO; Qing-Jian, LIANG; Hong-Lei, ZHU; Xiao-Qiu, ZHOU; Wei-Hong, ZHENG; Jin-Song, LIU

2014-01-01

To better understand the physiological characteristics of the silky starling (Sturnus sericeus), its body temperature (Tb), basal metabolic rate (BMR), evaporative water loss (EWL) and thermal conductance (C) elicited by different ambient temperatures (Ta) (5−30 ℃) were determined in the present study. Our results showed that they have a high Tb (41.6±0.1 ℃), a wide thermal neutral zone (TNZ) (20−27.5 ℃) and a relatively low BMR within the TNZ (3.37±0.17 mL O2/g·h). The EWL was nearly stable below the TNZ (0.91±0.07 mg H2O/g·h) but increased remarkably within and above the TNZ. The C was constant below the TNZ, with a minimum value of 0.14±0.01 mL O2/g·h·℃. These findings indicate that the BMR, Tb and EWL of the silky starling were all affected by Ta, especially when Ta was below 20 ℃ and the EWL plays an important role in thermal regulation. PMID:25017746

A calmodulin binding protein from Arabidopsis is induced by ethylene and contains a DNA-binding motif

NASA Technical Reports Server (NTRS)

Reddy, A. S.; Reddy, V. S.; Golovkin, M.

2000-01-01

Calmodulin (CaM), a key calcium sensor in all eukaryotes, regulates diverse cellular processes by interacting with other proteins. To isolate CaM binding proteins involved in ethylene signal transduction, we screened an expression library prepared from ethylene-treated Arabidopsis seedlings with 35S-labeled CaM. A cDNA clone, EICBP (Ethylene-Induced CaM Binding Protein), encoding a protein that interacts with activated CaM was isolated in this screening. The CaM binding domain in EICBP was mapped to the C-terminus of the protein. These results indicate that calcium, through CaM, could regulate the activity of EICBP. The EICBP is expressed in different tissues and its expression in seedlings is induced by ethylene. The EICBP contains, in addition to a CaM binding domain, several features that are typical of transcription factors. These include a DNA-binding domain at the N terminus, an acidic region at the C terminus, and nuclear localization signals. In database searches a partial cDNA (CG-1) encoding a DNA-binding motif from parsley and an ethylene up-regulated partial cDNA from tomato (ER66) showed significant similarity to EICBP. In addition, five hypothetical proteins in the Arabidopsis genome also showed a very high sequence similarity with EICBP, indicating that there are several EICBP-related proteins in Arabidopsis. The structural features of EICBP are conserved in all EICBP-related proteins in Arabidopsis, suggesting that they may constitute a new family of DNA binding proteins and are likely to be involved in modulating gene expression in the presence of ethylene.

Binding Preferences, Surface Attachment, Diffusivity, and Orientation of a Family 1 Carbohydrate-binding Module on Cellulose*

PubMed Central

Nimlos, Mark R.; Beckham, Gregg T.; Matthews, James F.; Bu, Lintao; Himmel, Michael E.; Crowley, Michael F.

2012-01-01

Cellulase enzymes often contain carbohydrate-binding modules (CBMs) for binding to cellulose. The mechanisms by which CBMs recognize specific surfaces of cellulose and aid in deconstruction are essential to understand cellulase action. The Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase, Cel7A, is known to selectively bind to hydrophobic surfaces of native cellulose. It is most commonly suggested that three aromatic residues identify the planar binding face of this CBM, but several recent studies have challenged this hypothesis. Here, we use molecular simulation to study the CBM binding orientation and affinity on hydrophilic and hydrophobic cellulose surfaces. Roughly 43 μs of molecular dynamics simulations were conducted, which enables statistically significant observations. We quantify the fractions of the CBMs that detach from crystal surfaces or diffuse to other surfaces, the diffusivity along the hydrophobic surface, and the overall orientation of the CBM on both hydrophobic and hydrophilic faces. The simulations demonstrate that there is a thermodynamic driving force for the Cel7A CBM to bind preferentially to the hydrophobic surface of cellulose relative to hydrophilic surfaces. In addition, the simulations demonstrate that the CBM can diffuse from hydrophilic surfaces to the hydrophobic surface, whereas the reverse transition is not observed. Lastly, our simulations suggest that the flat faces of Family 1 CBMs are the preferred binding surfaces. These results enhance our understanding of how Family 1 CBMs interact with and recognize specific cellulose surfaces and provide insights into the initial events of cellulase adsorption and diffusion on cellulose. PMID:22496371

Probing the electrostatics and pharmacologic modulation of sequence-specific binding by the DNA-binding domain of the ETS-family transcription factor PU.1: a binding affinity and kinetics investigation

PubMed Central

Munde, Manoj; Poon, Gregory M. K.; Wilson, W. David

2013-01-01

Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally-conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤105 M−1 s−1), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt-dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>107 M−1 s−1). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes. PMID:23416556

Computation of pH-Dependent Binding Free Energies

PubMed Central

Kim, M. Olivia; McCammon, J. Andrew

2015-01-01

Protein-ligand binding accompanies changes in the surrounding electrostatic environments of the two binding partners and may lead to changes in protonation upon binding. In cases where the complex formation results in a net transfer of protons, the binding process is pH-dependent. However, conventional free energy computations or molecular docking protocols typically employ fixed protonation states for the titratable groups in both binding partners set a priori, which are identical for the free and bound states. In this review, we draw attention to these important yet largely ignored binding-induced protonation changes in protein-ligand association by outlining physical origins and prevalence of the protonation changes upon binding. Following a summary of various theoretical methods for pKa prediction, we discuss the theoretical framework to examine the pH dependence of protein-ligand binding processes. PMID:26202905

The selectivity of the Na+/K+-pump is controlled by binding site protonation and self-correcting occlusion

PubMed Central

Rui, Huan; Artigas, Pablo; Roux, Benoît

2016-01-01

The Na+/K+-pump maintains the physiological K+ and Na+ electrochemical gradients across the cell membrane. It operates via an 'alternating-access' mechanism, making iterative transitions between inward-facing (E1) and outward-facing (E2) conformations. Although the general features of the transport cycle are known, the detailed physicochemical factors governing the binding site selectivity remain mysterious. Free energy molecular dynamics simulations show that the ion binding sites switch their binding specificity in E1 and E2. This is accompanied by small structural arrangements and changes in protonation states of the coordinating residues. Additional computations on structural models of the intermediate states along the conformational transition pathway reveal that the free energy barrier toward the occlusion step is considerably increased when the wrong type of ion is loaded into the binding pocket, prohibiting the pump cycle from proceeding forward. This self-correcting mechanism strengthens the overall transport selectivity and protects the stoichiometry of the pump cycle. DOI: http://dx.doi.org/10.7554/eLife.16616.001 PMID:27490484

Monoclonal antibodies to human vitamin D-binding protein.

PubMed Central

Pierce, E A; Dame, M C; Bouillon, R; Van Baelen, H; DeLuca, H F

1985-01-01

Monoclonal antibodies to vitamin D-binding protein isolated from human serum have been produced. The antibodies obtained have been shown to be specific for human vitamin D-binding protein by three independent assays. The antibodies recognize human vitamin D-binding protein specifically in an enzyme-linked immunosorbent assay. Human vitamin D-binding protein is detected specifically in both pure and crude samples by a radiometric immunosorbent assay (RISA) and by an immunoprecipitation assay. The anti-human vitamin D-binding protein antibodies cross-react with monkey and pig vitamin D-binding protein, but not with vitamin D-binding protein from rat, mouse, or chicken, as determined by the RISA and immunoprecipitation assays. Images PMID:3936035

Interaction entropy for protein-protein binding

NASA Astrophysics Data System (ADS)

Sun, Zhaoxi; Yan, Yu N.; Yang, Maoyou; Zhang, John Z. H.

2017-03-01

Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interaction entropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interaction entropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

Interaction entropy for protein-protein binding.

PubMed

Sun, Zhaoxi; Yan, Yu N; Yang, Maoyou; Zhang, John Z H

2017-03-28

Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interactionentropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interactionentropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

Etude du comportement sous charges laterales des ossatures de beton arme avec murs de remplissage de maconnerie, construites avant les annees 1960

NASA Astrophysics Data System (ADS)

Lefebvre, Karine

Reinforced concrete structures with unreinforced masonry infills (BMR) are considered vulnerable to earthquakes. Under seismic actions, infills could fail (causing injuries or death) and cause damages to columns. In Quebec and Canada, most of BMR structures have been constructed prior to the introduction of modern seismic design codes raising question on the contribution of the infill to the structure lateral resistance. The aim of this thesis is to improve modelling technique of BMR structures built in Quebec between 1915 and 1960. This type of structures is found in hospitals or schools buildings, which must comply with some post-earthquake functionality requirements. They could also be residential or office buildings. Actually, practicing engineers usually calculate seismic capacity of BMR structures without considering the infill's structural contribution to the lateral resistance. Yet, this contribution should not be omitted. The first part of the thesis investigates the construction techniques and material properties of the old BMR structures in the Province. The results are the material properties (concrete, reinforcing steel, brick, terra cotta tile, and mortar) and the characteristics of the assemblies (wall section, reinforcement details…). The second part of the thesis presents the results of series of parametric analyses to identify among modelling and geometric parameters, which ones are the most influent on the lateral load response (rigidity, fundamental period, normal modes). Linear and modal analyses were performed. The most influent parameters identified are: number of storeys, number of bays, bay's width, soft storey, openings, upper storeys modelized (instead of being replaced by punctual loads) and the modelization technique of infills panels (strut or shell). Nonlinear static analyses have been performed to identify the most influent parameters to be considered for evaluating the lateral resistance, the capacity (load / displacement) and the

Sex-hormone-binding globulin.

PubMed

Anderson, D C

1974-01-01

A review was made to understand how plasma binding protein might influence sex-hormone action in target tissues. Steroids are predominately bound to plasma proteins and only unbound steroids enter the cells. Sex-hormone-binding globulin (SHBG) binds to both the main circulating steroid T and E2 but changes in SHBG concentrations exert significant results. Increased SHBG levels increase estrogen production and decreases T activity; whereas, increased androgens increase T action and inhibit SHBG production. These disturbances in hormone maintenance may lead to abnormal adult sex differentiation such as hirsutism and forms of hynaecomastia. By developing SHBG concentration measurement methods-responses of hirsutism to glucocorticoid or estrogem may be assessed. In addition, the effect of thyroid hormones on SHBG may also have therapeutic implications in endocrine disease.

Binding Energy and Enzymatic Catalysis.

ERIC Educational Resources Information Center

Hansen, David E.; Raines, Ronald T.

1990-01-01

Discussed is the fundamental role that the favorable free energy of binding of the rate-determining transition state plays in catalysis. The principle that all of the catalytic factors discussed are realized by the use of this binding energy is reviewed. (CW)

Kinetics of phloretin binding to phosphatidylcholine vesicle membranes

PubMed Central

1980-01-01

The submillisecond kinetics for phloretin binding to unilamellar phosphatidylcholine (PC) vesicles was investigated using the temperature-jump technique. Spectrophotometric studies of the equilibrium binding performed at 328 nm demonstrated that phloretin binds to a single set of independent, equivalent sites on the vesicle with a dissociation constant of 8.0 microM and a lipid/site ratio of 4.0. The temperature of the phloretin-vesicle solution was jumped by 4 degrees C within 4 microseconds producing a monoexponential, concentration-dependent relaxation process with time constants in the 30--200-microseconds time range. An analysis of the concentration dependence of relaxation time constants at pH 7.30 and 24 degrees C yielded a binding rate constant of 2.7 X 10(8) M-1 s-1 and an unbinding constant of 2,900 s-1; approximately 66 percent of total binding sites are exposed at the outer vesicle surface. The value of the binding rate constant and three additional observations suggest that the binding kinetics are diffusion limited. The phloretin analogue, naringenin, which has a diffusion coefficient similar to phloretin yet a dissociation constant equal to 24 microM, bound to PC vesicle with the same rate constant as phloretin did. In addition, the phloretin-PC system was studied in buffers made one to six times more viscous than water by addition of sucrose or glycerol to the differ. The equilibrium affinity for phloretin binding to PC vesicles is independent of viscosity, yet the binding rate constant decreases with the expected dependence (kappa binding alpha 1/viscosity) for diffusion-limited processes. Thus, the binding rate constant is not altered by differences in binding affinity, yet depends upon the diffusion coefficient in buffer. Finally, studies of the pH dependence of the binding rate constant showed a dependence (kappa binding alpha [1 + 10pH-pK]) consistent with the diffusion-limited binding of a weak acid. PMID:7391812

Quantitative in vivo receptor binding. III. Tracer kinetic modeling of muscarinic cholinergic receptor binding

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

Frey, K.A.; Hichwa, R.D.; Ehrenkaufer, R.L.

1985-10-01

A tracer kinetic method is developed for the in vivo estimation of high-affinity radioligand binding to central nervous system receptors. Ligand is considered to exist in three brain pools corresponding to free, nonspecifically bound, and specifically bound tracer. These environments, in addition to that of intravascular tracer, are interrelated by a compartmental model of in vivo ligand distribution. A mathematical description of the model is derived, which allows determination of regional blood-brain barrier permeability, nonspecific binding, the rate of receptor-ligand association, and the rate of dissociation of bound ligand, from the time courses of arterial blood and tissue tracer concentrations.more » The term ''free receptor density'' is introduced to describe the receptor population measured by this method. The technique is applied to the in vivo determination of regional muscarinic acetylcholine receptors in the rat, with the use of (TH)scopolamine. Kinetic estimates of free muscarinic receptor density are in general agreement with binding capacities obtained from previous in vivo and in vitro equilibrium binding studies. In the striatum, however, kinetic estimates of free receptor density are less than those in the neocortex--a reversal of the rank ordering of these regions derived from equilibrium determinations. A simplified model is presented that is applicable to tracers that do not readily dissociate from specific binding sites during the experimental period.« less

Nickel binding to NikA: an additional binding site reconciles spectroscopy, calorimetry and crystallography.

PubMed

Addy, Christine; Ohara, Masato; Kawai, Fumihiro; Kidera, Akinori; Ikeguchi, Mitsunori; Fuchigami, Sotaro; Osawa, Masanori; Shimada, Ichio; Park, Sam-Yong; Tame, Jeremy R H; Heddle, Jonathan G

2007-02-01

Intracellular nickel is required by Escherichia coli as a cofactor for a number of enzymes and is necessary for anaerobic respiration. However, high concentrations of nickel are toxic, so both import and export systems have evolved to control the cellular level of the metal. The nik operon in E. coli encodes a nickel-uptake system that includes the periplasmic nickel-binding protein NikA. The crystal structures of wild-type NikA both bound to nickel and in the apo form have been solved previously. The liganded structure appeared to show an unusual interaction between the nickel and the protein in which no direct bonds are formed. The highly unusual nickel coordination suggested by the crystal structure contrasted strongly with earlier X-ray spectroscopic studies. The known nickel-binding site has been probed by extensive mutagenesis and isothermal titration calorimetry and it has been found that even large numbers of disruptive mutations appear to have little effect on the nickel affinity. The crystal structure of a binding-site mutant with nickel bound has been solved and it is found that nickel is bound to two histidine residues at a position distant from the previously characterized binding site. This novel site immediately resolves the conflict between the crystal structures and other biophysical analyses. The physiological relevance of the two binding sites is discussed.

Conformational selection in protein binding and function

PubMed Central

Weikl, Thomas R; Paul, Fabian

2014-01-01

Protein binding and function often involves conformational changes. Advanced nuclear magnetic resonance (NMR) experiments indicate that these conformational changes can occur in the absence of ligand molecules (or with bound ligands), and that the ligands may “select” protein conformations for binding (or unbinding). In this review, we argue that this conformational selection requires transition times for ligand binding and unbinding that are small compared to the dwell times of proteins in different conformations, which is plausible for small ligand molecules. Such a separation of timescales leads to a decoupling and temporal ordering of binding/unbinding events and conformational changes. We propose that conformational-selection and induced-change processes (such as induced fit) are two sides of the same coin, because the temporal ordering is reversed in binding and unbinding direction. Conformational-selection processes can be characterized by a conformational excitation that occurs prior to a binding or unbinding event, while induced-change processes exhibit a characteristic conformational relaxation that occurs after a binding or unbinding event. We discuss how the ordering of events can be determined from relaxation rates and effective on- and off-rates determined in mixing experiments, and from the conformational exchange rates measured in advanced NMR or single-molecule fluorescence resonance energy transfer experiments. For larger ligand molecules such as peptides, conformational changes and binding events can be intricately coupled and exhibit aspects of conformational-selection and induced-change processes in both binding and unbinding direction. PMID:25155241

Structure of the Mecl Repressor from Staphylococcus aureus in Complex with the Cognate DNA Operator of mec

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

Safo,M.; Ko, T.; Musayev, F.

The dimeric repressor MecI regulates the mecA gene that encodes the penicillin-binding protein PBP-2a in methicillin-resistant Staphylococcus aureus (MRSA). MecI is similar to BlaI, the repressor for the blaZ gene of {beta}-lactamase. MecI and BlaI can bind to both operator DNA sequences. The crystal structure of MecI in complex with the 32 base-pair cognate DNA of mec was determined to 3.8 Angstroms resolution. MecI is a homodimer and each monomer consists of a compact N-terminal winged-helix domain, which binds to DNA, and a loosely packed C-terminal helical domain, which intertwines with its counter-monomer. The crystal contains horizontal layers of virtualmore » DNA double helices extending in three directions, which are separated by perpendicular DNA segments. Each DNA segment is bound to two MecI dimers. Similar to the BlaI-mec complex, but unlike the MecI-bla complex, the MecI repressors bind to both sides of the mec DNA dyad that contains four conserved sequences of TACA/TGTA. The results confirm the up-and-down binding to the mec operator, which may account for cooperative effect of the repressor.« less

Systematic prediction of control proteins and their DNA binding sites

PubMed Central

Sorokin, Valeriy; Severinov, Konstantin; Gelfand, Mikhail S.

