Proline substitution independently enhances H-2D(b) complex stabilization and TCR recognition of melanoma-associated peptides.

PubMed

Uchtenhagen, Hannes; Abualrous, Esam T; Stahl, Evi; Allerbring, Eva B; Sluijter, Marjolein; Zacharias, Martin; Sandalova, Tatyana; van Hall, Thorbald; Springer, Sebastian; Nygren, Per-Åke; Achour, Adnane

2013-11-01

The immunogenicity of H-2D(b) (D(b)) restricted epitopes can be significantly increased by substituting peptide position 3 to a proline (p3P). The p3P modification enhances MHC stability without altering the conformation of the modified epitope allowing for T-cell cross-reactivity with the native peptide. The present study reveals how specific interactions between p3P and the highly conserved MHC heavy chain residue Y159 increase the stability of D(b) in complex with an optimized version of the melanoma-associated epitope gp10025-33 . Furthermore, the p3P modification directly increased the affinity of the D(b)/gp10025-33 -specific T-cell receptor (TCR) pMel. Surprisingly, the enhanced TCR binding was independent from the observed increased stability of the optimized D(b)/gp10025-33 complex and from the interactions formed between p3P and Y159, indicating a direct effect of the p3P modification on TCR recognition. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Interactions of cullin3/KCTD5 complexes with both cytoplasmic and nuclear proteins: Evidence for a role in protein stabilization

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

Rutz, Natalja; Heilbronn, Regine; Weger, Stefan, E-mail: stefan.weger@charite.de

2015-08-28

Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 inducedmore » polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability. - Highlights: • KCTD5 nuclear translocation depends upon M phase and protein oligomerization. • Identification of MCM7, ZNF711 and FAM193 as KCTD5 interaction partners. • Formation of trimeric complexes of KCTD5/cullin3 with MCM7, ZNF711 and FAM193B. • KCTD5 is not involved in polyubiquitylation of MCM7 replication factor. • The KCTD5/cullin3 complex stabilizes ZNF711 transcription factor.« less

Mechanism of protein precipitation and stabilization by co-solvents

NASA Astrophysics Data System (ADS)

Timasheff, Serge N.; Arakawa, Tsutomu

1988-07-01

The interactions between proteins and a number of substances which, when present at high concentration, stabilize or precipitate proteins, have been analyzed in terms of the preferential interactions of these co-solvents with proteins. In all cases, stabilization or precipitation was accompanied by preferential exclusion of the co-solvent from the immediate domain of the protein, i.e., preferential hydration of the protein. This means that addition of the co-solvent to the aqueous protein solution increased the chemical potentials of both components. The thermodynamic interaction parameters derived from such data make it possible to calculate the salting out constant, Ks, as well as to construct a phase isotherm for any given solvent mixture which indicates the limiting protein solubility. The salting-out effect can be decomposed into contributions from non-specific preferential exclusion and specific binding of the ligand to the protein, the balance leading to solubilization or precipitation. In reactions, such as denaturation, the effect of co-solvent on the reaction depends on the difference in the preferential interactions of the two end states of the protein. Principal sources of preferential exclusion have been identified as steric exclusion, increase of the surface tension of water by the co-solvent, repulsion by charged loci on the protein and solvophobicity.

Genome-Wide Analysis of Grain Yield Stability and Environmental Interactions in a Multiparental Soybean Population.

PubMed

Xavier, Alencar; Jarquin, Diego; Howard, Reka; Ramasubramanian, Vishnu; Specht, James E; Graef, George L; Beavis, William D; Diers, Brian W; Song, Qijian; Cregan, Perry B; Nelson, Randall; Mian, Rouf; Shannon, J Grover; McHale, Leah; Wang, Dechun; Schapaugh, William; Lorenz, Aaron J; Xu, Shizhong; Muir, William M; Rainey, Katy M

2018-02-02

Genetic improvement toward optimized and stable agronomic performance of soybean genotypes is desirable for food security. Understanding how genotypes perform in different environmental conditions helps breeders develop sustainable cultivars adapted to target regions. Complex traits of importance are known to be controlled by a large number of genomic regions with small effects whose magnitude and direction are modulated by environmental factors. Knowledge of the constraints and undesirable effects resulting from genotype by environmental interactions is a key objective in improving selection procedures in soybean breeding programs. In this study, the genetic basis of soybean grain yield responsiveness to environmental factors was examined in a large soybean nested association population. For this, a genome-wide association to performance stability estimates generated from a Finlay-Wilkinson analysis and the inclusion of the interaction between marker genotypes and environmental factors was implemented. Genomic footprints were investigated by analysis and meta-analysis using a recently published multiparent model. Results indicated that specific soybean genomic regions were associated with stability, and that multiplicative interactions were present between environments and genetic background. Seven genomic regions in six chromosomes were identified as being associated with genotype-by-environment interactions. This study provides insight into genomic assisted breeding aimed at achieving a more stable agronomic performance of soybean, and documented opportunities to exploit genomic regions that were specifically associated with interactions involving environments and subpopulations. Copyright © 2018 Xavier et al.

Competitive interactions and controlled release of a natural antioxidant from halloysite nanotubes.

PubMed

Hári, József; Gyürki, Ádám; Sárközi, Márk; Földes, Enikő; Pukánszky, Béla

2016-01-15

Halloysite nanotubes used as potential carrier material for a controlled release stabilizer in polyethylene were thoroughly characterized with several techniques including the measurement of specific surface area, pore volume and surface energy. The high surface energy of the halloysite results in the strong bonding of the additive to the surface. Dissolution experiments carried out with eight different solvents for the determination of the effect of solvent characteristics on the amount of irreversibly bonded quercetin proved that adsorption and dissolution depend on competitive interactions prevailing in the system. Solvents with low polarity dissolve only surplus quercetin adsorbed in multilayers. Polyethylene is a very apolar polymer forming weak interactions with every substance; quercetin dissolves into it from the halloysite surface only above a critical surface coverage. Stabilization experiments confirmed that strong adhesion prevents dissolution and results in limited stabilization efficiency. At larger adsorbed amounts better stability and extended effect were measured indicating dissolution and controlled release. Copyright © 2015 Elsevier Inc. All rights reserved.

Predicting suicide ideation through intrapersonal and interpersonal factors: The interplay of Big-Five personality traits and social support.

PubMed

Ayub, Nailah

2015-11-01

While a specific personality trait may escalate suicide ideation, contextual factors such as social support, when provided effectively, may alleviate the effects of such personality traits. This study examined the moderating role of social support in the relationship between the Big-Five personality traits and suicide ideation. Significant interactions were found between social support and extraversion and emotional stability. Specifically, the relationship between emotional stability and extraversion to suicide ideation was exacerbated when social support was low. Slope analysis showed openness also interacted with low social support. Results were computed for frequency, duration and attitude dimensions of suicide ideation. Extraversion interacted with social support to predict all three dimensions. Social support moderated emotional stability to predict frequency and duration, moderated conscientiousness towards frequency and attitude, and moderated openness towards attitude. The results imply that whereas personality traits may be difficult to alter, social support may play a significant role in saving a life. Psychologists should include family and friends when treating a suicidal youth, guiding them to awareness of one's personality and being more supportive. Copyright © 2015 John Wiley & Sons, Ltd.

Iron addition to soil specifically stabilized lignin

Treesearch

Steven J. Hall; Whendee L. Silver; Vitaliy I. Timokhin; Kenneth E. Hammel

2016-01-01

The importance of lignin as a recalcitrant constituent of soil organic matter (SOM) remains contested. Associations with iron (Fe) oxides have been proposed to specifically protect lignin from decomposition, but impacts of Fe-lignin interactions on mineralization rates remain unclear. Oxygen (O2) fluctuations characteristic of humid tropical...

Autoinhibition of ETV6 DNA Binding Is Established by the Stability of Its Inhibitory Helix

PubMed Central

De, Soumya; Okon, Mark; Graves, Barbara J.; McIntosh, Lawrence P.

2017-01-01

The ETS transcriptional repressor ETV6 (or TEL) is autoinhibited by an α-helix that sterically blocks its DNA-binding ETS domain. The inhibitory helix is marginally stable and unfolds when ETV6 binds to either specific or non-specific DNA. Using NMR spectroscopy, we show that folding of the inhibitory helix requires a buried charge–dipole interaction with helix H1 of the ETS domain. This interaction also contributes directly to autoinhibition by precluding a highly conserved dipole-enhanced hydrogen bond between the phosphodiester backbone of bound DNA and the N terminus of helix H1. To probe further the thermodynamic basis of autoinhibition, ETV6 variants were generated with amino acid substitutions introduced along the solvent exposed surface of the inhibitory helix. These changes were designed to increase the intrinsic helical propensity of the inhibitory helix without perturbing its packing interactions with the ETS domain. NMR-monitored amide hydrogen exchange measurements confirmed that the stability of the folded inhibitory helix increases progressively with added helix-promoting substitutions. This also results in progressively reinforced autoinhibition and decreased DNA-binding affinity. Surprisingly, locking the inhibitory helix onto the ETS domain by a disulfide bridge severely impairs, but does not abolish DNA binding. Weak interactions still occur via an interface displaced from the canonical ETS domain DNA-binding surface. Collectively, these studies establish a direct thermodynamic linkage between inhibitory helix stability and ETV6 autoinhibition, and demonstrate that helix unfolding does not strictly precede DNA binding. Modulating inhibitory helix stability provides a potential route for the in vivo regulation of ETV6 activity. PMID:26920109

The disordered C-terminal domain of human DNA glycosylase NEIL1 contributes to its stability via intramolecular interactions.