2009-01-01

We present here the results of a systematic bioinformatics analysis of control (C) proteins, a class of DNA-binding regulators that control time-delayed transcription of their own genes as well as restriction endonuclease genes in many type II restriction-modification systems. More than 290 C protein homologs were identified and DNA-binding sites for ∼70% of new and previously known C proteins were predicted by a combination of phylogenetic footprinting and motif searches in DNA upstream of C protein genes. Additional analysis revealed that a large proportion of C protein genes are translated from leaderless RNA, which may contribute to time-delayed nature of genetic switches operated by these proteins. Analysis of genetic contexts of newly identified C protein genes revealed that they are not exclusively associated with restriction-modification genes; numerous instances of associations with genes originating from mobile genetic elements were observed. These instances might be vestiges of ancient horizontal transfers and indicate that during evolution ancestral restriction-modification system genes were the sites of mobile elements insertions. PMID:19056824

Coordination of two sequential ester-transfer reactions: exogenous guanosine binding promotes the subsequent ωG binding to a group I intron

PubMed Central

Bao, Penghui; Wu, Qi-Jia; Yin, Ping; Jiang, Yanfei; Wang, Xu; Xie, Mao-Hua; Sun, Tao; Huang, Lin; Mo, Ding-Ding; Zhang, Yi

2008-01-01

Self-splicing of group I introns is accomplished by two sequential ester-transfer reactions mediated by sequential binding of two different guanosine ligands, but it is yet unclear how the binding is coordinated at a single G-binding site. Using a three-piece trans-splicing system derived from the Candida intron, we studied the effect of the prior GTP binding on the later ωG binding by assaying the ribozyme activity in the second reaction. We showed that adding GTP simultaneously with and prior to the esterified ωG in a substrate strongly accelerated the second reaction, suggesting that the early binding of GTP facilitates the subsequent binding of ωG. GTP-mediated facilitation requires C2 amino and C6 carbonyl groups on the Watson–Crick edge of the base but not the phosphate or sugar groups, suggesting that the base triple interactions between GTP and the binding site are important for the subsequent ωG binding. Strikingly, GTP binding loosens a few local structures of the ribozyme including that adjacent to the base triple, providing structural basis for a rapid exchange of ωG for bound GTP. PMID:18978026

Survey of immune-related, mannose/fucose-binding C-type lectin receptors reveals widely divergent sugar-binding specificities

PubMed Central

Lee, Reiko T; Hsu, Tsui-Ling; Huang, Shau Ku; Hsieh, Shie-Liang; Wong, Chi-Huey; Lee, Yuan C

2011-01-01

C-type lectins (CTLs) are proteins that contain one or more carbohydrate-recognition domains (CRDs) that require calcium for sugar binding and share high degree of sequence homology and tertiary structure. CTLs whose CRD contain EPN (Glu-Pro-Asn) tripeptide motifs have potential to bind mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc) and l-fucose (Fuc), whereas those with QPD (Glu-Pro-Asp) tripeptide motifs bind galactose (Gal) and N-acetylgalactosamine (GalNAc). We report here for the first time a direct comparison of monosaccharide (and some di- and trisaccharides)-binding characteristics of 11 EPX-containing (X = N, S or D) immune-related CTLs using a competition assay and an enzyme-linked immunosorbent assay, and neoglycoproteins as ligand. The EPX CTLs studied are DC-SIGN, L-SIGN, mSIGNR1, human and mouse mannose receptors, Langerin, BDCA-2, DCIR, dectin-2, MCL and MINCLE. We found that: (1) they all bound Man and Fuc; (2) binding of Glc and GlcNAc varied considerably among these lectins, but was always less than Man and Fuc; (3) in general, Gal and GalNAc were not bound. However, dectin-2, DCIR and MINCLE showed ability to bind Gal/GalNAc; (4) DC-SIGN, L-SIGN, mSIGNR1 and Langerin showed enhanced binding of Manα2Man over Man, whereas all others showed no enhancement; (5) DC-SIGN bound Lex trisaccharide structure, which has terminal Gal and Fuc residues, more avidly than Fuc, whereas L-SIGN, mSIGNR1, DCIR and MINCLE bound Lex less avidly than Fuc. BDCA-2, dectin-2, Langerin, MCL and mannose receptor did not bind Lex at all. PMID:21112966

Survey of immune-related, mannose/fucose-binding C-type lectin receptors reveals widely divergent sugar-binding specificities.

PubMed

Lee, Reiko T; Hsu, Tsui-Ling; Huang, Shau Ku; Hsieh, Shie-Liang; Wong, Chi-Huey; Lee, Yuan C

2011-04-01

C-type lectins (CTLs) are proteins that contain one or more carbohydrate-recognition domains (CRDs) that require calcium for sugar binding and share high degree of sequence homology and tertiary structure. CTLs whose CRD contain EPN (Glu-Pro-Asn) tripeptide motifs have potential to bind mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc) and l-fucose (Fuc), whereas those with QPD (Glu-Pro-Asp) tripeptide motifs bind galactose (Gal) and N-acetylgalactosamine (GalNAc). We report here for the first time a direct comparison of monosaccharide (and some di- and trisaccharides)-binding characteristics of 11 EPX-containing (X = N, S or D) immune-related CTLs using a competition assay and an enzyme-linked immunosorbent assay, and neoglycoproteins as ligand. The EPX CTLs studied are DC-SIGN, L-SIGN, mSIGNR1, human and mouse mannose receptors, Langerin, BDCA-2, DCIR, dectin-2, MCL and MINCLE. We found that: (1) they all bound Man and Fuc; (2) binding of Glc and GlcNAc varied considerably among these lectins, but was always less than Man and Fuc; (3) in general, Gal and GalNAc were not bound. However, dectin-2, DCIR and MINCLE showed ability to bind Gal/GalNAc; (4) DC-SIGN, L-SIGN, mSIGNR1 and Langerin showed enhanced binding of Manα2Man over Man, whereas all others showed no enhancement; (5) DC-SIGN bound Le(x) trisaccharide structure, which has terminal Gal and Fuc residues, more avidly than Fuc, whereas L-SIGN, mSIGNR1, DCIR and MINCLE bound Le(x) less avidly than Fuc. BDCA-2, dectin-2, Langerin, MCL and mannose receptor did not bind Le(x) at all.

Structure of the MecI repressor from Staphylococcus aureus in complex with the cognate DNA operator of mec

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

Safo, Martin K., E-mail: msafo@vcu.edu; Ko, Tzu-Ping; Musayev, Faik N.

The up-and-down binding of dimeric MecI to mecA dyad DNA may account for the cooperative effect of the repressor. The dimeric repressor MecI regulates the mecA gene that encodes the penicillin-binding protein PBP-2a in methicillin-resistant Staphylococcus aureus (MRSA). MecI is similar to BlaI, the repressor for the blaZ gene of β-lactamase. MecI and BlaI can bind to both operator DNA sequences. The crystal structure of MecI in complex with the 32 base-pair cognate DNA of mec was determined to 3.8 Å resolution. MecI is a homodimer and each monomer consists of a compact N-terminal winged-helix domain, which binds to DNA,more » and a loosely packed C-terminal helical domain, which intertwines with its counter-monomer. The crystal contains horizontal layers of virtual DNA double helices extending in three directions, which are separated by perpendicular DNA segments. Each DNA segment is bound to two MecI dimers. Similar to the BlaI–mec complex, but unlike the MecI–bla complex, the MecI repressors bind to both sides of the mec DNA dyad that contains four conserved sequences of TACA/TGTA. The results confirm the up-and-down binding to the mec operator, which may account for cooperative effect of the repressor.« less

Expensive Brains: "Brainy" Rodents have Higher Metabolic Rate.

PubMed

Sobrero, Raúl; May-Collado, Laura J; Agnarsson, Ingi; Hernández, Cristián E

2011-01-01

Brains are the centers of the nervous system of animals, controlling the organ systems of the body and coordinating responses to changes in the ecological and social environment. The evolution of traits that correlate with cognitive ability, such as relative brain size is thus of broad interest. Brain mass relative to body mass (BM) varies among mammals, and diverse factors have been proposed to explain this variation. A recent study provided evidence that energetics play an important role in brain evolution (Isler and van Schaik, 2006). Using composite phylogenies and data drawn from multiple sources, these authors showed that basal metabolic rate (BMR) correlates with brain mass across mammals. However, no such relationship was found within rodents. Here we re-examined the relationship between BMR and brain mass within Rodentia using a novel species-level phylogeny. Our results are sensitive to parameter evaluation; in particular how species mass is estimated. We detect no pattern when applying an approach used by previous studies, where each species BM is represented by two different numbers, one being the individual that happened to be used for BMR estimates of that species. However, this approach may compromise the analysis. When using a single value of BM for each species, whether representing a single individual, or available species mean, our findings provide evidence that brain mass (independent of BM) and BMR are correlated. These findings are thus consistent with the hypothesis that large brains evolve when the payoff for increased brain mass is greater than the energetic cost they incur.

Contaminants and energy expenditure in an Arctic seabird: Organochlorine pesticides and perfluoroalkyl substances are associated with metabolic rate in a contrasted manner.

PubMed

Blévin, Pierre; Tartu, Sabrina; Ellis, Hugh I; Chastel, Olivier; Bustamante, Paco; Parenteau, Charline; Herzke, Dorte; Angelier, Frédéric; Gabrielsen, Geir W

2017-08-01

Basal metabolic rate (BMR), the minimal energetic cost of living in endotherms, is known to be influenced by thyroid hormones (THs) which are known to stimulate in vitro oxygen consumption of tissues in birds and mammals. Several environmental contaminants may act on energy expenditure through their thyroid hormone-disrupting properties. However, the effect of contaminants on BMR is still poorly documented for wildlife. Here, we investigated the relationships between three groups of contaminants (organochlorines (OCs), perfluoroalkyl substances (PFASs), and mercury) with metabolic rate (MR), considered here as a proxy of BMR and also with circulating total THs (thyroxine (TT4) and triiodothyronine (TT3)) in Arctic breeding adult black-legged kittiwakes (Rissa tridactyla) from Svalbard, during the chick rearing period. Our results indicate a negative relationship between the sum of all detected chlordanes (∑CHLs) and MR in both sexes whereas perfluorotridecanoate (PFTrA) and MR were positively related in females only. MR was not associated with mercury. Additionally, levels of TT3 were negatively related to ∑CHLs but not to PFTrA. The findings from the present study indicate that some OCs (in both sexes) and some PFASs (only in females) could disrupt fine adjustment of BMR during reproduction in adult kittiwakes. Importantly, highly lipophilic OCs and highly proteinophilic PFASs appear, at least in females, to have the ability to disrupt the metabolic rate in an opposite way. Therefore, our study highlights the need for ecotoxicological studies to include a large variety of contaminants which can act in an antagonistic manner. Copyright © 2017 Elsevier Inc. All rights reserved.

Seasonal Metabolic Acclimatization Varies in Direction and Magnitude among Populations of an Afrotropical Passerine Bird.

PubMed

Noakes, Matthew J; Wolf, Blair O; McKechnie, Andrew E

Avian metabolic responses demonstrate considerable diversity under fluctuating environmental conditions, a well-studied example being the seasonal upregulation of basal metabolic rate (BMR) and summit metabolism (M sum ) in temperate species experiencing harsh winters. Fewer studies have examined seasonal metabolic acclimatization in subtropical or tropical species. We investigated seasonal metabolic variation in an Afrotropical ploceid passerine, the white-browed sparrow-weaver (Plocepasser mahali; ∼47 g), at three sites along a climatic gradient of approximately 7°C in winter minimum air temperature (T a ). We measured M sum (n ≥ 10 per site per season) in a helox atmosphere, BMR of the same birds at thermoneutrality (T a ≈ 30°C), and resting metabolic rates at 5°C ≤ T a ≤ 20°C. Patterns of seasonal adjustments in BMR varied among populations in a manner not solely related to variation in seasonal T a extremes, ranging from BMR ∼52% higher in winter than in summer to no seasonal difference. Greater cold tolerance was found in a population at a colder desert site, manifested as higher M sum (∼25% higher) and lower helox temperature at cold limit values compared with a milder, mesic site. Our results lend support to the idea that greater variance in the pattern of seasonal metabolic responses occurs in subtropical and tropical species compared with their temperate-zone counterparts and that factors other than T a extremes (e.g., food availability) may be important in determining the magnitude and direction of seasonal metabolic adjustments in subtropical birds.

A Coupled 2 × 2D Babcock-Leighton Solar Dynamo Model. II. Reference Dynamo Solutions

NASA Astrophysics Data System (ADS)

Lemerle, Alexandre; Charbonneau, Paul

2017-01-01

In this paper we complete the presentation of a new hybrid 2 × 2D flux transport dynamo (FTD) model of the solar cycle based on the Babcock-Leighton mechanism of poloidal magnetic field regeneration via the surface decay of bipolar magnetic regions (BMRs). This hybrid model is constructed by allowing the surface flux transport (SFT) simulation described in Lemerle et al. to provide the poloidal source term to an axisymmetric FTD simulation defined in a meridional plane, which in turn generates the BMRs required by the SFT. A key aspect of this coupling is the definition of an emergence function describing the probability of BMR emergence as a function of the spatial distribution of the internal axisymmetric magnetic field. We use a genetic algorithm to calibrate this function, together with other model parameters, against observed cycle 21 emergence data. We present a reference dynamo solution reproducing many solar cycle characteristics, including good hemispheric coupling, phase relationship between the surface dipole and the BMR-generating internal field, and correlation between dipole strength at cycle maximum and peak amplitude of the next cycle. The saturation of the cycle amplitude takes place through the quenching of the BMR tilt as a function of the internal field. The observed statistical scatter about the mean BMR tilt, built into the model, acts as a source of stochasticity which dominates amplitude fluctuations. The model thus can produce Dalton-like epochs of strongly suppressed cycle amplitude lasting a few cycles and can even shut off entirely following an unfavorable sequence of emergence events.

Computational search for aflatoxin binding proteins

NASA Astrophysics Data System (ADS)

Wang, Ying; Liu, Jinfeng; Zhang, Lujia; He, Xiao; Zhang, John Z. H.

2017-10-01

Aflatoxin is one of the mycotoxins that contaminate various food products. Among various aflatoxin types (B1, B2, G1, G2 and M1), aflatoxin B1 is the most important and the most toxic one. In this study, through computational screening, we found that several proteins may bind specifically with different type of aflatoxins. Combination of theoretical methods including target fishing, molecular docking, molecular dynamics (MD) simulation, MM/PBSA calculation were utilized to search for new aflatoxin B1 binding proteins. A recently developed method for calculating entropic contribution to binding free energy called interaction entropy (IE) was employed to compute the binding free energy between the protein and aflatoxin B1. Through comprehensive comparison, three proteins, namely, trihydroxynaphthalene reductase, GSK-3b, and Pim-1 were eventually selected as potent aflatoxin B1 binding proteins. GSK-3b and Pim-1 are drug targets of cancers or neurological diseases. GSK-3b is the strongest binder for aflatoxin B1.

BIND: the Biomolecular Interaction Network Database

PubMed Central

Bader, Gary D.; Betel, Doron; Hogue, Christopher W. V.

2003-01-01

The Biomolecular Interaction Network Database (BIND: http://bind.ca) archives biomolecular interaction, complex and pathway information. A web-based system is available to query, view and submit records. BIND continues to grow with the addition of individual submissions as well as interaction data from the PDB and a number of large-scale interaction and complex mapping experiments using yeast two hybrid, mass spectrometry, genetic interactions and phage display. We have developed a new graphical analysis tool that provides users with a view of the domain composition of proteins in interaction and complex records to help relate functional domains to protein interactions. An interaction network clustering tool has also been developed to help focus on regions of interest. Continued input from users has helped further mature the BIND data specification, which now includes the ability to store detailed information about genetic interactions. The BIND data specification is available as ASN.1 and XML DTD. PMID:12519993

Hyaluronate-binding proteins of murine brain.

PubMed

Marks, M S; Chi-Rosso, G; Toole, B P

1990-01-01

The distribution of hyaluronate-binding activity was determined in the soluble and membrane fractions derived from adult mouse brain by sonication in low-ionic-strength buffer. Approximately 60% of the total activity was recovered in the soluble fraction and 33% in membrane fractions. In both cases, the hyaluronate-binding activities were found to be of high affinity (KD = 10(-9) M), specific for hyaluronate, and glycoprotein in nature. Most of the hyaluronate-binding activity from the soluble fraction chromatographed in the void volume of Sepharose CL-4B and CL-6B. Approximately 50% of this activity was highly negatively charged, eluting from diethylaminoethyl (DEAE)-cellulose in 0.5 M NaCl, and contained chondroitin sulfate chains. This latter material also reacted with antibodies raised against cartilage link protein and the core protein of cartilage proteoglycan. Thus, the binding and physical characteristics of this hyaluronate-binding activity are consistent with those of a chondroitin sulfate proteoglycan aggregate similar to that found in cartilage. A 500-fold purification of this proteoglycan-like, hyaluronate-binding material was achieved by wheat germ agglutinin affinity chromatography, molecular sieve chromatography on Sepharose CL-6B, and ion exchange chromatography on DEAE-cellulose. Another class of hyaluronate-binding material (25-50% of that recovered) eluted from DEAE with 0.24 M NaCl; this material had the properties of a complex glycoprotein, did not contain chondroitin sulfate, and did not react with the antibodies against cartilage link protein and proteoglycan. Thus, adult mouse brain contains at least three different forms of hyaluronate-binding macromolecules. Two of these have properties similar to the link protein and proteoglycan of cartilage proteoglycan aggregates; the third is distinguishable from these entities.

Managing a Library Binding Program.

ERIC Educational Resources Information Center

Merrill-Oldham, Jan

Library binding is one of the activities typically included in newly created preservation departments, but librarians continue to discover that transforming a traditional binding program into one that better meets preservation objectives requires considerable investment of time. This resource guide is intended to help libraries review their…

The helical structure of DNA facilitates binding

NASA Astrophysics Data System (ADS)

Berg, Otto G.; Mahmutovic, Anel; Marklund, Emil; Elf, Johan

2016-09-01

The helical structure of DNA imposes constraints on the rate of diffusion-limited protein binding. Here we solve the reaction-diffusion equations for DNA-like geometries and extend with simulations when necessary. We find that the helical structure can make binding to the DNA more than twice as fast compared to a case where DNA would be reactive only along one side. We also find that this rate advantage remains when the contributions from steric constraints and rotational diffusion of the DNA-binding protein are included. Furthermore, we find that the association rate is insensitive to changes in the steric constraints on the DNA in the helix geometry, while it is much more dependent on the steric constraints on the DNA-binding protein. We conclude that the helical structure of DNA facilitates the nonspecific binding of transcription factors and structural DNA-binding proteins in general.

The Identity-Location Binding Problem.

PubMed

Howe, Piers D L; Ferguson, Adam

2015-09-01

The binding problem is fundamental to visual perception. It is the problem of associating an object's visual properties with itself and not with some other object. The problem is made particular difficult because different properties of an object, such as its color, shape, size, and motion, are often processed independently, sometimes in different cortical areas. The results of these separate analyses have to be combined before the object can be seen as a single coherent entity as opposed to a collection of unconnected features. Visual bindings are typically initiated and updated in a serial fashion, one object at a time. Here, we show that one type of binding, location-identity bindings, can be updated in parallel. We do this by using two complementary techniques, the simultaneous-sequential paradigm and systems factorial technology. These techniques make different assumptions and rely on different behavioral measures, yet both came to the same conclusion. Copyright © 2014 Cognitive Science Society, Inc.

Evaluating the binding efficiency of pheromone binding protein with its natural ligand using molecular docking and fluorescence analysis

NASA Astrophysics Data System (ADS)

Ilayaraja, Renganathan; Rajkumar, Ramalingam; Rajesh, Durairaj; Muralidharan, Arumugam Ramachandran; Padmanabhan, Parasuraman; Archunan, Govindaraju

2014-06-01

Chemosignals play a crucial role in social and sexual communication among inter- and intra-species. Chemical cues are bound with protein that is present in the pheromones irrespective of sex are commonly called as pheromone binding protein (PBP). In rats, the pheromone compounds are bound with low molecular lipocalin protein α2u-globulin (α2u). We reported farnesol is a natural endogenous ligand (compound) present in rat preputial gland as a bound volatile compound. In the present study, an attempt has been made through computational method to evaluating the binding efficiency of α2u with the natural ligand (farnesol) and standard fluorescent molecule (2-naphthol). The docking analysis revealed that the binding energy of farnesol and 2-naphthol was almost equal and likely to share some binding pocket of protein. Further, to extrapolate the results generated through computational approach, the α2u protein was purified and subjected to fluorescence titration and binding assay. The results showed that the farnesol is replaced by 2-naphthol with high hydrophobicity of TYR120 in binding sites of α2u providing an acceptable dissociation constant indicating the binding efficiency of α2u. The obtained results are in corroboration with the data made through computational approach.

Hardware device binding and mutual authentication

DOEpatents

Hamlet, Jason R; Pierson, Lyndon G

2014-03-04

Detection and deterrence of device tampering and subversion by substitution may be achieved by including a cryptographic unit within a computing device for binding multiple hardware devices and mutually authenticating the devices. The cryptographic unit includes a physically unclonable function ("PUF") circuit disposed in or on the hardware device, which generates a binding PUF value. The cryptographic unit uses the binding PUF value during an enrollment phase and subsequent authentication phases. During a subsequent authentication phase, the cryptographic unit uses the binding PUF values of the multiple hardware devices to generate a challenge to send to the other device, and to verify a challenge received from the other device to mutually authenticate the hardware devices.

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

E2 Proteins from High- and Low-Risk Human Papillomavirus Types Differ in Their Ability To Bind p53 and Induce Apoptotic Cell Death

PubMed Central

Parish, Joanna L.; Kowalczyk, Anna; Chen, Hsin-Tien; Roeder, Geraldine E.; Sessions, Richard; Buckle, Malcolm; Gaston, Kevin

2006-01-01

The E2 proteins from oncogenic (high-risk) human papillomaviruses (HPVs) can induce apoptotic cell death in both HPV-transformed and non-HPV-transformed cells. Here we show that the E2 proteins from HPV type 6 (HPV6) and HPV11, two nononcogenic (low-risk) HPV types, fail to induce apoptosis. Unlike the high-risk HPV16 E2 protein, these low-risk E2 proteins fail to bind p53 and fail to induce p53-dependent transcription activation. Interestingly, neither the ability of p53 to activate transcription nor the ability of p53 to bind DNA, are required for HPV16 E2-induced apoptosis in non-HPV-transformed cells. However, mutations that reduce the binding of the HPV16 E2 protein to p53 inhibit E2-induced apoptosis in non-HPV-transformed cells. In contrast, the interaction between HPV16 E2 and p53 is not required for this E2 protein to induce apoptosis in HPV-transformed cells. Thus, our data suggest that this high-risk HPV E2 protein induces apoptosis via two pathways. One pathway involves the binding of E2 to p53 and can operate in both HPV-transformed and non-HPV-transformed cells. The second pathway requires the binding of E2 to the viral genome and can only operate in HPV-transformed cells. PMID:16611918

Ethylene binding site affinity in ripening apples

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

Blankenship, S.M.; Sisler, E.C.

1993-09-01

Scatchard plots for ethylene binding in apples (Malus domestica Borkh.), which were harvested weekly for 5 weeks to include the ethylene climacteric rise, showed C[sub 50] values (concentration of ethylene needed to occupy 50% of the ethylene binding sites) of 0.10, 0.11, 0.34, 0.40, and 0.57 [mu]l ethylene/liter[sup [minus]1], respectively, for each of the 5 weeks. Higher ethylene concentrations were required to saturate the binding sites during the climacteric rise than at other times. Diffusion of [sup 14]C-ethylene from the binding sites was curvilinear and did not show any indication of multiple binding sites. Ethylene was not metabolized by applemore » tissue.« less

Hydrophobic interaction chromatography in dual salt system increases protein binding capacity.

PubMed

Senczuk, Anna M; Klinke, Ralph; Arakawa, Tsutomu; Vedantham, Ganesh; Yigzaw, Yinges

2009-08-01

Hydrophobic interaction chromatography (HIC) uses weakly hydrophobic resins and requires a salting-out salt to promote protein-resin interaction. The salting-out effects increase with protein and salt concentration. Dynamic binding capacity (DBC) is dependent on the binding constant, as well as on the flow characteristics during sample loading. DBC increases with the salt concentration but decreases with increasing flow rate. Dynamic and operational binding capacity have a major raw material cost/processing time impact on commercial scale production of monoclonal antibodies. In order to maximize DBC the highest salt concentration without causing precipitation is used. We report here a novel method to maintain protein solubility while increasing the DBC by using a combination of two salting-out salts (referred to as dual salt). In a series of experiments, we explored the dynamic capacity of a HIC resin (TosoBioscience Butyl 650M) with combinations of salts. Using a model antibody, we developed a system allowing us to increase the dynamic capacity up to twofold using the dual salt system over traditional, single salt system. We also investigated the application of this novel approach to several other proteins and salt combinations, and noted a similar protein solubility and DBC increase. The observed increase in DBC in the dual salt system was maintained at different linear flow rates and did not impact selectivity.

Competition for DNA binding sites using Promega DNA IQ™ paramagnetic beads.

PubMed

Frégeau, Chantal J; De Moors, Anick

2012-09-01

The Promega DNA IQ™ system is easily amenable to automation and has been an integral part of standard operating procedures for many forensic laboratories including those of the Royal Canadian Mounted Police (RCMP) since 2004. Due to some failure to extract DNA from samples that should have produced DNA using our validated automated DNA IQ™-based protocol, the competition for binding sites on the DNA IQ™ magnetic beads was more closely examined. Heme from heavily blooded samples interfered slightly with DNA binding. Increasing the concentration of Proteinase K during lysis of these samples did not enhance DNA recovery. However, diluting the sample lysate following lysis prior to DNA extraction overcame the reduction in DNA yield and preserved portions of the lysates for subsequent manual or automated extraction. Dye/chemicals from black denim lysates competed for binding sites on the DNA IQ™ beads and significantly reduced DNA recovery. Increasing the size or number of black denim cuttings during lysis had a direct adverse effect on DNA yield from various blood volumes. The dilution approach was successful on these samples and permitted the extraction of high DNA yields. Alternatively, shortening the incubation time for cell lysis to 30 min instead of the usual overnight at 56 °C prevented competition from black denim dye/chemicals and increased DNA yields. Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.

Rate constants for proteins binding to substrates with multiple binding sites using a generalized forward flux sampling expression

NASA Astrophysics Data System (ADS)

Vijaykumar, Adithya; ten Wolde, Pieter Rein; Bolhuis, Peter G.

2018-03-01

To predict the response of a biochemical system, knowledge of the intrinsic and effective rate constants of proteins is crucial. The experimentally accessible effective rate constant for association can be decomposed in a diffusion-limited rate at which proteins come into contact and an intrinsic association rate at which the proteins in contact truly bind. Reversely, when dissociating, bound proteins first separate into a contact pair with an intrinsic dissociation rate, before moving away by diffusion. While microscopic expressions exist that enable the calculation of the intrinsic and effective rate constants by conducting a single rare event simulation of the protein dissociation reaction, these expressions are only valid when the substrate has just one binding site. If the substrate has multiple binding sites, a bound enzyme can, besides dissociating into the bulk, also hop to another binding site. Calculating transition rate constants between multiple states with forward flux sampling requires a generalized rate expression. We present this expression here and use it to derive explicit expressions for all intrinsic and effective rate constants involving binding to multiple states, including rebinding. We illustrate our approach by computing the intrinsic and effective association, dissociation, and hopping rate constants for a system in which a patchy particle model enzyme binds to a substrate with two binding sites. We find that these rate constants increase as a function of the rotational diffusion constant of the particles. The hopping rate constant decreases as a function of the distance between the binding sites. Finally, we find that blocking one of the binding sites enhances both association and dissociation rate constants. Our approach and results are important for understanding and modeling association reactions in enzyme-substrate systems and other patchy particle systems and open the way for large multiscale simulations of such systems.

Mechanisms of zinc binding to the solute-binding protein AztC and transfer from the metallochaperone AztD.

PubMed

Neupane, Durga P; Avalos, Dante; Fullam, Stephanie; Roychowdhury, Hridindu; Yukl, Erik T

2017-10-20

Bacteria can acquire the essential metal zinc from extremely zinc-limited environments by using ATP-binding cassette (ABC) transporters. These transporters are critical virulence factors, relying on specific and high-affinity binding of zinc by a periplasmic solute-binding protein (SBP). As such, the mechanisms of zinc binding and release among bacterial SBPs are of considerable interest as antibacterial drug targets. Zinc SBPs are characterized by a flexible loop near the high-affinity zinc-binding site. The function of this structure is not always clear, and its flexibility has thus far prevented structural characterization by X-ray crystallography. Here, we present intact structures for the zinc-specific SBP AztC from the bacterium Paracoccus denitrificans in the zinc-bound and apo-states. A comparison of these structures revealed that zinc loss prompts significant structural rearrangements, mediated by the formation of a sodium-binding site in the apo-structure. We further show that the AztC flexible loop has no impact on zinc-binding affinity, stoichiometry, or protein structure, yet is essential for zinc transfer from the metallochaperone AztD. We also found that 3 His residues in the loop appear to temporarily coordinate zinc and then convey it to the high-affinity binding site. Thus, mutation of any of these residues to Ala abrogated zinc transfer from AztD. Our structural and mechanistic findings conclusively identify a role for the AztC flexible loop in zinc acquisition from the metallochaperone AztD, yielding critical insights into metal binding by AztC from both solution and AztD. These proteins are highly conserved in human pathogens, making this work potentially useful for the development of novel antibiotics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Unusual binding of ursodeoxycholic acid to ileal bile acid binding protein: role in activation of FXRα[S

PubMed Central

Fang, Changming; Filipp, Fabian V.; Smith, Jeffrey W.

2012-01-01

Ursodeoxycholic acid (UDCA, ursodiol) is used to prevent damage to the liver in patients with primary biliary cirrhosis. The drug also prevents the progression of colorectal cancer and the recurrence of high-grade colonic dysplasia. However, the molecular mechanism by which UDCA elicits its beneficial effects is not entirely understood. The aim of this study was to determine whether ileal bile acid binding protein (IBABP) has a role in mediating the effects of UDCA. We find that UDCA binds to a single site on IBABP and increases the affinity for major human bile acids at a second binding site. As UDCA occupies one of the bile acid binding sites on IBABP, it reduces the cooperative binding that is often observed for the major human bile acids. Furthermore, IBABP is necessary for the full activation of farnesoid X receptor α (FXRα) by bile acids, including UDCA. These observations suggest that IBABP may have a role in mediating some of the intestinal effects of UDCA. PMID:22223860

Nucleotide-dependent bisANS binding to tubulin.

PubMed

Chakraborty, S; Sarkar, N; Bhattacharyya, B

1999-07-13

Non-covalent hydrophobic probes such as 5, 5'-bis(8-anilino-1-naphthalenesulfonate) (bisANS) have become increasingly popular to gain information about protein structure and conformation. However, there are limitations as bisANS binds non-specifically at multiple sites of many proteins. Successful use of this probe depends upon the development of binding conditions where only specific dye-protein interaction will occur. In this report, we have shown that the binding of bisANS to tubulin occurs instantaneously, specifically at one high affinity site when 1 mM guanosine 5'-triphosphate (GTP) is included in the reaction medium. Substantial portions of protein secondary structure and colchicine binding activity of tubulin are lost upon bisANS binding in absence of GTP. BisANS binding increases with time and occurs at multiple sites in the absence of GTP. Like GTP, other analogs, guanosine 5'-diphosphate, guanosine 5'-monophosphate and adenosine 5'-triphosphate, also displace bisANS from the lower affinity sites of tubulin. We believe that these multiple binding sites are generated due to the bisANS-induced structural changes on tubulin and the presence of GTP and other nucleotides protect those structural changes.

Protein Binding: Do We Ever Learn?▿

PubMed Central

Zeitlinger, Markus A.; Derendorf, Hartmut; Mouton, Johan W.; Cars, Otto; Craig, William A.; Andes, David; Theuretzbacher, Ursula

2011-01-01

Although the influence of protein binding (PB) on antibacterial activity has been reported for many antibiotics and over many years, there is currently no standardization for pharmacodynamic models that account for the impact of protein binding of antimicrobial agents in vitro. This might explain the somewhat contradictory results obtained from different studies. Simple in vitro models which compare the MIC obtained in protein-free standard medium versus a protein-rich medium are prone to methodological pitfalls and may lead to flawed conclusions. Within in vitro test systems, a range of test conditions, including source of protein, concentration of the tested antibiotic, temperature, pH, electrolytes, and supplements may influence the impact of protein binding. As new antibiotics with a high degree of protein binding are in clinical development, attention and action directed toward the optimization and standardization of testing the impact of protein binding on the activity of antibiotics in vitro become even more urgent. In addition, the quantitative relationship between the effects of protein binding in vitro and in vivo needs to be established, since the physiological conditions differ. General recommendations for testing the impact of protein binding in vitro are suggested. PMID:21537013

Expression profile and ligand-binding characterization of odorant-binding protein 2 in Batocera horsfieldi (Hope)

USDA-ARS?s Scientific Manuscript database

Odorant-binding proteins (OBPs) are important components in insect olfactory systems that transport semiochemicals through the aqueous sensillum lymph to surface of olfactory receptor neurons. In this study, we cloned the cDNA of odorant-binding protein 2 (BhorOBP2) in Batocera horsfieldi (Hope) and...

Dissociation of binding and learning processes.

PubMed

Moeller, Birte; Frings, Christian

2017-11-01

A single encounter of a stimulus together with a response can result in a short-lived association between the stimulus and the response [sometimes called an event file, see Hommel, Müsseler, Aschersleben, & Prinz, (2001) Behavioral and Brain Sciences, 24, 910-926]. The repetition of stimulus-response pairings typically results in longer lasting learning effects indicating stimulus-response associations (e.g., Logan & Etherton, (1994) Journal of Experimental Psychology: Learning, Memory, and Cognition, 20, 1022-1050]. An important question is whether or not what has been described as stimulus-response binding in action control research is actually identical with an early stage of incidental learning (e.g., binding might be seen as single-trial learning). Here, we present evidence that short-lived binding effects can be distinguished from learning of longer lasting stimulus-response associations. In two experiments, participants always responded to centrally presented target letters that were flanked by response irrelevant distractor letters. Experiment 1 varied whether distractors flanked targets on the horizontal or vertical axis. Binding effects were larger for a horizontal than for a vertical distractor-target configuration, while stimulus configuration did not influence incidental learning of longer lasting stimulus-response associations. In Experiment 2, the duration of the interval between response n - 1 and presentation of display n (500 ms vs. 2000 ms) had opposing influences on binding and learning effects. Both experiments indicate that modulating factors influence stimulus-response binding and incidental learning effects in different ways. We conclude that distinct underlying processes should be assumed for binding and incidental learning effects.

Identification of AOSC-binding proteins in neurons

NASA Astrophysics Data System (ADS)

Liu, Ming; Nie, Qin; Xin, Xianliang; Geng, Meiyu

2008-11-01

Acidic oligosaccharide sugar chain (AOSC), a D-mannuronic acid oligosaccharide, derived from brown algae polysaccharide, has been completed Phase I clinical trial in China as an anti-Alzheimer’s Disease (AD) drug candidate. The identification of AOSC-binding protein(s) in neurons is very important for understanding its action mechanism. To determine the binding protein(s) of AOSC in neurons mediating its anti-AD activities, confocal microscopy, affinity chromatography, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were used. Confocal microscopy analysis shows that AOSC binds to SH-SY5Y cells in concentration-, time-, and temperature-dependent fashions. The AOSC binding proteins were purified by affinity chromatography and identified by LC-MS/MS analysis. The results showed that there are 349 proteins binding AOSC, including clathrin, adaptor protein-2 (AP-2) and amyloid precursor protein (APP). These results suggest that the binding/entrance of AOSC to neurons is probably responsible for anti-AD activities.

[Glutamate-binding membrane proteins from human platelets].

PubMed

Gurevich, V S; Popov, Iu G; Gorodinskiĭ, A I; Dambinova, S A

1991-09-01

Solubilization of the total membrane fraction of human platelets in a 2% solution of sodium deoxycholate and subsequent affinity chromatography on glutamate agarose resulted in two protein fractions possessing a glutamate-binding activity. As can be evidenced from radioligand binding data, the first fraction contains two types of binding sites (Kd1 = 1 microM, Bmax 1 = 100 pmol/mg of protein; Kd2 = 9.3 microMm Bmax2 = 395 pmol/mg of protein). The second fraction has only one type of binding sites (Kd = 1 microM, Bmax = = 110 pmol/mg of protein). SDS-PAAG electrophoresis revealed the presence in the first fraction of proteins with Mr of 14, 24, 56 and 155 kDa, whereas the second fraction was found to contain 14, 46, 71 and 155 kDa proteins. Solid phase immunoenzymatic analysis using poly- and monoclonal specific antibodies against mammalian brain glutamate-binding proteins revealed a marked immunochemical similarity of the isolated protein fractions with human brain synaptic membrane glutamate-binding proteins.

Unusual Characteristics of the DNA Binding Domain of Epigenetic Regulatory Protein MeCP2 Determine Its Binding Specificity

PubMed Central

2015-01-01

The protein MeCP2 mediates epigenetic regulation by binding methyl-CpG (mCpG) sites on chromatin. MeCP2 consists of six domains of which one, the methyl binding domain (MBD), binds mCpG sites in duplex DNA. We show that solution conditions with physiological or greater salt concentrations or the presence of nonspecific competitor DNA is necessary for the MBD to discriminate mCpG from CpG with high specificity. The specificity for mCpG over CpG is >100-fold under these solution conditions. In contrast, the MBD does not discriminate hydroxymethyl-CpG from CpG. The MBD is unusual among site-specific DNA binding proteins in that (i) specificity is not conferred by the enhanced affinity for the specific site but rather by suppression of its affinity for generic DNA, (ii) its specific binding to mCpG is highly electrostatic, and (iii) it takes up as well as displaces monovalent cations upon DNA binding. The MBD displays an unusually high affinity for single-stranded DNA independent of modification or sequence. In addition, the MBD forms a discrete dimer on DNA via a noncooperative binding pathway. Because the affinity of the second monomer is 1 order of magnitude greater than that of nonspecific binding, the MBD dimer is a unique molecular complex. The significance of these results in the context of neuronal function and development and MeCP2-related developmental disorders such as Rett syndrome is discussed. PMID:24828757

Mutation Induced Conformational Change In CaMKII Peptide Alters Binding Affinity to CaM Through Alternate Binding Site

NASA Astrophysics Data System (ADS)

Ezerski, Jacob; Cheung, Margaret

CaM forms distinct conformation states through modifications in its charge distribution upon binding to Ca2+ ions. The occurrence of protein structural change resulting from an altered charge distribution is paramount in the scheme of cellular signaling. Not only is charge induced structural change observed in CaM, it is also seen in an essential binding target: calmodulin-depended protein kinase II (CaMKII). In order to investigate the mechanism of selectivity in relation to changes in secondary structure, the CaM binding domain of CaMKII is isolated. Experimentally, charged residues of the CaMKII peptide are systematically mutated to alanine, resulting in altered binding kinetics between the peptide and the Ca2+ saturated state of CaM. We perform an all atom simulation of the wildtype (RRK) and mutated (AAA) CaMKII peptides and generate structures from the trajectory. We analyze RRK and AAA using DSSP and find significant structural differences due to the mutation. Structures from the RRK and AAA ensembles are then selected and docked onto the crystal structure of Ca2+ saturated CaM. We observe that RRK binds to CaM at the C-terminus, whereas the 3-residue mutation, AAA, shows increased patterns of binding to the N-terminus and linker regions of CaM. Due to the conformational change of the peptide ensemble from charged residue mutation, a distinct change in the binding site can be seen, which offers an explanation to experimentally observed changes in kinetic binding rates

THE EFFECTS OF TYPE II BINDING ON METABOLIC STABILITY AND BINDING AFFINITY IN CYTOCHROME P450 CYP3A4

PubMed Central

Peng, Chi-Chi; Pearson, Josh T.; Rock, Dan A.; Joswig-Jones, Carolyn A.; Jones, Jeffrey P.