PubMed

Hegde, Muralidhar L; Tsutakawa, Susan E; Hegde, Pavana M; Holthauzen, Luis Marcelo F; Li, Jing; Oezguen, Numan; Hilser, Vincent J; Tainer, John A; Mitra, Sankar

2013-07-10

NEIL1 [Nei (endonuclease VIII)-like protein 1], one of the five mammalian DNA glycosylases that excise oxidized DNA base lesions in the human genome to initiate base excision repair, contains an intrinsically disordered C-terminal domain (CTD; ~100 residues), not conserved in its Escherichia coli prototype Nei. Although dispensable for NEIL1's lesion excision and AP lyase activities, this segment is required for efficient in vivo enzymatic activity and may provide an interaction interface for many of NEIL1's interactions with other base excision repair proteins. Here, we show that the CTD interacts with the folded domain in native NEIL1 containing 389 residues. The CTD is poised for local folding in an ordered structure that is induced in the purified fragment by osmolytes. Furthermore, deletion of the disordered tail lacking both Tyr and Trp residues causes a red shift in NEIL1's intrinsic Trp-specific fluorescence, indicating a more solvent-exposed environment for the Trp residues in the truncated protein, which also exhibits reduced stability compared to the native enzyme. These observations are consistent with stabilization of the native NEIL1 structure via intramolecular, mostly electrostatic, interactions that were disrupted by mutating a positively charged (Lys-rich) cluster of residues (amino acids 355-360) near the C-terminus. Small-angle X-ray scattering (SAXS) analysis confirms the flexibility and dynamic nature of NEIL1's CTD, a feature that may be critical to providing specificity for NEIL1's multiple, functional interactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Implications of Network Topology on Stability

PubMed Central

Kinkhabwala, Ali

2015-01-01

In analogy to chemical reaction networks, I demonstrate the utility of expressing the governing equations of an arbitrary dynamical system (interaction network) as sums of real functions (generalized reactions) multiplied by real scalars (generalized stoichiometries) for analysis of its stability. The reaction stoichiometries and first derivatives define the network’s “influence topology”, a signed directed bipartite graph. Parameter reduction of the influence topology permits simplified expression of the principal minors (sums of products of non-overlapping bipartite cycles) and Hurwitz determinants (sums of products of the principal minors or the bipartite cycles directly) for assessing the network’s steady state stability. Visualization of the Hurwitz determinants over the reduced parameters defines the network’s stability phase space, delimiting the range of its dynamics (specifically, the possible numbers of unstable roots at each steady state solution). Any further explicit algebraic specification of the network will project onto this stability phase space. Stability analysis via this hierarchical approach is demonstrated on classical networks from multiple fields. PMID:25826219

Some remarks on the attractor behaviour in ELKO cosmology

NASA Astrophysics Data System (ADS)

Pereira, S. H.; A. Pinho S., S.; Hoff da Silva, J. M.

2014-08-01

Recent results on the dynamical stability of a system involving the interaction of the ELKO spinor field with standard matter in the universe have been reanalysed, and the conclusion is that such system does not exhibit isolated stable points that could alleviate the cosmic coincidence problem. When a constant parameter δ related to the potential of the ELKO field is introduced in the system however, stable fixed points are found for some specific types of interaction between the ELKO field and matter. Although the parameter δ is related to an unknown potential, in order to satisfy the stability conditions and also that the fixed points are real, the range of the constant parameter δ can be constrained for the present time and the coincidence problem can be alleviated for some specific interactions. Such restriction on the ELKO potential opens possibility to apply the ELKO field as a candidate to dark energy in the universe, and so explain the present phase of acceleration of the universe through the decay of the ELKO field into matter.

Gold nanoparticle assemblies stabilized by bis(phthalocyaninato)lanthanide(III) complexes through van der Waals interactions

NASA Astrophysics Data System (ADS)

Noda, Yuki; Noro, Shin-Ichiro; Akutagawa, Tomoyuki; Nakamura, Takayoshi

2014-01-01

Gold nanoparticle assemblies possess diverse application potential, ranging from industrial nanotechnology to medical biotechnology. Because the structures and properties of assemblies are directly affected by the stabilization mechanism between the organic molecules serving as protecting ligands and the gold nanoparticle surface, it is crucial to find and investigate new stabilization mechanisms. Here, we report that π-conjugated phthalocyanine rings can serve as stabilizing ligands for gold nanoparticles. Bis(phthalocyaninato)lutetium(III) (LuPc2) or bis(phthalocyaninato)terbium(III) (TbPc2), even though complex, do not have specific binding units and stabilize gold nanoparticles through van der Waals interaction between parallel adsorbed phthalocyanine ligands and the gold nanoparticle surface. AC magnetic measurements and the electron-transport properties of the assemblies give direct evidence that the phthalocyanines are isolated from each other. Each nanoparticle shows weak electronic coupling despite the short internanoparticle distance (~1 nm), suggesting Efros-Shklovskii-type variable-range hopping and collective single-electron tunnelling behaviours.

Protein Stabilization and Enzyme Activation in Ionic Liquids: Specific Ion Effects

PubMed Central

Zhao, Hua

2015-01-01

There are still debates on whether the hydration of ions perturbs the water structure, and what is the degree of such disturbance; therefore, the origin of Hofmeister effect on protein stabilization continues being questioned. For this reason, it is suggested to use the ‘specific ion effect’ instead of other misleading terms such as Hofmeister effect, Hofmeister series, lyotropic effect, and lyotropic series. In this review, we firstly discuss the controversial aspect of inorganic ion effects on water structures, and several possible contributors to the specific ion effect of protein stability. Due to recent overwhelming attraction of ionic liquids (ILs) as benign solvents in many enzymatic reactions, we further evaluate the structural properties and molecular-level interactions in neat ILs and their aqueous solutions. Next, we systematically compare the specific ion effects of ILs on enzyme stability and activity, and conclude that (a) the specificity of many enzymatic systems in diluted aqueous IL solutions is roughly in line with the traditional Hofmeister series albeit some exceptions; (b) however, the specificity follows a different track in concentrated or neat ILs because other factors (such as hydrogen-bond basicity, nucelophilicity, and hydrophobicity, etc) are playing leading roles. In addition, we demonstrate some examples of biocatalytic reactions in IL systems that are guided by the empirical specificity rule. PMID:26949281

Engineering Lipases: walking the fine line between activity and stability

NASA Astrophysics Data System (ADS)

Dasetty, Siva; Blenner, Mark A.; Sarupria, Sapna

2017-11-01

Lipases are enzymes that hydrolyze lipids and have several industrial applications. There is a tremendous effort in engineering the activity, specificity and stability of lipases to render them functional in a variety of environmental conditions. In this review, we discuss the recent experimental and simulation studies focused on engineering lipases. Experimentally, mutagenesis studies have demonstrated that the activity, stability, and specificity of lipases can be modulated by mutations. It has been particularly challenging however, to elucidate the underlying mechanisms through which these mutations affect the lipase properties. We summarize results from experiments and molecular simulations highlighting the emerging picture to this end. We end the review with suggestions for future research which underscores the delicate balance of various facets in the lipase that affect their activity and stability necessitating the consideration of the enzyme as a network of interactions.

Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation

NASA Astrophysics Data System (ADS)

Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

2016-11-01

Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD+-dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some “loose-binding” substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation.

PubMed

Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

2016-11-30

Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD + -dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some "loose-binding" substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

The role of glycosylation and domain interactions in the thermal stability of human angiotensin-converting enzyme.

PubMed

O'Neill, Hester G; Redelinghuys, Pierre; Schwager, Sylva L U; Sturrock, Edward D

2008-09-01

The N and C domains of somatic angiotensin-converting enzyme (sACE) differ in terms of their substrate specificity, inhibitor profiling, chloride dependency and thermal stability. The C domain is thermally less stable than sACE or the N domain. Since both domains are heavily glycosylated, the effect of glycosylation on their thermal stability was investigated by assessing their catalytic and physicochemical properties. Testis ACE (tACE) expressed in mammalian cells, mammalian cells in the presence of a glucosidase inhibitor and insect cells yielded proteins with altered catalytic and physicochemical properties, indicating that the more complex glycans confer greater thermal stabilization. Furthermore, a decrease in tACE and N-domain N-glycans using site-directed mutagenesis decreased their thermal stability, suggesting that certain N-glycans have an important effect on the protein's thermodynamic properties. Evaluation of the thermal stability of sACE domain swopover and domain duplication mutants, together with sACE expressed in insect cells, showed that the C domain contained in sACE is less dependent on glycosylation for thermal stabilization than a single C domain, indicating that stabilizing interactions between the two domains contribute to the thermal stability of sACE and are decreased in a C-domain-duplicating mutant.