2010-01-01

One goal in drug design is to decrease clearance due to metabolism. It has been suggested that a compound’s metabolic stability can be increased by incorporation of a sp2 nitrogen into an aromatic ring. Nitrogen incorporation is hypothesized to increase metabolic stability by coordination of nitrogen to the heme iron (termed type II binding). However, questions regarding binding affinity, metabolic stability, and how metabolism of type II binders occurs remain unanswered. Herein, we use pyridinyl quinoline-4-carboxamide analogs to answer these questions. We show that type II binding can have a profound influence on binding affinity for CYP3A4, and the difference in binding affinity can be as high as 1,200 fold. We also find that type II binding compounds can be extensively metabolized, which is not consistent with the dead-end complex kinetic model assumed for type II binders. Two alternate kinetic mechanisms are presented to explain the results. The first involves a rapid equilibrium between the type II bound substrate and a metabolically oriented binding mode. The second involves direct reduction of the nitrogen-coordinated heme followed by oxygen binding. PMID:20346909

Binding of DNA-binding alkaloids berberine and palmatine to tRNA and comparison to ethidium: Spectroscopic and molecular modeling studies

NASA Astrophysics Data System (ADS)

Islam, Md. Maidul; Pandya, Prateek; Chowdhury, Sebanti Roy; Kumar, Surat; Kumar, Gopinatha Suresh

2008-11-01

The interaction of two natural protoberberine plant alkaloids berberine and palmatine with tRNA phe was studied using various biophysical techniques and molecular modeling and the data were compared with the binding of the classical DNA intercalator, ethidium. Circular dichroic studies revealed that the tRNA conformation was moderately perturbed on binding of the alkaloids. The cooperative binding of both the alkaloids and ethidium to tRNA was revealed from absorbance and fluorescence studies. Fluorescence quenching studies advanced a conclusion that while berberine and palmatine are partially intercalated, ethidium is fully intercalated on the tRNA molecule. The binding of the alkaloids as well as ethidium stabilized the tRNA melting, and the binding constant evaluated from the averaged optical melting temperature data was in agreement with fluorescence spectral-binding data. Differential scanning calorimetry revealed that the tRNA melting showed three close transitions that were affected on binding of these small molecules. Molecular docking calculations performed showed the preferred regions of binding of these small molecules on the tRNA. Taken together, the results suggest that the binding of the alkaloids berberine and palmatine on the tRNA structure appears to be mostly by partial intercalation while ethidium intercalates fully on the tRNA. These results further advance our knowledge on the molecular aspects on the interaction of these alkaloids to tRNA.

Sugar-binding sites on the surface of the carbohydrate-binding module of CBH I from Trichoderma reesei.

PubMed

Tavagnacco, Letizia; Mason, Philip E; Schnupf, Udo; Pitici, Felicia; Zhong, Linghao; Himmel, Michael E; Crowley, Michael; Cesàro, Attilio; Brady, John W

2011-05-01

Molecular dynamics simulations were carried out for a system consisting of the carbohydrate-binding module (CBM) of the cellulase CBH I from Trichoderma reesei (Hypocrea jecorina) in a concentrated solution of β-D-glucopyranose, to determine whether there is any tendency for the sugar molecules to bind to the CBM. In spite of the general tendency of glucose to behave as an osmolyte, a marked tendency for the sugar molecules to bind to the protein was observed. However, the glucose molecules tended to bind only to specific sites on the protein. As expected, the hydrophobic face of the sugar molecules, comprising the axial H1, H3, and H5 aliphatic protons, tended to adhere to the flat faces of the three tyrosine side chains on the planar binding surface of the CBM. However, a significant tendency to bind to a groove-like feature on the upper surface of the CBM was also observed. These results would not be inconsistent with a model of the mechanism for this globular domain in which the cellodextrin chain being removed from the surface of crystalline cellulose passes over the upper surface of the CBM, presumably then available for hydrolysis in the active site tunnel of this processive cellulase. Copyright © 2011 Elsevier Ltd. All rights reserved.

Characterization of chlorophyll binding to LIL3.

PubMed

Mork-Jansson, Astrid Elisabeth; Eichacker, Lutz Andreas

2018-01-01

The light harvesting like protein 3 (LIL 3) from higher plants, has been linked to functions in chlorophyll and tocopherol biosynthesis, photo-protection and chlorophyll transfer. However, the binding of chlorophyll to LIL3 is unclear. We present a reconstitution protocol for chlorophyll binding to LIL3 in DDM micelles. It is shown in the absence of lipids and carotenoids that reconstitution of chlorophyll binding to in vitro expressed LIL3 requires pre-incubation of reaction partners at room temperature. We show chlorophyll a but not chlorophyll b binding to LIL3 at a molar ratio of 1:1. Neither dynamic light scattering nor native PAGE, enabled a discrimination between binding of chlorophyll a and/or b to LIL3.

Characterization of chlorophyll binding to LIL3

PubMed Central

Mork-Jansson, Astrid Elisabeth

2018-01-01

The light harvesting like protein 3 (LIL 3) from higher plants, has been linked to functions in chlorophyll and tocopherol biosynthesis, photo-protection and chlorophyll transfer. However, the binding of chlorophyll to LIL3 is unclear. We present a reconstitution protocol for chlorophyll binding to LIL3 in DDM micelles. It is shown in the absence of lipids and carotenoids that reconstitution of chlorophyll binding to in vitro expressed LIL3 requires pre-incubation of reaction partners at room temperature. We show chlorophyll a but not chlorophyll b binding to LIL3 at a molar ratio of 1:1. Neither dynamic light scattering nor native PAGE, enabled a discrimination between binding of chlorophyll a and/or b to LIL3. PMID:29390011

Antifreeze Protein Binds Irreversibly to Ice

NASA Astrophysics Data System (ADS)

Braslavsky, I.; Pertaya, N.; di Prinzio, C. L.; Wilen, L.; Thomson, E.; Wettlaufer, J. S.; Marshall, C. B.; Davies, P. L.

2006-03-01

Many organisms are protected from freezing by antifreeze proteins (AFPs), which bind to ice and prevent its growth by a mechanism not completely understood. Although it has been postulated that AFPs would have to bind irreversibly to arrest the growth of an ice crystal bathed in excess liquid water, the binding forces seem insufficient to support such a tight interaction. By putting a fluorescent tag on a fish AFP, we were able to visualize AFP binding to ice and demonstrate, by lack of recovery after photo-bleaching, that it is indeed irreversible. Because even the most avid protein/ligand interactions exhibit reversibility, this finding is key to understanding the mechanism of antifreeze proteins, which are becoming increasingly valuable in cryopreservation and improving the frost tolerance of crops.

Surface properties of adipocyte lipid-binding protein: Response to lipid binding, and comparison with homologous proteins.

PubMed

LiCata, V J; Bernlohr, D A

1998-12-01

Adipocyte lipid-binding protein (ALBP) is one of a family of intracellular lipid-binding proteins (iLBPs) that bind fatty acids, retinoids, and other hydrophobic ligands. The different members of this family exhibit a highly conserved three-dimensional structure; and where structures have been determined both with (holo) and without (apo) bound lipid, observed conformational changes are extremely small (Banaszak, et al., 1994, Adv. Prot. Chem. 45, 89; Bernlohr, et al., 1997, Annu. Rev. Nutr. 17, 277). We have examined the electrostatic, hydrophobic, and water accessible surfaces of ALBP in the apo form and of holo forms with a variety of bound ligands. These calculations reveal a number of previously unrecognized changes between apo and holo ALBP, including: 1) an increase in the overall protein surface area when ligand binds, 2) expansion of the binding cavity when ligand is bound, 3) clustering of individual residue exposure increases in the area surrounding the proposed ligand entry portal, and 4) ligand-binding dependent variation in the topology of the electrostatic potential in the area surrounding the ligand entry portal. These focused analyses of the crystallographic structures thus reveal a number of subtle but consistent conformational and surface changes that might serve as markers for differential targeting of protein-lipid complexes within the cell. Most changes are consistent from ligand to ligand, however there are some ligand-specific changes. Comparable calculations with intestinal fatty-acid-binding protein and other vertebrate iLBPs show differences in the electrostatic topology, hydrophobic topology, and in localized changes in solvent exposure near the ligand entry portal. These results provide a basis toward understanding the functional and mechanistic differences among these highly structurally homologous proteins. Further, they suggest that iLBPs from different tissues exhibit one of two predominant end-state structural distributions of the

Modified high-affinity binding of Ni2+, Ca2+ and Zn2+ to natural mutants of human serum albumin and proalbumin.

PubMed

Kragh-Hansen, U; Brennan, S O; Minchiotti, L; Galliano, M

1994-07-01

High-affinity binding of radioactive Ni2+, Ca2+ and Zn2+ to six genetic albumin variants and to normal albumin isolated from the same heterozygote carriers was studied by equilibrium dialysis at pH 7.4. The three cations bind differently to albumin. Ni2+ binds to a site in the N-terminal region of the protein which is partially blocked by the presence of a propeptide as in proalbumin (proAlb) Varese (Arg-2-->His), proAlb Christchurch (Arg-1-->Gln) and proAlb Blenheim (Asp1-->Val) and by the presence of only an extra Arg residue (Arg-1) as in Arg-Alb and albumin (Alb) Redhill. The association constants are decreased by more than one order of magnitude in these cases, suggesting biological consequences for the ligand. The additional structural changes in Alb Redhill have no effect on Ni2+ binding. Finally, the modification of Alb Blenheim (Asp1-->Val) reduces the binding constant to 50%. Ca2+ binding is decreased to about 60-80% by the presence of a propeptide and the mutation Asp1-->Val. Arg-1 alone does not affect binding, whereas Alb Redhill binds Ca2+ more strongly than the normal protein (125%). In contrast with binding of Ni2+ and Ca2+, albumin shows heterogeneity with regard to binding of Zn2+, i.e. the number of high-affinity sites was calculated to be, on average, 0.43. The binding constant for Zn2+ is increased to 125% in the case of proAlb Varese, decreased to 50-60% for proAlb Christchurch and Alb Redhill but is normal for proAlb Blenheim, Alb Blenheim and Arg-Alb. The effects of the mutations on binding of Ca2+ and Zn2+ indicate that primary binding, when operative, is to as yet unidentified sites in domain I of the albumin molecule.

Characterization of the dextran-binding domain in the glucan-binding protein C of Streptococcus mutans.

PubMed

Takashima, Y; Fujita, K; Ardin, A C; Nagayama, K; Nomura, R; Nakano, K; Matsumoto-Nakano, M

2015-10-01

Streptococcus mutans produces multiple glucan-binding proteins (Gbps), among which GbpC encoded by the gbpC gene is known to be a cell-surface-associated protein involved in dextran-induced aggregation. The purpose of the present study was to characterize the dextran-binding domain of GbpC using bioinformatics analysis and molecular techniques. Bioinformatics analysis specified five possible regions containing molecular binding sites termed GB1 through GB5. Next, truncated recombinant GbpC (rGbpC) encoding each region was produced using a protein expression vector and five deletion mutant strains were generated, termed CDGB1 through CDGB5 respectively. The dextran-binding rates of truncated rGbpC that included the GB1, GB3, GB4 and GB5 regions in the upstream sequences were higher than that of the construct containing GB2 in the downstream region. In addition, the rates of dextran-binding for strains CDGB4 and CD1, which was entire gbpC deletion mutant, were significantly lower than for the other strains, while those of all other deletion mutants were quite similar to that of the parental strain MT8148. Biofilm structures formed by CDGB4 and CD1 were not as pronounced as that of MT8148, while those formed by other strains had greater density as compared to that of CD1. Our results suggest that the dextran-binding domain may be located in the GB4 region in the interior of the gbpC gene. Bioinformatics analysis is useful for determination of functional domains in many bacterial species. © 2015 The Society for Applied Microbiology.

Untangling Locality and Orientation Constraints in the L2 Acquisition of Anaphoric Binding: A Feature-Based Approach

ERIC Educational Resources Information Center

Dominguez, Laura; Hicks, Glyn; Song, Hee-Jeong

2012-01-01

This study offers a Minimalist analysis of the L2 acquisition of binding properties whereby cross-linguistic differences arise from the interaction of anaphoric feature specifications and operations of the computational system (Reuland 2001, 2011; Hicks 2009). This analysis attributes difficulties in the L2 acquisition of locality and orientation…

Modification of opiate agonist binding by pertussis toxin

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

Abood, M.E.; Lee, N.M.; Loh, H.H.

1986-03-05

Opiate agonist binding is decreased by GTP, suggesting the possible involvement of GTP binding proteins in regulation of opiate receptor binding. This possibility was addressed by asking whether pertussis toxin treatment, which results in ADP-ribosylation and modification of G proteins, would alter opiate agonist binding. The striatum was chosen for the initial brain area to be studied, since regulation of opiate action in this area had been shown to be modified by pertussis toxin. Treatment of striatal membranes with pertussis toxin results in up to a 55% decrease in /sup 3/(H)-DADLE binding as compared with membranes treated identically without toxin.more » This corresponds to a near complete ADP-ribosylation of both G proteins in the striatal membrane. The decrease in agonist binding appears to be due to an altered affinity of the receptor for agonist as opposed to a decrease in the number of sites. This effect of pertussis toxin on opiate agonist binding demonstrates the actual involvement of G proteins in regulation of opiate receptor binding.« less

Aluminum binding by humus

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

Benedetti, M.F.; Hiemstra, T.; Riemsdijk, W. van

The need for qualitative and quantitative description of the chemical speciation of Al, in particular and other metal ions in general, is stressed by the increased mobilization of metal ions in water and soils due to acid rain deposition. In this paper we present new data of Al binding to two humic acids. These new data sets and the some previously published data will be analyzed with the NICA-Donnan model using one set of parameters to describe the Al binding to the different humic substances. Once the experimental data is described with the NICA-Donnan approach, we will show the effectmore » of Ca on Al binding and surface speciation as well as the effect of Al on the charge of the humic particles. The parameters derived from the laboratory experiments will be used to describe the variation of the field based Al partition coefficient.« less

Antigenic modulation of the cytophilic binding of guinea-pig IgG and IgM antibodies to homologous macrophages.

PubMed Central

Webster, R O; Lawrence, D A

1979-01-01

The cytophilic binding of immune complexes by peritoneal exudate cells (PEC) from adjuvant-stimulated guinea-pigs was studied using 125I-labelled guinea-pig IgG1, IgG2 and IgM antibodies to the dinitrophenyl (DNP) group. The influence of hapten density upon cytophilic activity was studied by the addition of DNP-conjugated antigens to antibody in 2-200 molar ratios of DNP:antibody. Only IgG2 binding was enhanced by immune complex formation, and the increased binding of IgG2 anti-DNP was dependent on the number of DNP determinants per antigen molecule. Cytophilic activity with epsilon-DNP-L-lysine (DNP-LYS), alpha,epsilon-di-DNP-L-lysine (DNP-LYS-DNP), or DNP1-8-BSA was no greater than that seen in the absence of hapten. Increased cytophilic binding was noted only with DNP20-41-BSA. The binding of IgG2 and IgG2 anti-DNP:DNP-bovine serum albumin (BSA) complexes was inhibited by monomeric IgG2. The relative cytophilic capacities of guinea-pig immunoglobulins appeared as follows: IgG greater than IgG1 greater than IgM. IgG1 and IgM binding of DNP conjugates did not enhance their cytophilic activity; therefore, IgG1 and IgM cytophilic binding to PEC was considered biologically insignificant. This investigation provides further evidence that cytophilic binding of immune complexes to macrophages is due to the co-operative action of multiple Fc sites rather than a conformational change in the IgG2 antibodies, and serum proteins, notably complement components, can alter the binding and/or phagocytosis of IgG2 anti-DNP:DNP-BSA complexes. PMID:86509

Clinical role of protein binding of quinolones.

PubMed

Bergogne-Bérézin, Eugénie

2002-01-01

Protein binding of antibacterials in plasma and tissues has long been considered a component of their pharmacokinetic parameters, playing a potential role in distribution, excretion and therapeutic effectiveness. Since the beginning of the 'antibacterial era', this factor has been extensively analysed for all antibacterial classes, showing that wide variations of the degree of protein binding occur even in the same antibacterial class, as with beta-lactams. As the understanding of protein binding grew, the complexity of the binding system was increasingly perceived and its dynamic character described. Studies of protein binding of the fluoroquinolones have shown that the great majority of these drugs exhibit low protein binding, ranging from approximately 20 to 40% in plasma, and that they are bound predominantly to albumin. The potential role in pharmacokinetics-pharmacodynamics of binding of fluoroquinolones to plasma, tissue and intracellular proteins has been analysed, but it has not been established that protein binding has any significant direct or indirect impact on therapeutic effectiveness. Regarding the factors influencing the tissue distribution of antibacterials, physicochemical characteristics and the small molecular size of fluoroquinolones permit a rapid penetration into extravascular sites and intracellularly, with a rapid equilibrium being established between intravascular and extravascular compartments. The high concentrations of these drugs achieved in tissues, body fluids and intracellularly, in addition to their wide antibacterial spectrum, mean that fluoroquinolones have therapeutic effectiveness in a large variety of infections. The tolerability of quinolones has generally been reported as good, based upon long experience in using pefloxacin, ciprofloxacin and ofloxacin in clinical practice. Among more recently developed molecules, good tolerability has been reported for levofloxacin, moxifloxacin and gatifloxacin, but certain other new

Enantioselective binding of L, D-phenylalanine to ct DNA

NASA Astrophysics Data System (ADS)

Zhang, Lijin; Xu, Jianhua; Huang, Yan; Min, Shungeng

2009-10-01

The enantioselective binding of L, D-phenylalanine to calf thymus DNA was studied by absorption, circular dichroism, fluorescence quenching, viscosity, salt effect and emission experiments. The results obtained from absorption, circular dichroism, fluorescence quenching and viscosity experiments excluded the intercalative binding and salt effect experiments did not support electrostatic binding. So the binding of L, D-phenylalanine to ct DNA should be groove binding. Furthermore, the emission spectra revealed that the binding is enantioselective.

Enantioselective binding of L,D-phenylalanine to ct DNA.

PubMed

Zhang, Lijin; Xu, Jianhua; Huang, Yan; Min, Shungeng

2009-10-15

The enantioselective binding of L,D-phenylalanine to calf thymus DNA was studied by absorption, circular dichroism, fluorescence quenching, viscosity, salt effect and emission experiments. The results obtained from absorption, circular dichroism, fluorescence quenching and viscosity experiments excluded the intercalative binding and salt effect experiments did not support electrostatic binding. So the binding of l,d-phenylalanine to ct DNA should be groove binding. Furthermore, the emission spectra revealed that the binding is enantioselective.

Glycosylation of Cblns attenuates their receptor binding.

PubMed

Rong, Yongqi; Bansal, Parmil K; Wei, Peng; Guo, Hong; Correia, Kristen; Parris, Jennifer; Morgan, James I

2018-05-18

Cbln1 is the prototype of a family (Cbln1-Cbln4) of secreted glycoproteins and is essential for normal synapse structure and function in cerebellum by bridging presynaptic Nrxn to postsynaptic Grid2. Here we report the effects of glycosylation on the in vitro receptor binding properties of Cblns. Cbln1, 2 and 4 harbor two N-linked glycosylation sites, one at the N-terminus is in a region implicated in Nrxn binding and the second is in the C1q domain, a region involved in Grid2 binding. Mutation (asparagine to glutamine) of the N-terminal site, increased neurexin binding whereas mutation of the C1q site markedly increased Grid2 binding. These mutations did not influence subunit composition of Cbln trimeric complexes (mediated through the C1q domain) nor their assembly into hexamers (mediated by the N-terminal region). Therefore, glycosylation likely masks the receptor binding interfaces of Cblns. As Cbln4 has undetectable Grid2 binding in vitro we assessed whether transgenic expression of wild type Cbln4 or its glycosylation mutants rescued the Cbln1-null phenotype in vivo. Cbln4 partially rescued and both glycosylation mutants completely rescued ataxia in cbln1-null mice. Thus Cbln4 has intrinsic Grid2 binding that is attenuated by glycosylation, and glycosylation mutants exhibit gain of function in vivo. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Investigation of glucose binding sites on insulin.