Disordered Cold Regulated15 Proteins Protect Chloroplast Membranes during Freezing through Binding and Folding, But Do Not Stabilize Chloroplast Enzymes in Vivo1[W][OPEN

PubMed Central

Thalhammer, Anja; Bryant, Gary; Sulpice, Ronan; Hincha, Dirk K.

2014-01-01

Freezing can severely damage plants, limiting geographical distribution of natural populations and leading to major agronomical losses. Plants native to cold climates acquire increased freezing tolerance during exposure to low nonfreezing temperatures in a process termed cold acclimation. This involves many adaptative responses, including global changes in metabolite content and gene expression, and the accumulation of cold-regulated (COR) proteins, whose functions are largely unknown. Here we report that the chloroplast proteins COR15A and COR15B are necessary for full cold acclimation in Arabidopsis (Arabidopsis thaliana). They protect cell membranes, as indicated by electrolyte leakage and chlorophyll fluorescence measurements. Recombinant COR15 proteins stabilize lactate dehydrogenase during freezing in vitro. However, a transgenic approach shows that they have no influence on the stability of selected plastidic enzymes in vivo, although cold acclimation results in increased enzyme stability. This indicates that enzymes are stabilized by other mechanisms. Recombinant COR15 proteins are disordered in water, but fold into amphipathic α-helices at high osmolyte concentrations in the presence of membranes, a condition mimicking molecular crowding induced by dehydration during freezing. X-ray scattering experiments indicate protein-membrane interactions specifically under such crowding conditions. The COR15-membrane interactions lead to liposome stabilization during freezing. Collectively, our data demonstrate the requirement for COR15 accumulation for full cold acclimation of Arabidopsis. The function of these intrinsically disordered proteins is the stabilization of chloroplast membranes during freezing through a folding and binding mechanism, but not the stabilization of chloroplastic enzymes. This indicates a high functional specificity of these disordered plant proteins. PMID:25096979

Molecular Dynamics Simulation Study on the Binding and Stabilization Mechanism of Antiprion Compounds to the "Hot Spot" Region of PrPC.

PubMed

Zhou, Shuangyan; Liu, Xuewei; An, Xiaoli; Yao, Xiaojun; Liu, Huanxiang

2017-11-15

Structural transitions in the prion protein from the cellular form, PrP C , into the pathological isoform, PrP Sc , are regarded as the main cause of the transmissible spongiform encephalopathies, also known as prion diseases. Hence, discovering and designing effective antiprion drugs that can inhibit PrP C to PrP Sc conversion is regarded as a promising way to cure prion disease. Among several strategies to inhibit PrP C to PrP Sc conversion, stabilizing the native PrP C via specific binding is believed to be one of the valuable approaches and many antiprion compounds have been reported based on this strategy. However, the detailed mechanism to stabilize the native PrP C is still unknown. As such, to unravel the stabilizing mechanism of these compounds to PrP C is valuable for the further design and discovery of antiprion compounds. In this study, by molecular dynamics simulation method, we investigated the stabilizing mechanism of several antiprion compounds on PrP C that were previously reported to have specific binding to the "hot spot" region of PrP C . Our simulation results reveal that the stabilization mechanism of specific binding compounds can be summarized as (I) to stabilize both the flexible C-terminal of α2 and the hydrophobic core, such as BMD42-29 and GN8; (II) to stabilize the hydrophobic core, such as J1 and GJP49; (III) to stabilize the overall structure of PrP C by high binding affinity, as NPR-056. In addition, as indicated by the H-bond analysis and decomposition analysis of binding free energy, the residues N159 and Q160 play an important role in the specific binding of the studied compounds and all these compounds interact with PrP C in a similar way with the key interacting residues L130 in the β1 strand, P158, N159, Q160, etc. in the α1-β2 loop, and H187, T190, T191, etc. in the α2 C-terminus although the compounds have large structural difference. As a whole, our obtained results can provide some insights into the specific binding mechanism of main antiprion compounds to the "hot spot" region of PrP C at the molecular level and also provide guidance for effective antiprion drug design in the future.

Facile Affinity Maturation of Antibody Variable Domains Using Natural Diversity Mutagenesis

PubMed Central

Tiller, Kathryn E.; Chowdhury, Ratul; Li, Tong; Ludwig, Seth D.; Sen, Sabyasachi; Maranas, Costas D.; Tessier, Peter M.

2017-01-01

The identification of mutations that enhance antibody affinity while maintaining high antibody specificity and stability is a time-consuming and laborious process. Here, we report an efficient methodology for systematically and rapidly enhancing the affinity of antibody variable domains while maximizing specificity and stability using novel synthetic antibody libraries. Our approach first uses computational and experimental alanine scanning mutagenesis to identify sites in the complementarity-determining regions (CDRs) that are permissive to mutagenesis while maintaining antigen binding. Next, we mutagenize the most permissive CDR positions using degenerate codons to encode wild-type residues and a small number of the most frequently occurring residues at each CDR position based on natural antibody diversity. This mutagenesis approach results in antibody libraries with variants that have a wide range of numbers of CDR mutations, including antibody domains with single mutations and others with tens of mutations. Finally, we sort the modest size libraries (~10 million variants) displayed on the surface of yeast to identify CDR mutations with the greatest increases in affinity. Importantly, we find that single-domain (VHH) antibodies specific for the α-synuclein protein (whose aggregation is associated with Parkinson’s disease) with the greatest gains in affinity (>5-fold) have several (four to six) CDR mutations. This finding highlights the importance of sampling combinations of CDR mutations during the first step of affinity maturation to maximize the efficiency of the process. Interestingly, we find that some natural diversity mutations simultaneously enhance all three key antibody properties (affinity, specificity, and stability) while other mutations enhance some of these properties (e.g., increased specificity) and display trade-offs in others (e.g., reduced affinity and/or stability). Computational modeling reveals that improvements in affinity are generally not due to direct interactions involving CDR mutations but rather due to indirect effects that enhance existing interactions and/or promote new interactions between the antigen and wild-type CDR residues. We expect that natural diversity mutagenesis will be useful for efficient affinity maturation of a wide range of antibody fragments and full-length antibodies. PMID:28928732

A Bacteriophage-Encoded J-Domain Protein Interacts with the DnaK/Hsp70 Chaperone and Stabilizes the Heat-Shock Factor σ32 of Escherichia coli

PubMed Central

Perrody, Elsa; Cirinesi, Anne-Marie; Desplats, Carine; Keppel, France; Schwager, Françoise; Tranier, Samuel; Georgopoulos, Costa; Genevaux, Pierre

2012-01-01

The universally conserved J-domain proteins (JDPs) are obligate cochaperone partners of the Hsp70 (DnaK) chaperone. They stimulate Hsp70's ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene 057w of the T4-related enterobacteriophage RB43 encodes a bona fide JDP protein, named Rki, which specifically interacts with the Escherichia coli host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ32, which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ32 facilitates RB43 bacteriophage proliferation is discussed. PMID:23133404

Impact of Cannabis Use During Stabilization on Methadone Maintenance Treatment

PubMed Central

Scavone, Jillian L.; Sterling, Robert C.; Weinstein, Stephen P.; Van Bockstaele, Elisabeth J.

2016-01-01

Background and Objectives Illicit drug use, particularly of cannabis, is common among opiate-dependent individuals, and has the potential to impact treatment in a negative manner. Methods To examine this, patterns of cannabis use prior to and during methadone maintenance treatment (MMT) were examined to assess possible cannabis-related effects on MMT, particularly during methadone stabilization. Retrospective chart analysis was used to examine outpatient records of patients undergoing MMT (n=91), focusing specifically on past and present cannabis use and its association with opiate abstinence, methadone dose stabilization, and treatment compliance. Results Objective rates of cannabis use were high during methadone induction, dropping significantly following dose stabilization. History of cannabis use correlated with cannabis use during MMT, but did not negatively impact the methadone induction process. Pilot data also suggested that objective ratings of opiate withdrawal decrease in MMT patients using cannabis during stabilization. Conclusions and Scientific Significance The present findings may point to novel interventions to be employed during treatment for opiate dependence that specifically target cannabinoid-opioid system interactions. PMID:23795873

Identification of proteins interacting with lactate dehydrogenase in claw muscle of the porcelain crab Petrolisthes cinctipes

PubMed Central

Cayenne, Andrea P.; Gabert, Beverly; Stillman, Jonathon H.