PubMed

Zoete, Vincent; Meuwly, Markus; Karplus, Martin

2004-05-15

Possible insulin binding sites for D-glucose have been investigated theoretically by docking and molecular dynamics (MD) simulations. Two different docking programs for small molecules were used; Multiple Copy Simultaneous Search (MCSS) and Solvation Energy for Exhaustive Docking (SEED) programs. The configurations resulting from the MCSS search were evaluated with a scoring function developed to estimate the binding free energy. SEED calculations were performed using various values for the dielectric constant of the solute. It is found that scores emphasizing non-polar interactions gave a preferential binding site in agreement with that inferred from recent fluorescence and NMR NOESY experiments. The calculated binding affinity of -1.4 to -3.5 kcal/mol is within the measured range of -2.0 +/- 0.5 kcal/mol. The validity of the binding site is suggested by the dynamical stability of the bound glucose when examined with MD simulations with explicit solvent. Alternative binding sites were found in the simulations and their relative stabilities were estimated. The motions of the bound glucose during molecular dynamics simulations are correlated with the motions of the insulin side chains that are in contact with it and with larger scale insulin motions. These results raise the question of whether glucose binding to insulin could play a role in its activity. The results establish the complementarity of molecular dynamics simulations and normal mode analyses with the search for binding sites proposed with small molecule docking programs. Copyright 2004 Wiley-Liss, Inc.

Ribosomal proteins L5 and L11 co-operatively inactivate c-Myc via RNA-induced silencing complex.

PubMed

Liao, J-M; Zhou, X; Gatignol, A; Lu, H

2014-10-09

Oncogene MYC is highly expressed in many human cancers and functions as a global regulator of ribosome biogenesis. Previously, we reported that ribosomal protein (RP) L11 binds to c-Myc and inhibits its transcriptional activity in response to ribosomal stress. Here, we show that RPL5, co-operatively with RPL11, guides the RNA-induced silencing complex (RISC) to c-Myc mRNA and mediates the degradation of the mRNA, consequently leading to inhibition of c-Myc activity. Knocking down of RPL5 induced c-Myc expression at both mRNA and protein levels, whereas overexpression of RPL5 suppressed c-Myc expression and activity. Immunoprecipitation revealed that RPL5 binds to 3'UTR of c-Myc mRNA and two subunits of RISC, TRBP (HIV-1 TAR RNA-binding protein) and Ago2, mediating the targeting of c-Myc mRNA by miRNAs. Interestingly, RPL5 and RPL11 co-resided on c-Myc mRNA and suppressed c-Myc expression co-operatively. These findings uncover a mechanism by which these two RPs can co-operatively suppress c-Myc expression, allowing a tightly controlled ribosome biogenesis in cells.

Tetrodotoxin- and tributyltin-binding abilities of recombinant pufferfish saxitoxin and tetrodotoxin binding proteins of Takifugu rubripes.

PubMed

Satone, Hina; Nonaka, Shohei; Lee, Jae Man; Shimasaki, Yohei; Kusakabe, Takahiro; Kawabata, Shun-Ichiro; Oshima, Yuji

2017-01-01

We investigated the ability of recombinant pufferfish saxitoxin and tetrodotoxin binding protein types 1 and 2 of Takifugu rubripes (rTrub.PSTBP1 and rTrub.PSTBP2) to bind to tetrodotoxin (TTX) and tributyltin. Both rTrub.PSTBPs bound to tributyltin in an ultrafiltration binding assay but lost this ability on heat denaturation. In contrast, only rTrub.PSTBP2 bound to TTX even heat denaturation. This result suggests that the amino acid sequence of PSTBP2 may be contributed for its affinity for TTX. Copyright © 2016 Elsevier Ltd. All rights reserved.

Energetics of Glutamate Binding to an Ionotropic Glutamate Receptor.

PubMed

Yu, Alvin; Lau, Albert Y

2017-11-22

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that are responsible for the majority of excitatory transmission at the synaptic cleft. Mechanically speaking, agonist binding to the ligand binding domain (LBD) activates the receptor by triggering a conformational change that is transmitted to the transmembrane region, opening the ion channel pore. We use fully atomistic molecular dynamics simulations to investigate the binding process in the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, an iGluR subtype. The string method with swarms of trajectories was applied to calculate the possible pathways glutamate traverses during ligand binding. Residues peripheral to the binding cleft are found to metastably bind the ligand prior to ligand entry into the binding pocket. Umbrella sampling simulations were performed to compute the free energy barriers along the binding pathways. The calculated free energy profiles demonstrate that metastable interactions contribute substantially to the energetics of ligand binding and form local minima in the overall free energy landscape. Protein-ligand interactions at sites outside of the orthosteric agonist-binding site may serve to lower the transition barriers of the binding process.

Identification of thyroid hormone receptor binding sites and target genes using ChIP-on-chip in developing mouse cerebellum.

PubMed

Dong, Hongyan; Yauk, Carole L; Rowan-Carroll, Andrea; You, Seo-Hee; Zoeller, R Thomas; Lambert, Iain; Wade, Michael G

2009-01-01

Thyroid hormone (TH) is critical to normal brain development, but the mechanisms operating in this process are poorly understood. We used chromatin immunoprecipitation to enrich regions of DNA bound to thyroid receptor beta (TRbeta) of mouse cerebellum sampled on post natal day 15. Enriched target was hybridized to promoter microarrays (ChIP-on-chip) spanning -8 kb to +2 kb of the transcription start site (TSS) of 5000 genes. We identified 91 genes with TR binding sites. Roughly half of the sites were located in introns, while 30% were located within 1 kb upstream (5') of the TSS. Of these genes, 83 with known function included genes involved in apoptosis, neurodevelopment, metabolism and signal transduction. Two genes, MBP and CD44, are known to contain TREs, providing validation of the system. This is the first report of TR binding for 81 of these genes. ChIP-on-chip results were confirmed for 10 of the 13 binding fragments using ChIP-PCR. The expression of 4 novel TH target genes was found to be correlated with TH levels in hyper/hypothyroid animals providing further support for TR binding. A TRbeta binding site upstream of the coding region of myelin associated glycoprotein was demonstrated to be TH-responsive using a luciferase expression system. Motif searches did not identify any classic binding elements, indicating that not all TR binding sites conform to variations of the classic form. These findings provide mechanistic insight into impaired neurodevelopment resulting from TH deficiency and a rich bioinformatics resource for developing a better understanding of TR binding.

A novel class of plant-specific zinc-dependent DNA-binding protein that binds to A/T-rich DNA sequences

PubMed Central

Nagano, Yukio; Furuhashi, Hirofumi; Inaba, Takehito; Sasaki, Yukiko

2001-01-01

Complementary DNA encoding a DNA-binding protein, designated PLATZ1 (plant AT-rich sequence- and zinc-binding protein 1), was isolated from peas. The amino acid sequence of the protein is similar to those of other uncharacterized proteins predicted from the genome sequences of higher plants. However, no paralogous sequences have been found outside the plant kingdom. Multiple alignments among these paralogous proteins show that several cysteine and histidine residues are invariant, suggesting that these proteins are a novel class of zinc-dependent DNA-binding proteins with two distantly located regions, C-x2-H-x11-C-x2-C-x(4–5)-C-x2-C-x(3–7)-H-x2-H and C-x2-C-x(10–11)-C-x3-C. In an electrophoretic mobility shift assay, the zinc chelator 1,10-o-phenanthroline inhibited DNA binding, and two distant zinc-binding regions were required for DNA binding. A protein blot with 65ZnCl2 showed that both regions are required for zinc-binding activity. The PLATZ1 protein non-specifically binds to A/T-rich sequences, including the upstream region of the pea GTPase pra2 and plastocyanin petE genes. Expression of the PLATZ1 repressed those of the reporter constructs containing the coding sequence of luciferase gene driven by the cauliflower mosaic virus (CaMV) 35S90 promoter fused to the tandem repeat of the A/T-rich sequences. These results indicate that PLATZ1 is a novel class of plant-specific zinc-dependent DNA-binding protein responsible for A/T-rich sequence-mediated transcriptional repression. PMID:11600698

Binding of perlecan to transthyretin in vitro.

PubMed Central

Smeland, S; Kolset, S O; Lyon, M; Norum, K R; Blomhoff, R

1997-01-01

Transthyretin is one of two specific proteins involved in the transport of thyroid hormones in plasma; it possesses two binding sites for serum retinol-binding protein. In the present study we demonstrate that transthyretin also interacts in vitro with [35S]sulphate-labelled material from the medium of HepG2 cells. By using the same strategy as for purifying serum retinol-binding protein, [35S]sulphate-labelled medium was specifically eluted from a transthyretin-affinity column. Ion-exchange chromatography showed that the material was highly polyanionic, and its size and alkali susceptibility suggested that it was a proteoglycan. Structural analyses with chondroitinase ABC lyase and nitrous acid revealed that approx. 20% was chondroitin sulphate and 80% heparan sulphate. Immunoprecipitation showed that the [35S]sulphate-labelled material contained perlecan. Further analysis by binding studies revealed specific and saturable binding of 125I-transthyretin to perlecan-enriched Matrigel. Because inhibition of sulphation by treating HepG2 cells with sodium chlorate increased the affinity of the perlecan for transthyretin, and [3H]heparin was not retained by the transthyretin affinity column, the binding is probably mediated by the core protein and is not a protein-glycosaminoglycan interaction. Because perlecan is released from transthyretin in water, the binding might be due to hydrophobic interactions. PMID:9307034

Improved flow cytometer measurement of binding assays

DOEpatents

Saunders, G.C.

1984-05-30

The invention relates to a method of measuring binding assays carried out with different size particles wherein the binding assay sample is run through a flow cytometer without separating the sample from the marking agent. The amount of a binding reactant present in a sample is determined by providing particles with a coating of binder and also a known quantity of smaller particles with a coating of binder reactant. The binding reactant is the same as the binding reactant present in the sample. The smaller particles also contain a fluorescent chemical. The particles are combined with the sample and the binding reaction is allowed to occur for a set length of time followed by combining the smaller particles with the mixture of the particles and the sample produced and allowing the binding reactions to proceed to equilibrium. The fluorescence and light scatter of the combined mixture is then measured as the combined mixture passes through a flow cytometer equipped with a laser to bring about fluorescence, and the number and strength of fluorescent events are compared. A similar method is also provided for determining the amount of antigen present in the sample by providing spheres with an antibody coating and some smaller spheres with an antigen coating. (LEW)

Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coli.

PubMed

Levin, M S; Locke, B; Yang, N C; Li, E; Gordon, J I

1988-11-25

Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) are 132-residue cytosolic proteins which have 56% amino acid sequence identity and bind all-trans-retinol as their endogenous ligand. They belong to a family of cytoplasmic proteins which have evolved to bind distinct hydrophobic ligands. Their patterns of tissue-specific and developmental regulation are distinct. We have compared the ligand binding properties of rat apo-CRBP and apo-CRBP II that have been expressed in Escherichia coli. Several observations indicate that the E. coli-derived apoproteins are structurally similar to the native rat proteins: they co-migrate on isoelectric focusing gels; and when complexed with all-trans-retinol, their absorption and excitation/emission spectra are nearly identical to those of the authentic rat holoproteins. Comparative lifetime and acrylamide quenching studies suggest that there are differences in the conformations of apo-CRBP and apo-CRBP II. The interaction of E. coli-derived apo-CRBP and apo-CRBP II with a variety of retinoids was analyzed using spectroscopic techniques. Both apoproteins formed high affinity complexes with all-trans-retinol (K'd approximately 10 nM). In direct binding assays, all-trans-retinal bound to both apoproteins (K'd approximately 50 nM for CRBP; K'd approximately 90 nM for CRBP II). However, all-trans-retinal could displace all-trans-retinol bound to CRBP II but not to CRBP. These observations suggests that there is a specific yet distinct interaction between these two proteins and all-trans-retinal. Apo-CRBP and apo-CRBP II did not demonstrate significant binding to either retinoic acid or methyl retinoate, an uncharged derivative of all-trans-retinoic acid. This indicates that the carboxymethyl group of methyl retinoate cannot be sterically accommodated in their binding pockets and that failure to bind retinoic acid probably is not simply due to the negative charge of its C-15 carboxylate group

BiodMHC: an online server for the prediction of MHC class II-peptide binding affinity.

PubMed

Wang, Lian; Pan, Danling; Hu, Xihao; Xiao, Jinyu; Gao, Yangyang; Zhang, Huifang; Zhang, Yan; Liu, Juan; Zhu, Shanfeng

2009-05-01

Effective identification of major histocompatibility complex (MHC) molecules restricted peptides is a critical step in discovering immune epitopes. Although many online servers have been built to predict class II MHC-peptide binding affinity, they have been trained on different datasets, and thus fail in providing a unified comparison of various methods. In this paper, we present our implementation of seven popular predictive methods, namely SMM-align, ARB, SVR-pairwise, Gibbs sampler, ProPred, LP-top2, and MHCPred, on a single web server named BiodMHC (http://biod.whu.edu.cn/BiodMHC/index.html, the software is available upon request). Using a standard measure of AUC (Area Under the receiver operating characteristic Curves), we compare these methods by means of not only cross validation but also prediction on independent test datasets. We find that SMM-align, ProPred, SVR-pairwise, ARB, and Gibbs sampler are the five best-performing methods. For the binding affinity prediction of class II MHC-peptide, BiodMHC provides a convenient online platform for researchers to obtain binding information simultaneously using various methods.

Principal component analysis of chemical shift perturbation data of a multiple-ligand-binding system for elucidation of respective binding mechanism.

PubMed

Konuma, Tsuyoshi; Lee, Young-Ho; Goto, Yuji; Sakurai, Kazumasa

2013-01-01

Chemical shift perturbations (CSPs) in NMR spectra provide useful information about the interaction of a protein with its ligands. However, in a multiple-ligand-binding system, determining quantitative parameters such as a dissociation constant (K(d) ) is difficult. Here, we used a method we named CS-PCA, a principal component analysis (PCA) of chemical shift (CS) data, to analyze the interaction between bovine β-lactoglobulin (βLG) and 1-anilinonaphthalene-8-sulfonate (ANS), which is a multiple-ligand-binding system. The CSP on the binding of ANS involved contributions from two distinct binding sites. PCA of the titration data successfully separated the CSP pattern into contributions from each site. Docking simulations based on the separated CSP patterns provided the structures of βLG-ANS complexes for each binding site. In addition, we determined the K(d) values as 3.42 × 10⁻⁴ M² and 2.51 × 10⁻³ M for Sites 1 and 2, respectively. In contrast, it was difficult to obtain reliable K(d) values for respective sites from the isothermal titration calorimetry experiments. Two ANS molecules were found to bind at Site 1 simultaneously, suggesting that the binding occurs cooperatively with a partial unfolding of the βLG structure. On the other hand, the binding of ANS to Site 2 was a simple attachment without a significant conformational change. From the present results, CS-PCA was confirmed to provide not only the positions and the K(d) values of binding sites but also information about the binding mechanism. Thus, it is anticipated to be a general method to investigate protein-ligand interactions. Copyright © 2012 Wiley Periodicals, Inc.

Crystal Structure of the Chromodomain Helicase DNA-binding Protein 1 (Chd1) DNA-binding Domain in Complex with DNA*

PubMed Central

Sharma, Amit; Jenkins, Katherine R.; Héroux, Annie; Bowman, Gregory D.

2011-01-01

Chromatin remodelers are ATP-dependent machines that dynamically alter the chromatin packaging of eukaryotic genomes by assembling, sliding, and displacing nucleosomes. The Chd1 chromatin remodeler possesses a C-terminal DNA-binding domain that is required for efficient nucleosome sliding and believed to be essential for sensing the length of DNA flanking the nucleosome core. The structure of the Chd1 DNA-binding domain was recently shown to consist of a SANT and SLIDE domain, analogous to the DNA-binding domain of the ISWI family, yet the details of how Chd1 recognized DNA were not known. Here we present the crystal structure of the Saccharomyces cerevisiae Chd1 DNA-binding domain in complex with a DNA duplex. The bound DNA duplex is straight, consistent with the preference exhibited by the Chd1 DNA-binding domain for extranucleosomal DNA. Comparison of this structure with the recently solved ISW1a DNA-binding domain bound to DNA reveals that DNA lays across each protein at a distinct angle, yet contacts similar surfaces on the SANT and SLIDE domains. In contrast to the minor groove binding seen for Isw1 and predicted for Chd1, the SLIDE domain of the Chd1 DNA-binding domain contacts the DNA major groove. The majority of direct contacts with the phosphate backbone occur only on one DNA strand, suggesting that Chd1 may not strongly discriminate between major and minor grooves. PMID:22033927

Effects of nucleoside analog incorporation on DNA binding to the DNA binding domain of the GATA-1 erythroid transcription factor.

PubMed

Foti, M; Omichinski, J G; Stahl, S; Maloney, D; West, J; Schweitzer, B I

1999-02-05

We investigate here the effects of the incorporation of the nucleoside analogs araC (1-beta-D-arabinofuranosylcytosine) and ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl] guanine) into the DNA binding recognition sequence for the GATA-1 erythroid transcription factor. A 10-fold decrease in binding affinity was observed for the ganciclovir-substituted DNA complex in comparison to an unmodified DNA of the same sequence composition. AraC substitution did not result in any changes in binding affinity. 1H-15N HSQC and NOESY NMR experiments revealed a number of chemical shift changes in both DNA and protein in the ganciclovir-modified DNA-protein complex when compared to the unmodified DNA-protein complex. These changes in chemical shift and binding affinity suggest a change in the binding mode of the complex when ganciclovir is incorporated into the GATA DNA binding site.

Hardware device to physical structure binding and authentication

DOEpatents

Hamlet, Jason R.; Stein, David J.; Bauer, Todd M.

2013-08-20

Detection and deterrence of device tampering and subversion may be achieved by including a cryptographic fingerprint unit within a hardware device for authenticating a binding of the hardware device and a physical structure. The cryptographic fingerprint unit includes an internal physically unclonable function ("PUF") circuit disposed in or on the hardware device, which generate an internal PUF value. Binding logic is coupled to receive the internal PUF value, as well as an external PUF value associated with the physical structure, and generates a binding PUF value, which represents the binding of the hardware device and the physical structure. The cryptographic fingerprint unit also includes a cryptographic unit that uses the binding PUF value to allow a challenger to authenticate the binding.