2011-01-01

Biochemical adaptation of enzymes involves conservation of activity, stability and affinity across a wide range of intracellular and environmental conditions. Enzyme adaptation by alteration of primary structure is well known, but the roles of protein-protein interactions in enzyme adaptation are less well understood. Interspecific differences in thermal stability of lactate dehydrogenase (LDH) in porcelain crabs (genus Petrolisthes) are related to intrinsic differences among LDH molecules and by interactions with other stabilizing proteins. Here, we identified proteins that interact with LDH in porcelain crab claw muscle tissue using co-immunoprecipitation, and showed LDH exists in high molecular weight complexes using size exclusion chromatography and Western blot analyses. Co-immunoprecipitated proteins were separated using 2D SDS PAGE and analyzed by LC/ESI using peptide MS/MS. Peptide MS/MS ions were compared to an EST database for Petrolisthes cinctipes to identify proteins. Identified proteins included cytoskeletal elements, glycolytic enzymes, a phosphagen kinase, and the respiratory protein hemocyanin. Our results support the hypothesis that LDH interacts with glycolytic enzymes in a metabolon structured by cytoskeletal elements that may also include the enzyme for transfer of the adenylate charge in glycolytically produced ATP. Those interactions may play specific roles in biochemical adaptation of glycolytic enzymes. PMID:21968246

Population-based 3D genome structure analysis reveals driving forces in spatial genome organization

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

Tjong, Harianto; Li, Wenyuan; Kalhor, Reza

Conformation capture technologies (e.g., Hi-C) chart physical interactions between chromatin regions on a genome-wide scale. However, the structural variability of the genome between cells poses a great challenge to interpreting ensemble-averaged Hi-C data, particularly for long-range and interchromosomal interactions. Here, we present a probabilistic approach for deconvoluting Hi-C data into a model population of distinct diploid 3D genome structures, which facilitates the detection of chromatin interactions likely to co-occur in individual cells. Here, our approach incorporates the stochastic nature of chromosome conformations and allows a detailed analysis of alternative chromatin structure states. For example, we predict and experimentally confirm themore » presence of large centromere clusters with distinct chromosome compositions varying between individual cells. The stability of these clusters varies greatly with their chromosome identities. We show that these chromosome-specific clusters can play a key role in the overall chromosome positioning in the nucleus and stabilizing specific chromatin interactions. By explicitly considering genome structural variability, our population-based method provides an important tool for revealing novel insights into the key factors shaping the spatial genome organization.« less

Population-based 3D genome structure analysis reveals driving forces in spatial genome organization

DOE PAGES

Tjong, Harianto; Li, Wenyuan; Kalhor, Reza; ...

2016-03-07

Conformation capture technologies (e.g., Hi-C) chart physical interactions between chromatin regions on a genome-wide scale. However, the structural variability of the genome between cells poses a great challenge to interpreting ensemble-averaged Hi-C data, particularly for long-range and interchromosomal interactions. Here, we present a probabilistic approach for deconvoluting Hi-C data into a model population of distinct diploid 3D genome structures, which facilitates the detection of chromatin interactions likely to co-occur in individual cells. Here, our approach incorporates the stochastic nature of chromosome conformations and allows a detailed analysis of alternative chromatin structure states. For example, we predict and experimentally confirm themore » presence of large centromere clusters with distinct chromosome compositions varying between individual cells. The stability of these clusters varies greatly with their chromosome identities. We show that these chromosome-specific clusters can play a key role in the overall chromosome positioning in the nucleus and stabilizing specific chromatin interactions. By explicitly considering genome structural variability, our population-based method provides an important tool for revealing novel insights into the key factors shaping the spatial genome organization.« less

StaRProtein, A Web Server for Prediction of the Stability of Repeat Proteins

PubMed Central

Xu, Yongtao; Zhou, Xu; Huang, Meilan

2015-01-01

Repeat proteins have become increasingly important due to their capability to bind to almost any proteins and the potential as alternative therapy to monoclonal antibodies. In the past decade repeat proteins have been designed to mediate specific protein-protein interactions. The tetratricopeptide and ankyrin repeat proteins are two classes of helical repeat proteins that form different binding pockets to accommodate various partners. It is important to understand the factors that define folding and stability of repeat proteins in order to prioritize the most stable designed repeat proteins to further explore their potential binding affinities. Here we developed distance-dependant statistical potentials using two classes of alpha-helical repeat proteins, tetratricopeptide and ankyrin repeat proteins respectively, and evaluated their efficiency in predicting the stability of repeat proteins. We demonstrated that the repeat-specific statistical potentials based on these two classes of repeat proteins showed paramount accuracy compared with non-specific statistical potentials in: 1) discriminate correct vs. incorrect models 2) rank the stability of designed repeat proteins. In particular, the statistical scores correlate closely with the equilibrium unfolding free energies of repeat proteins and therefore would serve as a novel tool in quickly prioritizing the designed repeat proteins with high stability. StaRProtein web server was developed for predicting the stability of repeat proteins. PMID:25807112

Molecular dynamics simulations of electrostatics and hydration distributions around RNA and DNA motifs

NASA Astrophysics Data System (ADS)

Marlowe, Ashley E.; Singh, Abhishek; Semichaevsky, Andrey V.; Yingling, Yaroslava G.

2009-03-01

Nucleic acid nanoparticles can self-assembly through the formation of complementary loop-loop interactions or stem-stem interactions. Presence and concentration of ions can significantly affect the self-assembly process and the stability of the nanostructure. In this presentation we use explicit molecular dynamics simulations to examine the variations in cationic distributions and hydration environment around DNA and RNA helices and loop-loop interactions. Our simulations show that the potassium and sodium ionic distributions are different around RNA and DNA motifs which could be indicative of ion mediated relative stability of loop-loop complexes. Moreover in RNA loop-loop motifs ions are consistently present and exchanged through a distinct electronegative channel. We will also show how we used the specific RNA loop-loop motif to design a RNA hexagonal nanoparticle.

The Importance of Water for High Fidelity Information Processing and for Life

NASA Technical Reports Server (NTRS)

Hoehler, Tori M.; Pohorille, Andrew

2011-01-01

Is water an absolute prerequisite for life? Life depends on a variety of non-covalent interactions among molecules, the nature of which is determined as much by the solvent in which they occur as by the molecules themselves. Catalysis and information processing, two essential functions of life, require non-covalent molecular recognition with very high specificity. For example, to correctly reproduce a string consisting of 600,000 units of information (e.g ., 600 kilobases, equivalent to the genome of the smallest free living terrestrial organisms) with a 90% success rate requires specificity > 107 : 1 for the target molecule vs. incorrect alternatives. Such specificity requires (i) that the correct molecular association is energetically stabilized by at least 40 kJ/mol relative to alternatives, and (ii) that the system is able to sample among possible states (alternative molecular associations) rapidly enough to allow the system to fall under thermodynamic control and express the energetic stabilization. We argue that electrostatic interactions are required to confer the necessary energetic stabilization vs. a large library of molecular alternatives, and that a solvent with polarity and dielectric properties comparable to water is required for the system to sample among possible states and express thermodynamic control. Electrostatic associations can be made in non-polar solvents, but the resulting complexes are too stable to be "unmade" with sufficient frequency to confer thermodynamic control on the system. An electrostatic molecular complex representing 3 units of information (e.g., 3 base pairs) with specificity > 107 per unit has a stability in non-polar solvent comparable to that of a carbon-carbon bond at room temperature. These considerations suggest that water, or a solvent with properties very like water, is necessary to support high-fidelity information processing, and can therefore be considered a critical prerequisite for life.

Mms1 is an assistant for regulating G-quadruplex DNA structures.

PubMed

Schwindt, Eike; Paeschke, Katrin

2018-06-01

The preservation of genome stability is fundamental for every cell. Genomic integrity is constantly challenged. Among those challenges are also non-canonical nucleic acid structures. In recent years, scientists became aware of the impact of G-quadruplex (G4) structures on genome stability. It has been shown that folded G4-DNA structures cause changes in the cell, such as transcriptional up/down-regulation, replication stalling, or enhanced genome instability. Multiple helicases have been identified to regulate G4 structures and by this preserve genome stability. Interestingly, although these helicases are mostly ubiquitous expressed, they show specificity for G4 regulation in certain cellular processes (e.g., DNA replication). To this date, it is not clear how this process and target specificity of helicases are achieved. Recently, Mms1, an ubiquitin ligase complex protein, was identified as a novel G4-DNA-binding protein that supports genome stability by aiding Pif1 helicase binding to these regions. In this perspective review, we discuss the question if G4-DNA interacting proteins are fundamental for helicase function and specificity at G4-DNA structures.

Effect of novel food processing methods on packaging: structure, composition, and migration properties.

PubMed

Guillard, V; Mauricio-Iglesias, M; Gontard, N

2010-11-01

Classical stabilization techniques (thermal treatments) usually involve food to be packed after being processed. On the contrary and increasingly, novel food processing methods, such as high pressure or microwaves, imply that both packaging and foodstuff undergo the stabilization treatment. Moreover, novel treatments (UV light, irradiation, ozone, cold plasma) are specifically used for disinfection and sterilization of the packaging material itself. Therefore, in the last several years a number of papers have focused on the effects of these new treatments on food-packaging interactions with a special emphasis on chemical migration and safety concerns. New packaging materials merged on the market with specific interest regarding the environment (i.e. bio-sourced materials) or mechanical and barrier properties (i.e. nanocomposites packaging materials). It is time to evaluate the knowledge about how these in-package food technologies affect food/packaging interactions, and especially for novel biodegradable and/or active materials. This article presents the effect of high pressure treatment, microwave heating, irradiation, UV-light, ozone and, cold plasma treatment on food/packaging interactions.