Kinetics of Binding of Caldesmon to Actin*

PubMed Central

Chalovich, Joseph M.; Chen, Yi-der; Dudek, Ronald; Luo, Hai

2005-01-01

The time course of interaction of caldesmon with actin may be monitored by fluorescence changes that occur upon the binding of 12-(N-methyl-N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl))-labeled caldesmon to actin or to acrylodan actin. The concentration dependence of the observed rate of caldesmon-actin binding was analyzed to a first approximation as a single-step reaction using a Monte Carlo simulation. The derived association and dissociation rates were 107 m−1 s−1 and 18.2 s−1, respectively. Smooth muscle tropomyosin enhances the binding of caldesmon to actin, and this was found to be due to a reduction in the rate of dissociation to 6.3 s −1. There is no evidence from this study for a different mechanism of binding in the presence of tropomyosin. The fluorescence changes that occurred with the binding of 12-(N-methyl-N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl))-labeled caldesmon to actin or actin-tropomyosin were reversed by the addition of myosin subfragment 1 as predicted by a competitive binding mechanism. PMID:7730374

Two potential calmodulin-binding sequences in the ryanodine receptor contribute to a mobile, intra-subunit calmodulin-binding domain

PubMed Central

Huang, Xiaojun; Liu, Ying; Wang, Ruiwu; Zhong, Xiaowei; Liu, Yingjie; Koop, Andrea; Chen, S. R. Wayne; Wagenknecht, Terence; Liu, Zheng

2013-01-01

Summary Calmodulin (CaM), a 16 kDa ubiquitous calcium-sensing protein, is known to bind tightly to the calcium release channel/ryanodine receptor (RyR), and modulate RyR function. CaM binding studies using RyR fragments or synthetic peptides have revealed the presence of multiple, potential CaM-binding regions in the primary sequence of RyR. In the present study, we inserted GFP into two of these proposed CaM-binding sequences and mapped them onto the three-dimensional structure of intact cardiac RyR2 by cryo-electron microscopy. Interestingly, we found that the two potential CaM-binding regions encompassing, Arg3595 and Lys4269, respectively, are in close proximity and are adjacent to the previously mapped CaM-binding sites. To monitor the conformational dynamics of these CaM-binding regions, we generated a fluorescence resonance energy transfer (FRET) pair, a dual CFP- and YFP-labeled RyR2 (RyR2R3595-CFP/K4269-YFP) with CFP inserted after Arg3595 and YFP inserted after Lys4269. We transfected HEK293 cells with the RyR2R3595-CFP/K4269-YFP cDNA, and examined their FRET signal in live cells. We detected significant FRET signals in transfected cells that are sensitive to the channel activator caffeine, suggesting that caffeine is able to induce conformational changes in these CaM-binding regions. Importantly, no significant FRET signals were detected in cells co-transfected with cDNAs encoding the single CFP (RyR2R3595-CFP) and single YFP (RyR2K4269-YFP) insertions, indicating that the FRET signal stemmed from the interaction between R3595–CFP and K4269–YFP that are in the same RyR subunit. These observations suggest that multiple regions in the RyR2 sequence may contribute to an intra-subunit CaM-binding pocket that undergoes conformational changes during channel gating. PMID:23868982

Operational Plasticity Enables Hsp104 to Disaggregate Diverse Amyloid and Non-Amyloid Clients

PubMed Central

DeSantis, Morgan E.; Leung, Eunice H.; Sweeny, Elizabeth A.; Jackrel, Meredith E.; Cushman-Nick, Mimi; Neuhaus-Follini, Alexandra; Vashist, Shilpa; Sochor, Matthew A.; Knight, M. Noelle; Shorter, James

2012-01-01

Summary It is not understood how Hsp104, a hexameric AAA+ ATPase from yeast, disaggregates diverse structures including stress-induced aggregates, prions, and α-synuclein conformers connected to Parkinson disease. Here, we establish that Hsp104 hexamers adapt different mechanisms of intersubunit collaboration to disaggregate stress-induced aggregates versus amyloid. To resolve disordered aggregates, Hsp104 subunits collaborate non-co-operatively via probabilistic substrate binding and ATP hydrolysis. To disaggregate amyloid, several subunits co-operatively engage substrate and hydrolyze ATP. Importantly, Hsp104 variants with impaired intersubunit communication dissolve disordered aggregates but not amyloid. Unexpectedly, prokaryotic ClpB subunits collaborate differently than Hsp104 and couple probabilistic substrate binding to cooperative ATP hydrolysis, which enhances disordered aggregate dissolution but sensitizes ClpB to inhibition and diminishes amyloid disaggregation. Finally, we establish that Hsp104 hexamers deploy more subunits to disaggregate Sup35 prion strains with more stable ‘cross-β’ cores. Thus, operational plasticity enables Hsp104 to robustly dissolve amyloid and non-amyloid clients, which impose distinct mechanical demands. PMID:23141537

Change of the binding mode of the DNA/proflavine system induced by ethanol.

PubMed

García, Begoña; Leal, José M; Ruiz, Rebeca; Biver, Tarita; Secco, Fernando; Venturini, M

2010-07-01

The equilibria and kinetics of the binding of proflavine to poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT) were investigated in ethanol/water mixtures using spectrophotometric, circular dichroism, viscometric, and T-jump methods. All methods concur in showing that two modes of interaction are operative: intercalation and surface binding. The latter mode is favored by increasing ethanol and/or the proflavine content. Both static and kinetic experiments show that, concerning the poly(dG-dC).poly(dG-dC)/proflavine system, intercalation largely prevails up to 20% EtOH. For higher EtOH levels surface binding becomes dominant. Concerning the poly(dA-dT).poly(dA-dT)/proflavine system, melting experiments show that addition of proflavine stabilizes the double stranded structure, but the effect is reduced in the presence of EtOH. The DeltaH degrees and DeltaS degrees values of the melting process, measured at different concentrations of added proflavine, are linearly correlated, revealing the presence of the enthalpy-entropy compensation phenomenon (EEC). The nonmonotonicity of the "entropic term" of the EEC reveals the transition between the two binding modes. T-jump experiments show two relaxation effects, but at the highest levels of EtOH (>25%) the kinetic curves become monophasic, confirming the prevalence of the surface complex. A branched mechanism is proposed where diffusion controlled formation of a precursor complex occurs in the early stage of the binding process. This evolves toward the surface and/or the intercalated complex according to two rate-determining parallel steps. CD spectra suggest that, in the surface complex, proflavine is bound to DNA in the form of an aggregate.

The complexity of minocycline serum protein binding.

PubMed

Zhou, Jian; Tran, Brian T; Tam, Vincent H

2017-06-01

Serum protein binding is critical for understanding the pharmacology of antimicrobial agents. Tigecycline and eravacycline were previously reported to have atypical non-linear protein binding; the percentage of free fraction decreased with increasing total concentration. In this study, we extended the investigation to other tetracyclines and examined the factors that might impact protein binding. Different minocycline concentrations (0.5-50 mg/L) and perfusion media (saline, 0.1 M HEPES buffer and 0.1 and 1 M PBS) were examined by in vitro microdialysis. After equilibration, two dialysate samples were taken from each experiment and the respective antimicrobial agent concentrations were analysed by validated LC-MS/MS methods. For comparison, the serum protein bindings of doxycycline and levofloxacin were also determined. The free fraction of minocycline decreased with increasing total concentration, and the results depended on the perfusion media used. The trends of minocycline protein binding in mouse and human sera were similar. In addition, serum protein binding of doxycycline showed the same concentration-dependent trend as minocycline, while the results of levofloxacin were concentration independent. The serum protein bindings of minocycline and doxycycline are negatively correlated with their total concentrations. It is possible that all tetracyclines share the same pharmacological property. Moreover, the specific perfusion media used could also impact the results of microdialysis. Additional studies are warranted to understand the mechanism(s) and clinical implications of serum protein binding of tetracyclines. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Minimized virus binding for tests of barrier materials.

PubMed Central

Lytle, C D; Routson, L B

1995-01-01

Viruses are used to test the barrier properties of materials. Binding of virus particles during passage through holes in the material may yield misleading test results. The choices of challenge virus and suspending medium may be important for minimizing confounding effects that might arise from such binding. In this study, different surrogate viruses, as well as different support media, were evaluated to determine optimal test parameters. Two membranes with high-binding properties (nitrocellulose and cationic polysulfone) were used as filters to compare binding activities of different surrogate challenge viruses (MS2, phi X174, T7, PRD1, and phi 6) in different media. The media consisted of buffered saline with surfactants, serum, or culture broth as additives. In addition, elution rates of viruses that bound to the membranes were determined. The results suggest that viruses can bind by hydrophobic and electrostatic interactions, with phi X174 displaying the lowest level of binding by either process. The nonionic detergents Triton X-100 and Tween 80 (0.1%) equally minimized hydrophobic interactions. Neither anionic nor cationic surfactants were as effective at nontoxic levels. Serum was effective at reducing both hydrophobic and electrostatic binding, with 2% being sufficient for eliminating binding under our test conditions. Thus, phi X174 remains the best choice as a surrogate virus to test barrier materials, and Triton X-100 (0.1%) remains a good choice for reducing hydrophobic binding. In addition, binding of viruses by barrier materials is unlikely to prevent passage of blood-borne pathogens. PMID:7574603

Molecular Evolution of the Oxygen-Binding Hemerythrin Domain

PubMed Central

Alvarez-Carreño, Claudia; Becerra, Arturo; Lazcano, Antonio

2016-01-01

Background The evolution of oxygenic photosynthesis during Precambrian times entailed the diversification of strategies minimizing reactive oxygen species-associated damage. Four families of oxygen-carrier proteins (hemoglobin, hemerythrin and the two non-homologous families of arthropodan and molluscan hemocyanins) are known to have evolved independently the capacity to bind oxygen reversibly, providing cells with strategies to cope with the evolutionary pressure of oxygen accumulation. Oxygen-binding hemerythrin was first studied in marine invertebrates but further research has made it clear that it is present in the three domains of life, strongly suggesting that its origin predated the emergence of eukaryotes. Results Oxygen-binding hemerythrins are a monophyletic sub-group of the hemerythrin/HHE (histidine, histidine, glutamic acid) cation-binding domain. Oxygen-binding hemerythrin homologs were unambiguously identified in 367/2236 bacterial, 21/150 archaeal and 4/135 eukaryotic genomes. Overall, oxygen-binding hemerythrin homologues were found in the same proportion as single-domain and as long protein sequences. The associated functions of protein domains in long hemerythrin sequences can be classified in three major groups: signal transduction, phosphorelay response regulation, and protein binding. This suggests that in many organisms the reversible oxygen-binding capacity was incorporated in signaling pathways. A maximum-likelihood tree of oxygen-binding hemerythrin homologues revealed a complex evolutionary history in which lateral gene transfer, duplications and gene losses appear to have played an important role. Conclusions Hemerythrin is an ancient protein domain with a complex evolutionary history. The distinctive iron-binding coordination site of oxygen-binding hemerythrins evolved first in prokaryotes, very likely prior to the divergence of Firmicutes and Proteobacteria, and spread into many bacterial, archaeal and eukaryotic species. The later

Dual chain synthetic heparin-binding growth factor analogs

DOEpatents

Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY

2012-04-24

The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

Dual chain synthetic heparin-binding growth factor analogs

DOEpatents

Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY

2009-10-06

The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

NMR Chemical Exchange as a Probe for Ligand-Binding Kinetics in a Theophylline-Binding RNA Aptamer

PubMed Central

Latham, Michael P.; Zimmermann, Grant R.; Pardi, Arthur

2009-01-01

The apparent on- and off-rate constants for theophylline binding to its RNA aptamer in the absence of Mg2+ were determined here by 2D 1H-1H NMR ZZ-exchange spectroscopy. Analysis of the build-up rate of the exchange cross peaks for several base-paired imino protons in the RNA yielded an apparent kon of 600 M-1 s-1. This small apparent kon results from the free RNA existing as a dynamic equilibrium of inactive states rapidly interconverting with a low population of active species. The data here indicate that the RNA aptamer employs a conformational selection mechanism for binding theophylline in the absence of Mg2+. The kinetic data here also explain a very unusual property of this RNA-theophylline system, slow exchange on the NMR chemical shift timescale for a weak-binding complex. To our knowledge, it is unprecedented to have such a weak binding complex (Kd ≈ 3.0 mM at 15 °C) show slow exchange on the NMR chemical shift timescale, but the results clearly demonstrate that slow exchange and weak binding are readily rationalized by a small kon. Comparisons with other ligand-receptor interactions are presented. PMID:19317486

Genetics Home Reference: inherited thyroxine-binding globulin deficiency

MedlinePlus

... Health Conditions Inherited thyroxine-binding globulin deficiency Inherited thyroxine-binding globulin deficiency Printable PDF Open All Close ... to view the expand/collapse boxes. Description Inherited thyroxine-binding globulin deficiency is a genetic condition that ...

Binding of alkylphenols and alkylated non-phenolics to the rainbow trout (Oncorhynchus mykiss) plasma sex steroid-binding protein.

PubMed

Tollefsen, K-E

2007-09-01

Alkylphenols are well-known endocrine disrupters, mediating effects through the estrogen receptor (ER). Although the estrogenic properties of the alkylphenols are well documented, alternative mechanisms of action are poorly described. In the present work, the interaction of a range of alkyl-substituted phenols and alkyl-substituted non-phenolics with the rainbow trout (Oncorhynchus mykiss) sex steroid-binding protein (rtSBP) were determined by competitive ligand-binding studies. The role of alkyl chain length and branching, substituent position, number of alkylated groups, and the requirement of a phenolic ring structure were assessed. The results showed that the rtSBP binds to most chemical structures tested, although the highest affinity was obtained for mono-substituted alkylphenols with a chain length of four to eight methyl groups. Interestingly, rtSBP binding was also observed for non-phenolic compounds such as 4-t-butylcyclohexanol and 4-t-butylnitrobenzene suggesting that the rtSBP has a broad binding specificity for alkylphenols and alkylated non-phenolics.

Flexibility of KorA, a plasmid-encoded, global transcription regulator, in the presence and the absence of its operator

PubMed Central

Rajasekar, Karthik V.; Lovering, Andrew L.; Dancea, Felician; Scott, David J.; Harris, Sarah A.; Bingle, Lewis E.H.; Roessle, Manfred; Thomas, Christopher M.; Hyde, Eva I.; White, Scott A.

2016-01-01

Abstract The IncP (Incompatibility group P) plasmids are important carriers in the spread of antibiotic resistance across Gram-negative bacteria. Gene expression in the IncP-1 plasmids is stringently controlled by a network of four global repressors, KorA, KorB, TrbA and KorC interacting cooperatively. Intriguingly, KorA and KorB can act as co-repressors at varying distances between their operators, even when they are moved to be on opposite sides of the DNA. KorA is a homodimer with the 101-amino acid subunits, folding into an N-terminal DNA-binding domain and a C-terminal dimerization domain. In this study, we have determined the structures of the free KorA repressor and two complexes each bound to a 20-bp palindromic DNA duplex containing its consensus operator sequence. Using a combination of X-ray crystallography, nuclear magnetic resonance spectroscopy, SAXS and molecular dynamics calculations, we show that the linker between the two domains is very flexible and the protein remains highly mobile in the presence of DNA. This flexibility allows the DNA-binding domains of the dimer to straddle the operator DNA on binding and is likely to be important in cooperative binding to KorB. Unexpectedly, the C-terminal domain of KorA is structurally similar to the dimerization domain of the tumour suppressor p53. PMID:27016739

Measurement of free glucocorticoids: quantifying corticosteroid-binding globulin binding affinity and its variation within and among mammalian species.

PubMed

Delehanty, Brendan; Hossain, Sabrina; Jen, Chao Ching; Crawshaw, Graham J; Boonstra, Rudy

2015-01-01

Plasma glucocorticoids (GCs) are commonly used as measures of stress in wildlife. A great deal of evidence indicates that only free GC (GC not bound by the specific binding protein, corticosteroid-binding globulin, CBG) leaves the circulation and exerts biological effects on GC-sensitive tissues. Free hormone concentrations are difficult to measure directly, so researchers estimate free GC using two measures: the binding affinity and the binding capacity in plasma. We provide an inexpensive saturation binding method for calculating the binding affinity (equilibrium dissociation constant, K d) of CBG that can be run without specialized laboratory equipment. Given that other plasma proteins, such as albumin, also bind GCs, the method compensates for this non-specific binding. Separation of bound GC from free GC was achieved with dextran-coated charcoal. The method provides repeatable estimates (12% coefficient of variation in the red squirrel, Tamiasciurus hudsonicus), and there is little evidence of inter-individual variation in K d (range 2.0-7.3 nM for 16 Richardson's ground squirrels, Urocitellus richardsonii). The K d values of 28 mammalian species we assessed were mostly clustered around a median of 4 nM, but five species had values between 13 and 61 nM. This pattern may be distinct from birds, for which published values are more tightly distributed (1.5-5.1 nM). The charcoal separation method provides a reliable and robust method for measuring the K d in a wide range of species. It uses basic laboratory equipment to provide rapid results at very low cost. Given the importance of CBG in regulating the biological activity of GCs, this method is a useful tool for physiological ecologists.

The human mitochondrial single-stranded DNA-binding protein displays distinct kinetics and thermodynamics of DNA binding and exchange

PubMed Central

Qian, Yufeng; Johnson, Kenneth A.

2017-01-01

The human mitochondrial ssDNA-binding protein (mtSSB) is a homotetrameric protein, involved in mtDNA replication and maintenance. Although mtSSB is structurally similar to SSB from Escherichia coli (EcoSSB), it lacks the C-terminal disordered domain, and little is known about the biophysics of mtSSB–ssDNA interactions. Here, we characterized the kinetics and thermodynamics of mtSSB binding to ssDNA by equilibrium titrations and stopped-flow kinetic measurements. We show that the mtSSB tetramer can bind to ssDNA in two distinct binding modes: (SSB)30 and (SSB)60, defined by DNA binding site sizes of 30 and 60 nucleotides, respectively. We found that the binding mode is modulated by magnesium ion and NaCl concentration, but unlike EcoSSB, the mtSSB does not show negative intersubunit cooperativity. Global fitting of both the equilibrium and kinetic data afforded estimates for the rate and equilibrium constants governing the formation of (SSB)60 and (SSB)30 complexes and for the transitions between the two binding modes. We found that the mtSSB tetramer binds to ssDNA with a rate constant near the diffusion limit (2 × 109 m−1 s−1) and that longer DNA (≥60 nucleotides) rapidly wraps around all four monomers, as revealed by FRET assays. We also show that the mtSSB tetramer can directly transfer from one ssDNA molecule to another via an intermediate with two DNA molecules bound to the mtSSB. In conclusion, our results indicate that human mtSSB shares many physicochemical properties with EcoSSB and that the differences may be explained by the lack of an acidic, disordered C-terminal tail in human mtSSB protein. PMID:28615444

iDNA-Prot: Identification of DNA Binding Proteins Using Random Forest with Grey Model

PubMed Central

Lin, Wei-Zhong; Fang, Jian-An; Xiao, Xuan; Chou, Kuo-Chen

2011-01-01

DNA-binding proteins play crucial roles in various cellular processes. Developing high throughput tools for rapidly and effectively identifying DNA-binding proteins is one of the major challenges in the field of genome annotation. Although many efforts have been made in this regard, further effort is needed to enhance the prediction power. By incorporating the features into the general form of pseudo amino acid composition that were extracted from protein sequences via the “grey model” and by adopting the random forest operation engine, we proposed a new predictor, called iDNA-Prot, for identifying uncharacterized proteins as DNA-binding proteins or non-DNA binding proteins based on their amino acid sequences information alone. The overall success rate by iDNA-Prot was 83.96% that was obtained via jackknife tests on a newly constructed stringent benchmark dataset in which none of the proteins included has pairwise sequence identity to any other in a same subset. In addition to achieving high success rate, the computational time for iDNA-Prot is remarkably shorter in comparison with the relevant existing predictors. Hence it is anticipated that iDNA-Prot may become a useful high throughput tool for large-scale analysis of DNA-binding proteins. As a user-friendly web-server, iDNA-Prot is freely accessible to the public at the web-site on http://icpr.jci.edu.cn/bioinfo/iDNA-Prot or http://www.jci-bioinfo.cn/iDNA-Prot. Moreover, for the convenience of the vast majority of experimental scientists, a step-by-step guide is provided on how to use the web-server to get the desired results. PMID:21935457

Structural Analysis of Botulinum Neurotoxin Type G Receptor Binding

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

Schmitt, John; Karalewitz, Andrew; Benefield, Desire A.