Electrostatic interactions guide the active site face of a structure-specific ribonuclease to its RNA substrate.

PubMed

Plantinga, Matthew J; Korennykh, Alexei V; Piccirilli, Joseph A; Correll, Carl C

2008-08-26

Restrictocin, a member of the alpha-sarcin family of site-specific endoribonucleases, uses electrostatic interactions to bind to the ribosome and to RNA oligonucleotides, including the minimal specific substrate, the sarcin/ricin loop (SRL) of 23S-28S rRNA. Restrictocin binds to the SRL by forming a ground-state E:S complex that is stabilized predominantly by Coulomb interactions and depends on neither the sequence nor structure of the RNA, suggesting a nonspecific complex. The 22 cationic residues of restrictocin are dispersed throughout this protein surface, complicating a priori identification of a Coulomb interacting surface. Structural studies have identified an enzyme-substrate interface, which is expected to overlap with the electrostatic E:S interface. Here, we identified restrictocin residues that contribute to binding in the E:S complex by determining the salt dependence [partial differential log(k 2/ K 1/2)/ partial differential log[KCl

Differential Modulation of Functional Dynamics and Allosteric Interactions in the Hsp90-Cochaperone Complexes with p23 and Aha1: A Computational Study

PubMed Central

Blacklock, Kristin; Verkhivker, Gennady M.

2013-01-01

Allosteric interactions of the molecular chaperone Hsp90 with a large cohort of cochaperones and client proteins allow for molecular communication and event coupling in signal transduction networks. The integration of cochaperones into the Hsp90 system is driven by the regulatory mechanisms that modulate the progression of the ATPase cycle and control the recruitment of the Hsp90 clientele. In this work, we report the results of computational modeling of allosteric regulation in the Hsp90 complexes with the cochaperones p23 and Aha1. By integrating protein docking, biophysical simulations, modeling of allosteric communications, protein structure network analysis and the energy landscape theory we have investigated dynamics and stability of the Hsp90-p23 and Hsp90-Aha1 interactions in direct comparison with the extensive body of structural and functional experiments. The results have revealed that functional dynamics and allosteric interactions of Hsp90 can be selectively modulated by these cochaperones via specific targeting of the regulatory hinge regions that could restrict collective motions and stabilize specific chaperone conformations. The protein structure network parameters have quantified the effects of cochaperones on conformational stability of the Hsp90 complexes and identified dynamically stable communities of residues that can contribute to the strengthening of allosteric interactions. According to our results, p23-mediated changes in the Hsp90 interactions may provide “molecular brakes” that could slow down an efficient transmission of the inter-domain allosteric signals, consistent with the functional role of p23 in partially inhibiting the ATPase cycle. Unlike p23, Aha1-mediated acceleration of the Hsp90-ATPase cycle may be achieved via modulation of the equilibrium motions that facilitate allosteric changes favoring a closed dimerized form of Hsp90. The results of our study have shown that Aha1 and p23 can modulate the Hsp90-ATPase activity and direct the chaperone cycle by exerting the precise control over structural stability, global movements and allosteric communications in Hsp90. PMID:23977182

Polymer/Solvent and Polymer/Polymer Interaction Studies

DTIC Science & Technology

1980-09-01

temperatures up to 450 12 before serious degradation occurs. They have good hydrolytic stability, good solvent resistance, and excellent thermo- oxidative ...Concentration for Sorption in Glassy PVC 5 Temperature Dependence of the Flory-Huggins Interaction Parameters 115 6 Solubility of Dichloromethane in Polysulfone...116 7 Test of Applicability of the Langmuir Equation for Describing Sorption Data 117 8 Temperature Dependence of the Specific Volume of an Amorphous

Micropatterned cell-cell interactions enable functional encapsulation of primary hepatocytes in hydrogel microtissues.

PubMed

Li, Cheri Y; Stevens, Kelly R; Schwartz, Robert E; Alejandro, Brian S; Huang, Joanne H; Bhatia, Sangeeta N

2014-08-01

Drug-induced liver injury is a major cause of drug development failures and postmarket withdrawals. In vitro models that incorporate primary hepatocytes have been shown to be more predictive than model systems which rely on liver microsomes or hepatocellular carcinoma cell lines. Methods to phenotypically stabilize primary hepatocytes ex vivo often rely on mimicry of hepatic microenvironmental cues such as cell-cell interactions and cell-matrix interactions. In this work, we sought to incorporate phenotypically stable hepatocytes into three-dimensional (3D) microtissues, which, in turn, could be deployed in drug-screening platforms such as multiwell plates and diverse organ-on-a-chip devices. We first utilize micropatterning on collagen I to specify cell-cell interactions in two-dimensions, followed by collagenase digestion to produce well-controlled aggregates for 3D encapsulation in polyethylene glycol (PEG) diacrylate. Using this approach, we examined the influence of homotypic hepatocyte interactions and composition of the encapsulating hydrogel, and achieved the maintenance of liver-specific function for over 50 days. Optimally preaggregated structures were subsequently encapsulated using a microfluidic droplet-generator to produce 3D microtissues. Interactions of engineered hepatic microtissues with drugs was characterized by flow cytometry, and yielded both induction of P450 enzymes in response to prototypic small molecules and drug-drug interactions that give rise to hepatotoxicity. Collectively, this study establishes a pipeline for the manufacturing of 3D hepatic microtissues that exhibit stabilized liver-specific functions and can be incorporated into a wide array of emerging drug development platforms.

π-Cation Interactions in Molecular Recognition: Perspectives on Pharmaceuticals and Pesticides.

PubMed

Liang, Zhibin; Li, Qing X

2018-04-04

The π-cation interaction that differs from the cation-π interaction is a valuable concept in molecular design of pharmaceuticals and pesticides. In this Perspective we present an up-to-date review (from 1995 to 2017) on bioactive molecules involving π-cation interactions with the recognition site, and categorize into systems of inhibitor-enzyme, ligand-receptor, ligand-transporter, and hapten-antibody. The concept of π-cation interactions offers use of π systems in a small molecule to enhance the binding affinity, specificity, selectivity, lipophilicity, bioavailability, and metabolic stability, which are physiochemical features desired for drugs and pesticides.

Specific interactions between amyloid-β peptides in an amyloid-β hexamer with three-fold symmetry: Ab initio fragment molecular orbital calculations in water

NASA Astrophysics Data System (ADS)

Ishimura, Hiromi; Tomioka, Shogo; Kadoya, Ryushi; Shimamura, Kanako; Okamoto, Akisumi; Shulga, Sergiy; Kurita, Noriyuki

2017-03-01

The accumulation of amyloid-beta (Aβ) aggregates in brain contributes to the onset of Alzheimer's disease (AD). Recent structural analysis for the tissue obtained from AD patients revealed that Aβ aggregates have a single structure with three-fold symmetry. To explain why this structure possesses significant stability, we here investigated the specific interactions between Aβ peptides in the aggregate, using ab initio fragment molecular orbital calculations. The results indicate that the interactions between the Aβ peptides of the stacked Aβ pair are stronger than those between the Aβ peptides of the trimer with three-fold symmetry and that the charged amino-acids are important.

The DenA/DEN1 Interacting Phosphatase DipA Controls Septa Positioning and Phosphorylation-Dependent Stability of Cytoplasmatic DenA/DEN1 during Fungal Development

PubMed Central

Schinke, Josua; Kolog Gulko, Miriam; Christmann, Martin; Valerius, Oliver; Stumpf, Sina Kristin; Stirz, Margarita; Braus, Gerhard H.

2016-01-01

DenA/DEN1 and the COP9 signalosome (CSN) represent two deneddylases which remove the ubiquitin-like Nedd8 from modified target proteins and are required for distinct fungal developmental programmes. The cellular DenA/DEN1 population is divided into a nuclear and a cytoplasmatic subpopulation which is especially enriched at septa. DenA/DEN1 stability control mechanisms are different for the two cellular subpopulations and depend on different physical interacting proteins and the C-terminal DenA/DEN1 phosphorylation pattern. Nuclear DenA/DEN1 is destabilized during fungal development by five of the eight CSN subunits which target nuclear DenA/DEN1 for degradation. DenA/DEN1 becomes stabilized as a phosphoprotein at S243/S245 during vegetative growth, which is necessary to support further asexual development. After the initial phase of development, the newly identified cytoplasmatic DenA/DEN1 interacting phosphatase DipA and an additional developmental specific C-terminal phosphorylation site at serine S253 destabilize DenA/DEN1. Outside of the nucleus, DipA is co-transported with DenA/DEN1 in the cytoplasm between septa and nuclei. Deletion of dipA resulted in increased DenA/DEN1 stability in a strain which is unresponsive to illumination. The mutant strain is dysregulated in cytokinesis and impaired in asexual development. Our results suggest a dual phosphorylation-dependent DenA/DEN1 stability control with stabilizing and destabilizing modifications and physical interaction partner proteins which function as control points in the nucleus and the cytoplasm. PMID:27010942

Supramolecular PEGylation of biopharmaceuticals

PubMed Central

Webber, Matthew J.; Vinciguerra, Brittany; Cortinas, Abel B.; Thapa, Lavanya S.; Jhunjhunwala, Siddharth; Isaacs, Lyle; Langer, Robert; Anderson, Daniel G.

2016-01-01

The covalent modification of therapeutic biomolecules has been broadly explored, leading to a number of clinically approved modified protein drugs. These modifications are typically intended to address challenges arising in biopharmaceutical practice by promoting improved stability and shelf life of therapeutic proteins in formulation, or modifying pharmacokinetics in the body. Toward these objectives, covalent modification with poly(ethylene glycol) (PEG) has been a common direction. Here, a platform approach to biopharmaceutical modification is described that relies on noncovalent, supramolecular host–guest interactions to endow proteins with prosthetic functionality. Specifically, a series of cucurbit[7]uril (CB[7])–PEG conjugates are shown to substantially increase the stability of three distinct protein drugs in formulation. Leveraging the known and high-affinity interaction between CB[7] and an N-terminal aromatic residue on one specific protein drug, insulin, further results in altering of its pharmacological properties in vivo by extending activity in a manner dependent on molecular weight of the attached PEG chain. Supramolecular modification of therapeutic proteins affords a noncovalent route to modify its properties, improving protein stability and activity as a formulation excipient. Furthermore, this offers a modular approach to append functionality to biopharmaceuticals by noncovalent modification with other molecules or polymers, for applications in formulation or therapy. PMID:27911829

Electrostatics, structure prediction, and the energy landscapes for protein folding and binding.

PubMed

Tsai, Min-Yeh; Zheng, Weihua; Balamurugan, D; Schafer, Nicholas P; Kim, Bobby L; Cheung, Margaret S; Wolynes, Peter G

2016-01-01

While being long in range and therefore weakly specific, electrostatic interactions are able to modulate the stability and folding landscapes of some proteins. The relevance of electrostatic forces for steering the docking of proteins to each other is widely acknowledged, however, the role of electrostatics in establishing specifically funneled landscapes and their relevance for protein structure prediction are still not clear. By introducing Debye-Hückel potentials that mimic long-range electrostatic forces into the Associative memory, Water mediated, Structure, and Energy Model (AWSEM), a transferable protein model capable of predicting tertiary structures, we assess the effects of electrostatics on the landscapes of thirteen monomeric proteins and four dimers. For the monomers, we find that adding electrostatic interactions does not improve structure prediction. Simulations of ribosomal protein S6 show, however, that folding stability depends monotonically on electrostatic strength. The trend in predicted melting temperatures of the S6 variants agrees with experimental observations. Electrostatic effects can play a range of roles in binding. The binding of the protein complex KIX-pKID is largely assisted by electrostatic interactions, which provide direct charge-charge stabilization of the native state and contribute to the funneling of the binding landscape. In contrast, for several other proteins, including the DNA-binding protein FIS, electrostatics causes frustration in the DNA-binding region, which favors its binding with DNA but not with its protein partner. This study highlights the importance of long-range electrostatics in functional responses to problems where proteins interact with their charged partners, such as DNA, RNA, as well as membranes. © 2015 The Protein Society.

AKAP200 promotes Notch stability by protecting it from Cbl/lysosome-mediated degradation in Drosophila melanogaster.

PubMed

Bala Tannan, Neeta; Collu, Giovanna; Humphries, Ashley C; Serysheva, Ekatherina; Weber, Ursula; Mlodzik, Marek

2018-01-01

AKAP200 is a Drosophila melanogaster member of the "A Kinase Associated Protein" family of scaffolding proteins, known for their role in the spatial and temporal regulation of Protein Kinase A (PKA) in multiple signaling contexts. Here, we demonstrate an unexpected function of AKAP200 in promoting Notch protein stability. In Drosophila, AKAP200 loss-of-function (LOF) mutants show phenotypes that resemble Notch LOF defects, including eye patterning and sensory organ specification defects. Through genetic interactions, we demonstrate that AKAP200 interacts positively with Notch in both the eye and the thorax. We further show that AKAP200 is part of a physical complex with Notch. Biochemical studies reveal that AKAP200 stabilizes endogenous Notch protein, and that it limits ubiquitination of Notch. Specifically, our genetic and biochemical evidence indicates that AKAP200 protects Notch from the E3-ubiquitin ligase Cbl, which targets Notch to the lysosomal pathway. Indeed, we demonstrate that the effect of AKAP200 on Notch levels depends on the lysosome. Interestingly, this function of AKAP200 is fully independent of its role in PKA signaling and independent of its ability to bind PKA. Taken together, our data indicate that AKAP200 is a novel tissue specific posttranslational regulator of Notch, maintaining high Notch protein levels and thus promoting Notch signaling.

Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains

PubMed Central

Yang, Zhengrong; Wang, Chi; Zhou, Qingxian; An, Jianli; Hildebrandt, Ellen; Aleksandrov, Luba A; Kappes, John C; DeLucas, Lawrence J; Riordan, John R; Urbatsch, Ina L; Hunt, John F; Brouillette, Christie G

2014-01-01

Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide-binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Notably, the detergents show equivalent trends in their influence on the stability of isolated NBD1 and full-length CFTR. We used differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy to monitor changes in NBD1 stability and secondary structure, respectively, during titration with a series of detergents. Their effective harshness in these assays mirrors that widely accepted for their interaction with IMPs, i.e., anionic > zwitterionic > nonionic. It is noteworthy that including lipids or nonionic detergents is shown to mitigate detergent harshness, as will limiting contact time. We infer three thermodynamic mechanisms from the observed thermal destabilization by monomer or micelle: (i) binding to the unfolded state with no change in the native structure (all detergent classes); (ii) native state binding that alters thermodynamic properties and perhaps conformation (nonionic detergents); and (iii) detergent binding that directly leads to denaturation of the native state (anionic and zwitterionic). These results demonstrate that the accepted model for the harshness of detergents applies to their interaction with an ESD. It is concluded that destabilization of extramembranous soluble domains by specific detergents will influence the stability of some IMPs during purification. PMID:24652590

A method to determine residue-specific unfolded-state pKa values from analysis of stability changes in single mutant cycles.

PubMed

Shen, Jana K

2010-06-02

It is now widely recognized that the unfolded state of a protein in equilibrium with the native state under folding conditions may contain significant residual structures. However, due to technical difficulties residue-specific interactions in the unfolded state remain elusive. Here we introduce a method derived from the Wyman-Tanford theory to determine residue-specific pK(a)'s in the unfolded state. This method requires equilibrium stability measurements of the wild type and single-point mutants in which titrable residues are replaced with charge-neutral ones under two pH conditions. Application of the proposed approach reveals a highly depressed pK(a) for Asp8 in the unfolded state of the NTL9 protein. Knowledge of unfolded-state pK(a)'s enables quantitative estimation of the unfolded-state electrostatic effects on protein stability. It also provides valuable benchmarks for the improvement of force fields and validation of microscopic information from molecular dynamics simulations.

Effect of Dendritic Polymer Architecture on Biological Behaviors of Self-Assembled Nanocarriers

NASA Astrophysics Data System (ADS)

Hsu, Hao-Jui

Polymeric self-assembled nanocarriers represent one of the most versatile platforms for drug delivery. Through tailoring the physiochemical properties of amphiphilic block copolymers, self-assembled nanocarriers with great thermodynamic stability and desired biological properties could be achieved. The PEGylated dendron-based copolymers (PDCs) are one of the novel amphiphilic copolymers that have attracted a great deal of scientific interest due to their unique dendritic structure and properties. While the dendritic polymer architecture of PDC has been shown to enhance the thermodynamic stability of the self-assembling PDCs, dendron micelles, the effect of this polymer architecture on the biological properties of dendron micelles has not yet been studied. Therefore, this dissertation research is focused on understanding the role of dendritic polymer structure on moderating the biological properties of various self-assembled nanocarriers. To systematically investigate this, three studies have been designed and performed. First, we studied whether the dendritic structure of PDC allows dendron micelles to behave non-specific cellular interactions in a similar way that dendrimers would do. Second, cell-specific interactions of dendron micelles mediated by conjugated ligands were investigated. Third, we investigated the influence of dendritic PEG outer shell on micelle-serum protein interactions and its subsequent implication. Our results revealed that both non-specific and specific cellular interactions of dendron micelles were controllable through modulation of the PEG corona length. While the non-specific charge-dependent cellular interactions of dendron micelles were tunable through controlling the length of PEG corona, the use of long PEG tether was found to enhance the ligand-mediated cellular interactions of dendron micelles. With the ligand tethers, a 27-fold enhancement in ligand-mediated cellular interactions can be achieved, compared to non-targeted dendron micelles. Furthermore, we demonstrate that the dense PEG outer shell introduced by its dendritic structure reduced non-specific micelle-serum protein interactions and suppressed the subsequent micelle disintegration or premature drug release, which was not the case for linear block copolymer (LBC)-based micelles. Molecular dynamic (MD) simulation results also supported that dendron micelles exhibited a weaker interaction with serum albumin compared to LBC-based micelles. In the presence of serum proteins, the half-life of dendron micelles was 2-fold longer than that of LBC-based micelles, which could be attributed to their low serum protein interactions. In conclusion, our results provide fundamental understanding on the role of PEG corona and the effect of polymeric architecture on biological properties of polymer micelles, all indicating that dendron micelles have great potential as a novel drug delivery platform.