2010-10-19

Botulinum neurotoxin (BoNT) binds peripheral neurons at the neuromuscular junction through a dual-receptor mechanism that includes interactions with ganglioside and protein receptors. The receptor identities vary depending on BoNT serotype (A-G). BoNT/B and BoNT/G bind the luminal domains of synaptotagmin I and II, homologous synaptic vesicle proteins. We observe conditions under which BoNT/B binds both Syt isoforms, but BoNT/G binds only SytI. Both serotypes bind ganglioside G{sub T1b}. The BoNT/G receptor-binding domain crystal structure provides a context for examining these binding interactions and a platform for understanding the physiological relevance of different Syt receptor isoforms in vivo.

Increased thyrotropin binding in hyperfunctioning thyroid nodules.

PubMed

Müller-Gärtner, H W; Schneider, C; Bay, V; Tadt, A; Rehpenning, W; de Heer, K; Jessel, M

1987-08-01

The object of this study was to investigate TSH receptors in hyperfunctioning thyroid nodules (HFN). In HFN, obtained from seven patients, 125-I-TSH binding as determined by equilibrium binding analysis on particulate membrane preparations, was found to be significantly increased as compared with normal thyroid tissues (five patients; P less than 0.001). Scatchard analysis of TSH-binding revealed two kinds of binding sites for both normal thyroid tissue and HFN, and displayed significantly increased association constants of high- and low-affinity binding sites in HFN (Ka = 11.75 +/- 6.8 10(9) M-1, P less than 0.001 and Ka = 2.1 +/- 1.0 10(7) M-1, P less than 0.025; x +/- SEM) as compared with normal thyroid tissue (Ka = 0.25 +/- 0.06 10(9) M-1, Ka = 0.14 +/- 0.03 10(7) M-1; x +/- SEM). The capacity of the high-affinity binding sites in HFN was found to be decreased (1.8 +/- 1.1 pmol/mg protein, x +/- SEM) in comparison with normal thyroid tissue (4.26 +/- 1.27 pmol/mg protein; x +/- SEM). TSH-receptor autoradiography applied to cryostatic tissue sections confirmed increased TSH binding of the follicular epithelium in HFN. These data suggest that an increased affinity of TSH-receptor sites in HFN in iodine deficient areas may be an important event in thyroid autonomy.

Lis1 acts as a "clutch" between the ATPase and microtubule-binding domains of the dynein motor.

PubMed

Huang, Julie; Roberts, Anthony J; Leschziner, Andres E; Reck-Peterson, Samara L

2012-08-31

The lissencephaly protein Lis1 has been reported to regulate the mechanical behavior of cytoplasmic dynein, the primary minus-end-directed microtubule motor. However, the regulatory mechanism remains poorly understood. Here, we address this issue using purified proteins from Saccharomyces cerevisiae and a combination of techniques, including single-molecule imaging and single-particle electron microscopy. We show that rather than binding to the main ATPase site within dynein's AAA+ ring or its microtubule-binding stalk directly, Lis1 engages the interface between these elements. Lis1 causes individual dynein motors to remain attached to microtubules for extended periods, even during cycles of ATP hydrolysis that would canonically induce detachment. Thus, Lis1 operates like a "clutch" that prevents dynein's ATPase domain from transmitting a detachment signal to its track-binding domain. We discuss how these findings provide a conserved mechanism for dynein functions in living cells that require prolonged microtubule attachments. Copyright © 2012 Elsevier Inc. All rights reserved.

C60 fullerene binding to DNA

NASA Astrophysics Data System (ADS)

Alshehri, Mansoor H.; Cox, Barry J.; Hill, James M.

2014-09-01

Fullerenes have attracted considerable attention in various areas of science and technology. Owing to their exceptional physical, chemical, and biological properties, they have many applications, particularly in cosmetic and medical products. Using the Lennard-Jones 6-12 potential function and the continuum approximation, which assumes that intermolecular interactions can be approximated by average atomic surface densities, we determine the binding energies of a C60 fullerene with respect to both single-strand and double-strand DNA molecules. We assume that all configurations are in a vacuum and that the C60 fullerene is initially at rest. Double integrals are performed to determine the interaction energy of the system. We find that the C60 fullerene binds to the double-strand DNA molecule, at either the major or minor grooves, with binding energies of -4.7 eV or -2.3 eV, respectively, and that the C60 molecule binds to the single-strand DNA molecule with a binding energy of -1.6 eV. Our results suggest that the C60 molecule is most likely to be linked to the major groove of the dsDNA molecule.

Periplasmic Binding Protein Dimer Has a Second Allosteric Event Tied to Ligand Binding

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

Li, Le; Ghimire-Rijal, Sudipa; Lucas, Sarah L.

Here, the ligand-induced conformational changes of periplasmic binding proteins (PBP) play a key role in the acquisition of metabolites in ATP binding cassette (ABC) transport systems. This conformational change allows for differential recognition of the ligand occupancy of the PBP by the ABC transporter. This minimizes futile ATP hydrolysis in the transporter, a phenomenon in which ATP hydrolysis is not coupled to metabolite transport. In many systems, the PBP conformational change is insufficient at eliminating futile ATP hydrolysis. Here we identify an additional state of the PBP that is also allosterically regulated by the ligand. Ligand binding to the homodimericmore » apo PBP leads to a tightening of the interface alpha-helices so that the hydrogen bonding pattern shifts to that of a 3 10 helix, in-turn altering the contacts and the dynamics of the protein interface so that the monomer exists in the presence of ligand.« less

Periplasmic Binding Protein Dimer Has a Second Allosteric Event Tied to Ligand Binding

DOE PAGES

Li, Le; Ghimire-Rijal, Sudipa; Lucas, Sarah L.; ...

2017-09-06

Here, the ligand-induced conformational changes of periplasmic binding proteins (PBP) play a key role in the acquisition of metabolites in ATP binding cassette (ABC) transport systems. This conformational change allows for differential recognition of the ligand occupancy of the PBP by the ABC transporter. This minimizes futile ATP hydrolysis in the transporter, a phenomenon in which ATP hydrolysis is not coupled to metabolite transport. In many systems, the PBP conformational change is insufficient at eliminating futile ATP hydrolysis. Here we identify an additional state of the PBP that is also allosterically regulated by the ligand. Ligand binding to the homodimericmore » apo PBP leads to a tightening of the interface alpha-helices so that the hydrogen bonding pattern shifts to that of a 3 10 helix, in-turn altering the contacts and the dynamics of the protein interface so that the monomer exists in the presence of ligand.« less

Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

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

Su, Xiaoyun; Agarwal, Vinayak; Dodd, Dylan

2010-11-22

Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues thatmore » flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.« less

Binding biological motion and visual features in working memory.

PubMed

Ding, Xiaowei; Zhao, Yangfan; Wu, Fan; Lu, Xiqian; Gao, Zaifeng; Shen, Mowei

2015-06-01

Working memory mechanisms for binding have been examined extensively in the last decade, yet few studies have explored bindings relating to human biological motion (BM). Human BM is the most salient and biologically significant kinetic information encountered in everyday life and is stored independently from other visual features (e.g., colors). The current study explored 3 critical issues of BM-related binding in working memory: (a) how many BM binding units can be retained in working memory, (b) whether involuntarily object-based binding occurs during BM binding, and (c) whether the maintenance of BM bindings in working memory requires attention above and beyond that needed to maintain the constituent dimensions. We isolated motion signals of human BM from non-BM sources by using point-light displays as to-be-memorized BM and presented the participants colored BM in a change detection task. We found that working memory capacity for BM-color bindings is rather low; only 1 or 2 BM-color bindings could be retained in working memory regardless of the presentation manners (Experiments 1-3). Furthermore, no object-based encoding took place for colored BM stimuli regardless of the processed dimensions (Experiments 4 and 5). Central executive attention contributes to the maintenance of BM-color bindings, yet maintaining BM bindings in working memory did not require more central attention than did maintaining the constituent dimensions in working memory (Experiment 6). Overall, these results suggest that keeping BM bindings in working memory is a fairly resource-demanding process, yet central executive attention does not play a special role in this cross-module binding. (c) 2015 APA, all rights reserved).

Water binding in legume seeds

NASA Technical Reports Server (NTRS)

Vertucci, C. W.; Leopold, A. C.

1987-01-01

The physical status of water in seeds has a pivotal role in determining the physiological reactions that can take place in the dry state. Using water sorption isotherms from cotyledon and axis tissue of five leguminous seeds, the strength of water binding and the numbers of binding sites have been estimated using van't Hoff analyses and the D'Arcy/Watt equation. These parameters of water sorption are calculated for each of the three regions of water binding and for a range of temperatures. Water sorption characteristics are reflective of the chemical composition of the biological materials as well as the temperature at which hydration takes place. Changes in the sorption characteristics with temperature and hydration level may suggest hydration-induced structural changes in cellular components.

Dimerization-induced corepressor binding and relaxed DNA-binding specificity are critical for PML/RARA-induced immortalization

PubMed Central

Zhou, Jun; Pérès, Laurent; Honoré, Nicole; Nasr, Rihab; Zhu, Jun; de Thé, Hugues

2006-01-01

The pathogenesis of acute promyelocytic leukemia involves the transcriptional repression of master genes of myeloid differentiation by the promyelocytic leukemia–retinoic acid receptor α (PML/RARA) oncogene. PML-enforced RARA homodimerization allows the tighter binding of corepressors, silencing RARA target genes. In addition, homodimerization dramatically extends the spectrum of DNA-binding sites of the fusion protein compared with those of normal RARA. Yet, any contribution of these two properties of PML/RARA to differentiation arrest and immortalization of primary mouse hematopoietic progenitors was unknown. We demonstrate that dimerization-induced silencing mediator of retinoid and thyroid receptors (SMRT)-enhanced binding and relaxed DNA-binding site specificity are both required for efficient immortalization. Thus, enforced RARA dimerization is critical not only for triggering transcriptional repression but also for extending the repertoire of target genes. Our studies exemplify how dimerization-induced gain of functions converts an unessential transcription factor into a dominant oncogenic protein. PMID:16757557

Improved sugar yields from biomass sorghum feedstocks: comparing low-lignin mutants and pretreatment chemistries.

PubMed

Godin, Bruno; Nagle, Nick; Sattler, Scott; Agneessens, Richard; Delcarte, Jérôme; Wolfrum, Edward

2016-01-01

For biofuel production processes to be economically efficient, it is essential to maximize the production of monomeric carbohydrates from the structural carbohydrates of feedstocks. One strategy for maximizing carbohydrate production is to identify less recalcitrant feedstock cultivars by performing some type of experimental screening on a large and diverse set of candidate materials, or by identifying genetic modifications (random or directed mutations or transgenic plants) that provide decreased recalcitrance. Economic efficiency can also be increased using additional pretreatment processes such as deacetylation, which uses dilute NaOH to remove the acetyl groups of hemicellulose prior to dilute acid pretreatment. In this work, we used a laboratory-scale screening tool that mimics relevant thermochemical pretreatment conditions to compare the total sugar yield of three near-isogenic brown midrib ( bmr ) mutant lines and the wild-type (WT) sorghum cultivar. We then compared results obtained from the laboratory-scale screening pretreatment assay to a large-scale pretreatment system. After pretreatment and enzymatic hydrolysis, the bmr mutants had higher total sugar yields than the WT sorghum cultivar. Increased pretreatment temperatures increased reactivity for all sorghum samples reducing the differences observed at lower reaction temperatures. Deacetylation prior to dilute acid pretreatment increased the total sugar yield for all four sorghum samples, and reduced the differences in total sugar yields among them, but solubilized a sizable fraction of the non-structural carbohydrates. The general trends of increased total sugar yield in the bmr mutant compared to the WT seen at the laboratory scale were observed at the large-scale system. However, in the larger reactor system, the measured total sugar yields were lower and the difference in total sugar yield between the WT and bmr sorghum was larger. Sorghum bmr mutants, which have a reduced lignin content showed

Improved sugar yields from biomass sorghum feedstocks: comparing low-lignin mutants and pretreatment chemistries

DOE PAGES

Godin, Bruno; Nagle, Nick; Sattler, Scott; ...

2016-11-21

For biofuel production processes to be economically efficient, it is essential to maximize the production of monomeric carbohydrates from the structural carbohydrates of feedstocks. One strategy for maximizing carbohydrate production is to identify less recalcitrant feedstock cultivars by performing some type of experimental screening on a large and diverse set of candidate materials, or by identifying genetic modifications (random or directed mutations or transgenic plants) that provide decreased recalcitrance. Economic efficiency can also be increased using additional pretreatment processes such as deacetylation, which uses dilute NaOH to remove the acetyl groups of hemicellulose prior to dilute acid pretreatment. In thismore » work, we used a laboratory-scale screening tool that mimics relevant thermochemical pretreatment conditions to compare the total sugar yield of three near-isogenic brown midrib (bmr) mutant lines and the wild-type (WT) sorghum cultivar. We then compared results obtained from the laboratory-scale screening pretreatment assay to a large-scale pretreatment system. After pretreatment and enzymatic hydrolysis, the bmr mutants had higher total sugar yields than the WT sorghum cultivar. Increased pretreatment temperatures increased reactivity for all sorghum samples reducing the differences observed at lower reaction temperatures. Deacetylation prior to dilute acid pretreatment increased the total sugar yield for all four sorghum samples, and reduced the differences in total sugar yields among them, but solubilized a sizable fraction of the non-structural carbohydrates. The general trends of increased total sugar yield in the bmr mutant compared to the WT seen at the laboratory scale were observed at the large-scale system. However, in the larger reactor system, the measured total sugar yields were lower and the difference in total sugar yield between the WT and bmr sorghum was larger. Sorghum bmr mutants, which have a reduced lignin content

Improved sugar yields from biomass sorghum feedstocks: comparing low-lignin mutants and pretreatment chemistries

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

Godin, Bruno; Nagle, Nick; Sattler, Scott

For biofuel production processes to be economically efficient, it is essential to maximize the production of monomeric carbohydrates from the structural carbohydrates of feedstocks. One strategy for maximizing carbohydrate production is to identify less recalcitrant feedstock cultivars by performing some type of experimental screening on a large and diverse set of candidate materials, or by identifying genetic modifications (random or directed mutations or transgenic plants) that provide decreased recalcitrance. Economic efficiency can also be increased using additional pretreatment processes such as deacetylation, which uses dilute NaOH to remove the acetyl groups of hemicellulose prior to dilute acid pretreatment. In thismore » work, we used a laboratory-scale screening tool that mimics relevant thermochemical pretreatment conditions to compare the total sugar yield of three near-isogenic brown midrib (bmr) mutant lines and the wild-type (WT) sorghum cultivar. We then compared results obtained from the laboratory-scale screening pretreatment assay to a large-scale pretreatment system. After pretreatment and enzymatic hydrolysis, the bmr mutants had higher total sugar yields than the WT sorghum cultivar. Increased pretreatment temperatures increased reactivity for all sorghum samples reducing the differences observed at lower reaction temperatures. Deacetylation prior to dilute acid pretreatment increased the total sugar yield for all four sorghum samples, and reduced the differences in total sugar yields among them, but solubilized a sizable fraction of the non-structural carbohydrates. The general trends of increased total sugar yield in the bmr mutant compared to the WT seen at the laboratory scale were observed at the large-scale system. However, in the larger reactor system, the measured total sugar yields were lower and the difference in total sugar yield between the WT and bmr sorghum was larger. Sorghum bmr mutants, which have a reduced lignin content

Is Maximum Food Intake in Endotherms Constrained by Net or Factorial Aerobic Scope? Lessons from the Leaf-Eared Mouse.

PubMed

Maldonado, Karin; Sabat, Pablo; Piriz, Gabriela; Bogdanovich, José M; Nespolo, Roberto F; Bozinovic, Francisco

2016-01-01

Food availability varies substantially throughout animals' lifespans, thus the ability to profit from high food levels may directly influence animal fitness. Studies exploring the link between basal metabolic rate (BMR), growth, reproduction, and other fitness traits have shown varying relationships in terms of both magnitude and direction. The diversity of results has led to the hypothesis that these relationships are modulated by environmental conditions (e.g., food availability), suggesting that the fitness consequences of a given BMR may be context-dependent. In turn, there is indirect evidence that individuals with an increased capacity for aerobic work also have a high capacity for acquiring energy from food. Surprisingly, very few studies have explored the correlation between maximum rates of energy acquisition and BMR in endotherms, and to the best of our knowledge, none have attempted to elucidate relationships between the former and aerobic capacity [e.g., maximum metabolic rate (MMR), aerobic scope (Factorial aerobic scope, FAS; Net aerobic scope, NAS)]. In this study, we measured BMR, MMR, maximum food intake (recorded under low ambient temperature and ad libitum food conditions; MFI), and estimated aerobic scope in the leaf-eared mouse ( Phyllotis darwini ). We, then, examined correlations among these variables to determine whether metabolic rates and aerobic scope are functionally correlated, and whether an increased aerobic capacity is related to a higher MFI. We found that aerobic capacity measured as NAS is positively correlated with MFI in endotherms, but with neither FAS nor BMR. Therefore, it appears plausible that the capacity for assimilating energy under conditions of abundant resources is determined adaptively by NAS, as animals with higher NAS would be promoted by selection. In theory, FAS is an invariant measurement of the extreme capacity for energy turnover in relation to resting expenditure, whereas NAS represents the maximum capacity

Polymorphism of the beta3-adrenergic receptor gene affects basal metabolic rate in obese Finns.

PubMed

Sipiläinen, R; Uusitupa, M; Heikkinen, S; Rissanen, A; Laakso, M

1997-01-01

Low basal metabolic rate (BMR) is a risk factor for weight gain and obesity. The polymorphism at codon 64 of the beta3-adrenergic receptor gene has been suggested to be associated with BMR. We investigated the frequency of the Trp64Arg of the beta3-adrenergic receptor gene and the effects of this polymorphism on BMR in obese Finns. Altogether, 170 obese subjects (29 men, 141 women, BMI 34.7 +/- 3.8 kg/m2, mean +/- SD) participated in the study. The frequency of the Trp64Arg polymorphism was 19%. None of the obese subjects were homozygous for the Arg-encoding allele. The frequency of the Trp64Arg polymorphism in obese Finns did not differ from nonobese and normoglycemic control subjects. BMR adjusted for lean body mass and age was lower in subjects with the Trp64Arg polymorphism (n = 20) than in normal homozygotes Trp64Trp (n = 99) (1,569 +/- 73 vs. 1,635 +/- 142 kcal/day, P = 0.004). For the female group (n = 98), the respective values were 1,501 +/- 66 kcal/day vs. 1,568 +/- 127 kcal/day (P = 0.004). There were no significant differences in weight, BMI, waist-to-hip ratio, lean body mass, percentage of fat, and respiratory quotient between the groups with or without the Trp64Arg polymorphism. Neither serum glucose nor insulin levels differed between the two groups. We conclude that the Trp64Arg polymorphism of the beta3-adrenergic receptor gene affects basal metabolic rate in obese Finns but does not have significant effect on glucose metabolism.

Brown midrib corn silage and Tifton 85 bermudagrass in rations for early-lactation cows.

PubMed

Castro, J J; Bernard, J K; Mullis, N A; Eggleston, R B

2010-05-01

Forty Holstein cows were used in an 8-wk randomized trial to evaluate the effects of feeding combinations of forages with improved fiber digestibility on performance during early lactation. Treatments were arranged as a 2 x 2 factorial to include silage from normal (NCS) or brown midrib (BMR) corn silage with or without 10% Tifton 85 bermudagrass hay (T85). In a simultaneous digestion trial, degradation and passage kinetics and ruminal fermentation parameters were evaluated in a 4 x 4 Latin square design trial using late-lactation Holstein cows fitted with ruminal cannulas. Dry matter intake (DMI) and neutral detergent fiber (NDF) intake were greater with BMR than with NCS; however, milk yield and composition were similar among corn silage types. Inclusion of T85 reduced milk yield but supported higher milk fat percentage, resulting in similar yields of energy-corrected milk. Blood glucose concentrations were higher for BMR compared with NCS, and inclusion of T85 increased blood urea N concentrations. Treatments did not alter liquid or solid phase passage rates or rumen turnover. Corn silage type did not affect ruminal pH or volatile fatty acid concentrations, but inclusion of T85 increased pH and molar proportion of acetate but decreased butyrate. Molar proportions of propionate were greater for NCS and T85 compared with BMR and T85, resulting in an interaction. Results of this trial indicate that combinations of forages with improved fiber digestibility can be used to support intake and performance of cows during early lactation. Copyright 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Changes in Energy Metabolism after Continuous Positive Airway Pressure for Obstructive Sleep Apnea.

PubMed

Tachikawa, Ryo; Ikeda, Kaori; Minami, Takuma; Matsumoto, Takeshi; Hamada, Satoshi; Murase, Kimihiko; Tanizawa, Kiminobu; Inouchi, Morito; Oga, Toru; Akamizu, Takashi; Mishima, Michiaki; Chin, Kazuo

2016-09-15

Disrupted energy homeostasis in obstructive sleep apnea (OSA) may lead to weight gain. Paradoxically, treating OSA with continuous positive airway pressure (CPAP) may also promote weight gain, although the underlying mechanism remains unclear. To explore the underlying mechanism by which patients with OSA gain weight after CPAP. A comprehensive assessment of energy metabolism was performed in 63 newly diagnosed OSA study participants (51 men; 60.8 ± 10.1 yr; apnea-hypopnea index >20 h(-1)) at baseline, CPAP initiation, and at a 3-month follow-up. Measurements included polysomnography, body weight, body composition, basal metabolic rate (BMR), hormones (norepinephrine, cortisol, leptin, ghrelin, insulin-like growth factor-1), dietary intake, eating behavior, and physical activity. BMR significantly decreased after CPAP (1,584 kcal/d at baseline, 1,561 kcal/d at CPAP initiation, and 1,508 kcal/d at follow-up; P < 0.001), whereas physical activity and total caloric intake did not significantly change. In multivariate regression, baseline apnea-hypopnea index, Δurine norepinephrine, and CPAP adherence were significant predictors of ΔBMR. The weight gainers had higher leptin levels, lower ghrelin levels, and higher eating behavior scores than the non-weight gainers, indicating a positive energy balance and disordered eating behavior among the weight gainers. Among the parameters related to energy metabolism, increased caloric intake was a particularly significant predictor of weight gain. Although a reduction in BMR after CPAP predisposes to a positive energy balance, dietary intake and eating behavior had greater impacts on weight change. These findings highlight the importance of lifestyle modifications combined with CPAP. Clinical trial registered with http://www.umin.ac.jp/english/ (UMIN000012639).

A COUPLED 2 × 2D BABCOCK–LEIGHTON SOLAR DYNAMO MODEL. II. REFERENCE DYNAMO SOLUTIONS

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

Lemerle, Alexandre; Charbonneau, Paul, E-mail: lemerle@astro.umontreal.ca, E-mail: paulchar@astro.umontreal.ca

In this paper we complete the presentation of a new hybrid 2 × 2D flux transport dynamo (FTD) model of the solar cycle based on the Babcock–Leighton mechanism of poloidal magnetic field regeneration via the surface decay of bipolar magnetic regions (BMRs). This hybrid model is constructed by allowing the surface flux transport (SFT) simulation described in Lemerle et al. to provide the poloidal source term to an axisymmetric FTD simulation defined in a meridional plane, which in turn generates the BMRs required by the SFT. A key aspect of this coupling is the definition of an emergence function describing the probabilitymore » of BMR emergence as a function of the spatial distribution of the internal axisymmetric magnetic field. We use a genetic algorithm to calibrate this function, together with other model parameters, against observed cycle 21 emergence data. We present a reference dynamo solution reproducing many solar cycle characteristics, including good hemispheric coupling, phase relationship between the surface dipole and the BMR-generating internal field, and correlation between dipole strength at cycle maximum and peak amplitude of the next cycle. The saturation of the cycle amplitude takes place through the quenching of the BMR tilt as a function of the internal field. The observed statistical scatter about the mean BMR tilt, built into the model, acts as a source of stochasticity which dominates amplitude fluctuations. The model thus can produce Dalton-like epochs of strongly suppressed cycle amplitude lasting a few cycles and can even shut off entirely following an unfavorable sequence of emergence events.« less

A dye-binding assay for measurement of the binding of Cu(II) to proteins.

PubMed

Wilkinson-White, Lorna E; Easterbrook-Smith, Simon B

2008-10-01

We analysed the theory of the coupled equilibria between a metal ion, a metal ion-binding dye and a metal ion-binding protein in order to develop a procedure for estimating the apparent affinity constant of a metal ion:protein complex. This can be done by analysing from measurements of the change in the concentration of the metal ion:dye complex with variation in the concentration of either the metal ion or the protein. Using experimentally determined values for the affinity constant of Cu(II) for the dye, 2-(5-bromo-2-pyridylaxo)-5-(N-propyl-N-sulfopropylamino) aniline (5-Br-PSAA), this procedure was used to estimate the apparent affinity constants for formation of Cu(II):transthyretin, yielding values which were in agreement with literature values. An apparent affinity constant for Cu(II) binding to alpha-synuclein of approximately 1 x 10(9)M(-1) was obtained from measurements of tyrosine fluorescence quenching by Cu(II). This value was in good agreement with that obtained using 5-Br-PSAA. Our analysis and data therefore show that measurement of changes in the equilibria between Cu(II) and 5-Br-PSAA by Cu(II)-binding proteins provides a general procedure for estimating the affinities of proteins for Cu(II).

Microbubble Enzyme-Linked Immunosorbent Assay for the Detection of Targeted Microbubbles in in Vitro Static Binding Assays.

PubMed

Wischhusen, Jennifer; Padilla, Frederic

2017-07-01

Targeted microbubbles (MBs) are ultrasound contrast agents that are functionalized with a ligand for ultrasound molecular imaging of endothelial markers. Novel targeted MBs are characterized in vitro by incubation in protein-coated wells, followed by binding quantification by microscopy or ultrasound imaging. Both methods provide operator-dependent results: Between 3 and 20 fields of view from a heterogeneous sample are typically selected for analysis by microscopy, and in ultrasound imaging, different acoustic settings affect signal intensities. This study proposes a new method to reproducibly quantify MB binding based on enzyme-linked immunosorbent assay (ELISA), in which bound MBs are revealed with an enzyme-linked antibody. MB-ELISA was adapted to in vitro static binding assays, incubating the MBs in inverted position or by agitation, and compared with microscopy. The specificity and sensitivity of MB-ELISA enable the reliable quantification of MB binding in a rapid, high-throughput and whole-well analysis, facilitating the characterization of new targeted contrast agents. Copyright © 2017 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Electrostatically Biased Binding of Kinesin to Microtubules

PubMed Central

Zheng, Wenjun; Alonso, Maria; Huber, Gary; Dlugosz, Maciej; McCammon, J. Andrew; Cross, Robert A.

2011-01-01

The minimum motor domain of kinesin-1 is a single head. Recent evidence suggests that such minimal motor domains generate force by a biased binding mechanism, in which they preferentially select binding sites on the microtubule that lie ahead in the progress direction of the motor. A specific molecular mechanism for biased binding has, however, so far been lacking. Here we use atomistic Brownian dynamics simulations combined with experimental mutagenesis to show that incoming kinesin heads undergo electrostatically guided diffusion-to-capture by microtubules, and that this produces directionally biased binding. Kinesin-1 heads are initially rotated by the electrostatic field so that their tubulin-binding sites face inwards, and then steered towards a plus-endwards binding site. In tethered kinesin dimers, this bias is amplified. A 3-residue sequence (RAK) in kinesin helix alpha-6 is predicted to be important for electrostatic guidance. Real-world mutagenesis of this sequence powerfully influences kinesin-driven microtubule sliding, with one mutant producing a 5-fold acceleration over wild type. We conclude that electrostatic interactions play an important role in the kinesin stepping mechanism, by biasing the diffusional association of kinesin with microtubules. PMID:22140358

Odorant-binding proteins from a primitive termite.

PubMed

Ishida, Yuko; Chiang, Vicky P; Haverty, Michael I; Leal, Walter S

2002-09-01

Hitherto, odorant-binding proteins (OBPs) have been identified from insects belonging to more highly evolved insect orders (Lepidoptera, Coleoptera, Diptera, Hymenoptera, and Hemiptera), whereas only chemosensory proteins have been identified from more primitive species, such as orthopteran and phasmid species. Here, we report for the first time the isolation and cloning of odorant-binding proteins from a primitive termite species, the dampwood termite. Zootermopsis nevadensis nevadensis (Isoptera: Termopsidae). A major antennae-specific protein was detected by native PAGE along with four other minor proteins, which were also absent in the extract from control tissues (hindlegs). Multiple cDNA cloning led to the full characterization of the major antennae-specific protein (ZnevOBP1) and to the identification of two other antennae-specific cDNAs, encoding putative odorant-binding proteins (ZnevOBP2 and ZnevOBP3). N-terminal amino acid sequencing of the minor antennal bands and cDNA cloning showed that olfaction in Z. n. nevadensis may involve multiple odorant-binding proteins. Database searches suggest that the OBPs from this primitive termite are homologues of the pheromone-binding proteins from scarab beetles and antennal-binding proteins from moths.

Lost time: Bindings do not represent temporal order information.

PubMed

Moeller, Birte; Frings, Christian

2018-06-04

Many accounts of human action control assume bindings between features of stimuli and responses of individual events. One widely accepted assumption about these bindings is that they do not contain temporal-order representations regarding the integrated elements. Even though several theories either explicitly or implicitly include it, this assumption has never been tested directly. One reason for this lack of evidence is likely that typical stimulus-response binding paradigms are inapt for such a test. Adapting a new paradigm of response-response binding to include order switches between response integration and retrieval, we were able to analyze possible representation of order information in bindings for the first time. Binding effects were identical for intact and switched response orders, indicating that bindings indeed include no temporal-order information.

Somatotype and body composition analysis of Korean youth soccer players according to playing position for sports physiotherapy research

PubMed Central

Noh, Ji-Woong; Kim, Mee-Young; Lee, Lim-Kyu; Park, Byoung-Sun; Yang, Seung-Min; Jeon, Hye-Joo; Lee, Won-Deok; Kim, Ju-Hyun; Lee, Jeong-Uk; Kwak, Taek-Yong; Lee, Tae-Hyun; Kim, Ju-Young; Kim, Junghwan

2015-01-01

[Purpose] The purpose of this study was to investigate the somatotype and physical characteristic differences among elite youth soccer players. [Subjects and Methods] In the present study, we evaluated twenty-two Korean youth soccer players in different playing positions. The playing positions were divided into forward (FW), midfielder (MF), defender (DF), and goalkeeper (GK). The participants’ lean body mass (LBM), fat free mass (FFM), fat mass (FM), and basal metabolic rate (BMR) were measured and their somatotype determined according to the Heath-Carter method. [Results] The youth soccer players had twelve ectomorphic, eight mesomorphic, and two central predominant types. The DFs were taller than, but otherwise similar in physical characteristics to the FWs and MFs. The GKs were taller and heavier than the other players; however, their somatotype components were not significantly different. LBM, FFM, and BMR were significantly higher in GKs than in FWs and MFs. Although LBM, FFM, and BMR values between GKs and DFs showed large differences, they were not statistically significant. [Conclusion] The present study may contribute to our understanding of the differences in somatotype and body composition of Korean youth soccer players involved in sports physiotherapy research. PMID:25995545

Somatotype and body composition analysis of Korean youth soccer players according to playing position for sports physiotherapy research.

PubMed

Noh, Ji-Woong; Kim, Mee-Young; Lee, Lim-Kyu; Park, Byoung-Sun; Yang, Seung-Min; Jeon, Hye-Joo; Lee, Won-Deok; Kim, Ju-Hyun; Lee, Jeong-Uk; Kwak, Taek-Yong; Lee, Tae-Hyun; Kim, Ju-Young; Kim, Junghwan

2015-04-01

[Purpose] The purpose of this study was to investigate the somatotype and physical characteristic differences among elite youth soccer players. [Subjects and Methods] In the present study, we evaluated twenty-two Korean youth soccer players in different playing positions. The playing positions were divided into forward (FW), midfielder (MF), defender (DF), and goalkeeper (GK). The participants' lean body mass (LBM), fat free mass (FFM), fat mass (FM), and basal metabolic rate (BMR) were measured and their somatotype determined according to the Heath-Carter method. [Results] The youth soccer players had twelve ectomorphic, eight mesomorphic, and two central predominant types. The DFs were taller than, but otherwise similar in physical characteristics to the FWs and MFs. The GKs were taller and heavier than the other players; however, their somatotype components were not significantly different. LBM, FFM, and BMR were significantly higher in GKs than in FWs and MFs. Although LBM, FFM, and BMR values between GKs and DFs showed large differences, they were not statistically significant. [Conclusion] The present study may contribute to our understanding of the differences in somatotype and body composition of Korean youth soccer players involved in sports physiotherapy research.

Short photoperiod increases energy intake, metabolic thermogenesis and organ mass in silky starlings Sturnus sericeus

PubMed Central

WANG, Jia-Qi; WANG, Jia-Jia; WU, Xu-Jian; ZHENG, Wei-Hong; LIU, Jin-Song

2016-01-01

Environmental cues play important roles in the regulation of an animal’s physiology and behavior. One such cue, photoperiod, plays an important role in the seasonal acclimatization of birds. It has been demonstrated that an animal’s body mass, basal metabolic rate (BMR), and energy intake, are all affected by photoperiod. The present study was designed to examine photoperiod induced changes in the body mass, metabolism and metabolic organs of the silky starling, Sturnus sericeus. Captive silky starlings increased their body mass and BMR during four weeks of acclimation to a short photoperiod. Birds acclimated to a short photoperiod also increased the mass of certain organs (liver, gizzard and small intestine), and both gross energy intake (GEI) and digestible energy intake (DEI), relative to those acclimated to a long photoperiod. Furthermore, BMR was positively correlated with body mass, liver mass, GEI and DEI. These results suggest that silky starlings increase metabolic thermogenesis when exposed to a short photoperiod by increasing their body and metabolic organ mass, and their GEI and DEI. These findings support the hypothesis that bird species from temperate climates typically display high phenotypic flexibility in thermogenic capacity. PMID:27029864

Reaction Norms in Natural Conditions: How Does Metabolic Performance Respond to Weather Variations in a Small Endotherm Facing Cold Environments?

PubMed Central

Petit, Magali; Vézina, François

2014-01-01

Reaction norms reflect an organisms' capacity to adjust its phenotype to the environment and allows for identifying trait values associated with physiological limits. However, reaction norms of physiological parameters are mostly unknown for endotherms living in natural conditions. Black-capped chickadees (Poecile atricapillus) increase their metabolic performance during winter acclimatization and are thus good model to measure reaction norms in the wild. We repeatedly measured basal (BMR) and summit (Msum) metabolism in chickadees to characterize, for the first time in a free-living endotherm, reaction norms of these parameters across the natural range of weather variation. BMR varied between individuals and was weakly and negatively related to minimal temperature. Msum varied with minimal temperature following a Z-shape curve, increasing linearly between 24°C and −10°C, and changed with absolute humidity following a U-shape relationship. These results suggest that thermal exchanges with the environment have minimal effects on maintenance costs, which may be individual-dependent, while thermogenic capacity is responding to body heat loss. Our results suggest also that BMR and Msum respond to different and likely independent constraints. PMID:25426860

Calculation of Relative Binding Free Energy in the Water-Filled Active Site of Oligopeptide-Binding Protein A.

PubMed

Maurer, Manuela; de Beer, Stephanie B A; Oostenbrink, Chris

2016-04-15

The periplasmic oligopeptide binding protein A (OppA) represents a well-known example of water-mediated protein-ligand interactions. Here, we perform free-energy calculations for three different ligands binding to OppA, using a thermodynamic integration approach. The tripeptide ligands share a high structural similarity (all have the sequence KXK), but their experimentally-determined binding free energies differ remarkably. Thermodynamic cycles were constructed for the ligands, and simulations conducted in the bound and (freely solvated) unbound states. In the unbound state, it was observed that the difference in conformational freedom between alanine and glycine leads to a surprisingly slow convergence, despite their chemical similarity. This could be overcome by increasing the softness parameter during alchemical transformations. Discrepancies remained in the bound state however, when comparing independent simulations of the three ligands. These difficulties could be traced to a slow relaxation of the water network within the active site. Fluctuations in the number of water molecules residing in the binding cavity occur mostly on a timescale larger than the simulation time along the alchemical path. After extensive simulations, relative binding free energies that were converged to within thermal noise could be obtained, which agree well with available experimental data.

RNA binding and replication by the poliovirus RNA polymerase

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

Oberste, M.S.

1988-01-01

RNA binding and RNA synthesis by the poliovirus RNA-dependent RNA polymerase were studied in vitro using purified polymerase. Templates for binding and RNA synthesis studies were natural RNAs, homopolymeric RNAs, or subgenomic poliovirus-specific RNAs synthesized in vitro from cDNA clones using SP6 or T7 RNA polymerases. The binding of the purified polymerase to poliovirion and other RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in the viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. The binding of purified polymerase to {sup 32}P-labeled ribohomopolymeric RNAs wasmore » examined, and the order of binding observed was poly(G) >>> poly(U) > poly(C) > poly(A). The K{sub a} for polymerase binding to poliovirion RNA and to a full-length negative strand transcript was about 1 {times} 10{sup 9} M{sup {minus}1}. The polymerase binds to a subgenomic RNAs which contain the 3{prime} end of the genome with a K{sub a} similar to that for virion RNA, but binds less well to 18S rRNA, globin mRNA, and subgenomic RNAs which lack portions of the 3{prime} noncoding region.« less

Complex binding of the FabR repressor of bacterial unsaturated fatty acid biosynthesis to its cognate promoters.

PubMed

Feng, Youjun; Cronan, John E

2011-04-01

Two transcriptional regulators, the FadR activator and the FabR repressor, control biosynthesis of unsaturated fatty acids in Escherichia coli. FabR represses expression of the two genes, fabA and fabB, required for unsaturated fatty acid synthesis and has been reported to require the presence of an unsaturated thioester (of either acyl carrier protein or CoA) in order to bind the fabA and fabB promoters in vitro. We report in vivo experiments in which unsaturated fatty acid synthesis was blocked in the absence of exogenous unsaturated fatty acids in a ΔfadR strain and found that the rates of transcription of fabA and fabB were unaffected by the lack of unsaturated thioesters. To examine the discrepancy between our in vivo results and the prior in vitro results we obtained active, natively folded forms of the E. coli and Vibrio cholerae FabRs by use of an in vitro transcription-translation system. We report that FabR bound the intact promoter regions of both fabA and fabB in the absence of unsaturated acyl thioesters, but bound the two promoters differently. Native FabR bound the fabA promoter region provided that the canonical FabR binding site is extended by inclusion of flanking sequences that overlap the neighbouring FadR binding site. In contrast, although binding to the fabB operator also required a flanking sequence, a non-specific sequence could suffice. However, unsaturated thioesters did allow FabR binding to the minimal FabR operator sites of both promoters which otherwise were not bound. Thus unsaturated thioester ligands were not essential for FabR/target DNA interaction, but acted to enhance binding. The gel mobility shift data plus in vivo expression data indicate that despite the remarkably similar arrangements of promoter elements, FadR predominately regulates fabA expression whereas FabR is the dominant regulator of fabB expression. We also report that E. coli fabR expression is not autoregulated. Complementation, qRT-PCR and fatty acid