Study of a tri-trophic prey-dependent food chain model of interacting populations.

PubMed

Haque, Mainul; Ali, Nijamuddin; Chakravarty, Santabrata

2013-11-01

The current paper accounts for the influence of intra-specific competition among predators in a prey dependent tri-trophic food chain model of interacting populations. We offer a detailed mathematical analysis of the proposed food chain model to illustrate some of the significant results that has arisen from the interplay of deterministic ecological phenomena and processes. Biologically feasible equilibria of the system are observed and the behaviours of the system around each of them are described. In particular, persistence, stability (local and global) and bifurcation (saddle-node, transcritical, Hopf-Andronov) analysis of this model are obtained. Relevant results from previous well known food chain models are compared with the current findings. Global stability analysis is also carried out by constructing appropriate Lyapunov functions. Numerical simulations show that the present system is capable enough to produce chaotic dynamics when the rate of self-interaction is very low. On the other hand such chaotic behaviour disappears for a certain value of the rate of self interaction. In addition, numerical simulations with experimented parameters values confirm the analytical results and shows that intra-specific competitions bears a potential role in controlling the chaotic dynamics of the system; and thus the role of self interactions in food chain model is illustrated first time. Finally, a discussion of the ecological applications of the analytical and numerical findings concludes the paper. Copyright © 2013 Elsevier Inc. All rights reserved.

Optimization of Processing Parameters for Extraction of Amylase Enzyme from Dragon (Hylocereus polyrhizus) Peel Using Response Surface Methodology

PubMed Central

Abdul Manap, Mohd Yazid; Zohdi, Norkhanani

2014-01-01

The main goal of this study was to investigate the effect of extraction conditions on the enzymatic properties of thermoacidic amylase enzyme derived from dragon peel. The studied extraction variables were the buffer-to-sample (B/S) ratio (1 : 2 to 1 : 6, w/w), temperature (−18°C to 25°), mixing time (60 to 180 seconds), and the pH of the buffer (2.0 to 8.0). The results indicate that the enzyme extraction conditions exhibited the least significant (P < 0.05) effect on temperature stability. Conversely, the extraction conditions had the most significant (P < 0.05) effect on the specific activity and pH stability. The results also reveal that the main effect of the B/S ratio, followed by its interaction with the pH of the buffer, was significant (P < 0.05) among most of the response variables studied. The optimum extraction condition caused the amylase to achieve high enzyme activity (648.4 U), specific activity (14.2 U/mg), temperature stability (88.4%), pH stability (85.2%), surfactant agent stability (87.2%), and storage stability (90.3%). PMID:25050403

Emergence of synchronization and regularity in firing patterns in time-varying neural hypernetworks

NASA Astrophysics Data System (ADS)

Rakshit, Sarbendu; Bera, Bidesh K.; Ghosh, Dibakar; Sinha, Sudeshna

2018-05-01

We study synchronization of dynamical systems coupled in time-varying network architectures, composed of two or more network topologies, corresponding to different interaction schemes. As a representative example of this class of time-varying hypernetworks, we consider coupled Hindmarsh-Rose neurons, involving two distinct types of networks, mimicking interactions that occur through the electrical gap junctions and the chemical synapses. Specifically, we consider the connections corresponding to the electrical gap junctions to form a small-world network, while the chemical synaptic interactions form a unidirectional random network. Further, all the connections in the hypernetwork are allowed to change in time, modeling a more realistic neurobiological scenario. We model this time variation by rewiring the links stochastically with a characteristic rewiring frequency f . We find that the coupling strength necessary to achieve complete neuronal synchrony is lower when the links are switched rapidly. Further, the average time required to reach the synchronized state decreases as synaptic coupling strength and/or rewiring frequency increases. To quantify the local stability of complete synchronous state we use the Master Stability Function approach, and for global stability we employ the concept of basin stability. The analytically derived necessary condition for synchrony is in excellent agreement with numerical results. Further we investigate the resilience of the synchronous states with respect to increasing network size, and we find that synchrony can be maintained up to larger network sizes by increasing either synaptic strength or rewiring frequency. Last, we find that time-varying links not only promote complete synchronization, but also have the capacity to change the local dynamics of each single neuron. Specifically, in a window of rewiring frequency and synaptic coupling strength, we observe that the spiking behavior becomes more regular.

Fe-C interactions and soil organic matter stability in two tropical soils of contrasting parent materials

NASA Astrophysics Data System (ADS)

Coward, E.; Thompson, A.; Plante, A. F.

2014-12-01

The long residence time of soil organic matter (SOM) is a dynamic property, reflecting the diversity of stabilization mechanisms active within the soil matrix. Climate and ecosystem properties act at the broadest scale, while biochemical recalcitrance, physical occlusion and mineral association drive stability at the microscale. Increasing evidence suggests that the stability of SOM is dominated by organo-mineral interactions. However, the 2:1 clays that provide much of the stabilization capacity in temperate soils are typically absent in tropical soils due to weathering. In contrast, these soils may contain an abundance of iron and aluminium oxides and oxyhydroxides, known as short-range-order (SRO) minerals. These SRO minerals are capable of SOM stabilization through adsorption or co-precipitation, a faculty largely enabled by their high specific surface area (SSA). As such, despite their relatively small mass, SRO minerals may contribute substantially to the SOM stabilization capacity of tropical soils. The objective of this work is to characterize and quantify these Fe-C interactions. Surface (0-20 cm) soil samples were taken from 20 quantitative soil pits dug within the Luquillo Critical Zone Observatory in northeast Puerto Rico. Soils were stratified across granodiorite and volcaniclastic parent materials. Four extraction procedures were used to isolate three different forms of Fe-C interactions: sodium pyrophosphate to isolate organo-metallic complexes, hydroxylamine and oxalate to isolate SRO Fe- and Al-hydroxides, and dithionite to isolate crystalline Fe-oxyhydroxides. Extracts were analysed for DOC and Fe and Al concentrations to estimate the amount of SOM associated with each mineral type. Soils were subjected to SSA and solid-phase C analyses before and after extraction to determine the contribution of the various Fe mineral types to soil SSA, and therefore to potential stabilization capacity through organo-mineral complexation. Preliminary results suggest that extracts from granodiorite parent material contain on average twice the Fe than those from volcaniclastic parent material. The removal of SRO minerals reduced SSA in both soil types, and appear to contribute substantially to SOM stabilization compared to the bulk mineral matrix.

SH2 domains: modulators of nonreceptor tyrosine kinase activity.

PubMed

Filippakopoulos, Panagis; Müller, Susanne; Knapp, Stefan

2009-12-01

The Src homology 2 (SH2) domain is a sequence-specific phosphotyrosine-binding module present in many signaling molecules. In cytoplasmic tyrosine kinases, the SH2 domain is located N-terminally to the catalytic kinase domain (SH1) where it mediates cellular localization, substrate recruitment, and regulation of kinase activity. Initially, structural studies established a role of the SH2 domain stabilizing the inactive state of Src family members. However, biochemical characterization showed that the presence of the SH2 domain is frequently required for catalytic activity, suggesting a crucial function stabilizing the active state of many nonreceptor tyrosine kinases. Recently, the structure of the SH2-kinase domain of Fes revealed that the SH2 domain stabilizes the active kinase conformation by direct interactions with the regulatory helix alphaC. Stabilizing interactions between the SH2 and the kinase domains have also been observed in the structures of active Csk and Abl. Interestingly, mutations in the SH2 domain found in human disease can be explained by SH2 domain destabilization or incorrect positioning of the SH2. Here we summarize our understanding of mechanisms that lead to tyrosine kinase activation by direct interactions mediated by the SH2 domain and discuss how mutations in the SH2 domain trigger kinase inactivation.

Allosteric Regulation of the Hsp90 Dynamics and Stability by Client Recruiter Cochaperones: Protein Structure Network Modeling

PubMed Central

Blacklock, Kristin; Verkhivker, Gennady M.

2014-01-01

The fundamental role of the Hsp90 chaperone in supporting functional activity of diverse protein clients is anchored by specific cochaperones. A family of immune sensing client proteins is delivered to the Hsp90 system with the aid of cochaperones Sgt1 and Rar1 that act cooperatively with Hsp90 to form allosterically regulated dynamic complexes. In this work, functional dynamics and protein structure network modeling are combined to dissect molecular mechanisms of Hsp90 regulation by the client recruiter cochaperones. Dynamic signatures of the Hsp90-cochaperone complexes are manifested in differential modulation of the conformational mobility in the Hsp90 lid motif. Consistent with the experiments, we have determined that targeted reorganization of the lid dynamics is a unifying characteristic of the client recruiter cochaperones. Protein network analysis of the essential conformational space of the Hsp90-cochaperone motions has identified structurally stable interaction communities, interfacial hubs and key mediating residues of allosteric communication pathways that act concertedly with the shifts in conformational equilibrium. The results have shown that client recruiter cochaperones can orchestrate global changes in the dynamics and stability of the interaction networks that could enhance the ATPase activity and assist in the client recruitment. The network analysis has recapitulated a broad range of structural and mutagenesis experiments, particularly clarifying the elusive role of Rar1 as a regulator of the Hsp90 interactions and a stability enhancer of the Hsp90-cochaperone complexes. Small-world organization of the interaction networks in the Hsp90 regulatory complexes gives rise to a strong correspondence between highly connected local interfacial hubs, global mediator residues of allosteric interactions and key functional hot spots of the Hsp90 activity. We have found that cochaperone-induced conformational changes in Hsp90 may be determined by specific interaction networks that can inhibit or promote progression of the ATPase cycle and thus control the recruitment of client proteins. PMID:24466147

Allosteric regulation of the Hsp90 dynamics and stability by client recruiter cochaperones: protein structure network modeling.

PubMed

Blacklock, Kristin; Verkhivker, Gennady M

2014-01-01

The fundamental role of the Hsp90 chaperone in supporting functional activity of diverse protein clients is anchored by specific cochaperones. A family of immune sensing client proteins is delivered to the Hsp90 system with the aid of cochaperones Sgt1 and Rar1 that act cooperatively with Hsp90 to form allosterically regulated dynamic complexes. In this work, functional dynamics and protein structure network modeling are combined to dissect molecular mechanisms of Hsp90 regulation by the client recruiter cochaperones. Dynamic signatures of the Hsp90-cochaperone complexes are manifested in differential modulation of the conformational mobility in the Hsp90 lid motif. Consistent with the experiments, we have determined that targeted reorganization of the lid dynamics is a unifying characteristic of the client recruiter cochaperones. Protein network analysis of the essential conformational space of the Hsp90-cochaperone motions has identified structurally stable interaction communities, interfacial hubs and key mediating residues of allosteric communication pathways that act concertedly with the shifts in conformational equilibrium. The results have shown that client recruiter cochaperones can orchestrate global changes in the dynamics and stability of the interaction networks that could enhance the ATPase activity and assist in the client recruitment. The network analysis has recapitulated a broad range of structural and mutagenesis experiments, particularly clarifying the elusive role of Rar1 as a regulator of the Hsp90 interactions and a stability enhancer of the Hsp90-cochaperone complexes. Small-world organization of the interaction networks in the Hsp90 regulatory complexes gives rise to a strong correspondence between highly connected local interfacial hubs, global mediator residues of allosteric interactions and key functional hot spots of the Hsp90 activity. We have found that cochaperone-induced conformational changes in Hsp90 may be determined by specific interaction networks that can inhibit or promote progression of the ATPase cycle and thus control the recruitment of client proteins.

The nucleolar ubiquitin-specific protease USP36 deubiquitinates and stabilizes c-Myc

PubMed Central

Sun, Xiao-Xin; He, Xia; Yin, Li; Komada, Masayuki; Sears, Rosalie C.; Dai, Mu-Shui

2015-01-01

c-Myc protein stability and activity are tightly regulated by the ubiquitin-proteasome system. Aberrant stabilization of c-Myc contributes to many human cancers. c-Myc is ubiquitinated by SCFFbw7 (a SKP1-cullin-1-F-box complex that contains the F-box and WD repeat domain-containing 7, Fbw7, as the F-box protein) and several other ubiquitin ligases, whereas it is deubiquitinated and stabilized by ubiquitin-specific protease (USP) 28. The bulk of c-Myc degradation appears to occur in the nucleolus. However, whether c-Myc is regulated by deubiquitination in the nucleolus is not known. Here, we report that the nucleolar deubiquitinating enzyme USP36 is a novel c-Myc deubiquitinase. USP36 interacts with and deubiquitinates c-Myc in cells and in vitro, leading to the stabilization of c-Myc. This USP36 regulation of c-Myc occurs in the nucleolus. Interestingly, USP36 interacts with the nucleolar Fbw7γ but not the nucleoplasmic Fbw7α. However, it abolished c-Myc degradation mediated both by Fbw7γ and by Fbw7α. Consistently, knockdown of USP36 reduces the levels of c-Myc and suppresses cell proliferation. We further show that USP36 itself is a c-Myc target gene, suggesting that USP36 and c-Myc form a positive feedback regulatory loop. High expression levels of USP36 are found in a subset of human breast and lung cancers. Altogether, these results identified USP36 as a crucial and bono fide deubiquitinating enzyme controlling c-Myc’s nucleolar degradation pathway. PMID:25775507

Making the Bend: DNA Tertiary Structure and Protein-DNA Interactions

PubMed Central

Harteis, Sabrina; Schneider, Sabine

2014-01-01

DNA structure functions as an overlapping code to the DNA sequence. Rapid progress in understanding the role of DNA structure in gene regulation, DNA damage recognition and genome stability has been made. The three dimensional structure of both proteins and DNA plays a crucial role for their specific interaction, and proteins can recognise the chemical signature of DNA sequence (“base readout”) as well as the intrinsic DNA structure (“shape recognition”). These recognition mechanisms do not exist in isolation but, depending on the individual interaction partners, are combined to various extents. Driving force for the interaction between protein and DNA remain the unique thermodynamics of each individual DNA-protein pair. In this review we focus on the structures and conformations adopted by DNA, both influenced by and influencing the specific interaction with the corresponding protein binding partner, as well as their underlying thermodynamics. PMID:25026169

Micropatterned Cell–Cell Interactions Enable Functional Encapsulation of Primary Hepatocytes in Hydrogel Microtissues

PubMed Central

Li, Cheri Y.; Stevens, Kelly R.; Schwartz, Robert E.; Alejandro, Brian S.; Huang, Joanne H.

2014-01-01

Drug-induced liver injury is a major cause of drug development failures and postmarket withdrawals. In vitro models that incorporate primary hepatocytes have been shown to be more predictive than model systems which rely on liver microsomes or hepatocellular carcinoma cell lines. Methods to phenotypically stabilize primary hepatocytes ex vivo often rely on mimicry of hepatic microenvironmental cues such as cell–cell interactions and cell–matrix interactions. In this work, we sought to incorporate phenotypically stable hepatocytes into three-dimensional (3D) microtissues, which, in turn, could be deployed in drug-screening platforms such as multiwell plates and diverse organ-on-a-chip devices. We first utilize micropatterning on collagen I to specify cell–cell interactions in two-dimensions, followed by collagenase digestion to produce well-controlled aggregates for 3D encapsulation in polyethylene glycol (PEG) diacrylate. Using this approach, we examined the influence of homotypic hepatocyte interactions and composition of the encapsulating hydrogel, and achieved the maintenance of liver-specific function for over 50 days. Optimally preaggregated structures were subsequently encapsulated using a microfluidic droplet-generator to produce 3D microtissues. Interactions of engineered hepatic microtissues with drugs was characterized by flow cytometry, and yielded both induction of P450 enzymes in response to prototypic small molecules and drug–drug interactions that give rise to hepatotoxicity. Collectively, this study establishes a pipeline for the manufacturing of 3D hepatic microtissues that exhibit stabilized liver-specific functions and can be incorporated into a wide array of emerging drug development platforms. PMID:24498910

Forces Driving Chaperone Action

PubMed Central

Koldewey, Philipp; Stull, Frederick; Horowitz, Scott; Martin, Raoul; Bardwell, James C. A.

2016-01-01

SUMMARY It is still unclear what molecular forces drive chaperone-mediated protein folding. Here, we obtain a detailed mechanistic understanding of the forces that dictate the four key steps of chaperone-client interaction: initial binding, complex stabilization, folding, and release. Contrary to the common belief that chaperones recognize unfolding intermediates by their hydrophobic nature, we discover that the model chaperone Spy uses long-range electrostatic interactions to rapidly bind to its unfolded client protein Im7. Short-range hydrophobic interactions follow, which serve to stabilize the complex. Hydrophobic collapse of the client protein then drives its folding. By burying hydrophobic residues in its core, the client’s affinity to Spy decreases, which causes client release. By allowing the client to fold itself, Spy circumvents the need for client-specific folding instructions. This mechanism might help explain how chaperones can facilitate the folding of various unrelated proteins. PMID:27293188

Computational design of a pH stable enzyme: understanding molecular mechanism of penicillin acylase's adaptation to alkaline conditions.

PubMed

Suplatov, Dmitry; Panin, Nikolay; Kirilin, Evgeny; Shcherbakova, Tatyana; Kudryavtsev, Pavel; Svedas, Vytas

2014-01-01

Protein stability provides advantageous development of novel properties and can be crucial in affording tolerance to mutations that introduce functionally preferential phenotypes. Consequently, understanding the determining factors for protein stability is important for the study of structure-function relationship and design of novel protein functions. Thermal stability has been extensively studied in connection with practical application of biocatalysts. However, little work has been done to explore the mechanism of pH-dependent inactivation. In this study, bioinformatic analysis of the Ntn-hydrolase superfamily was performed to identify functionally important subfamily-specific positions in protein structures. Furthermore, the involvement of these positions in pH-induced inactivation was studied. The conformational mobility of penicillin acylase in Escherichia coli was analyzed through molecular modeling in neutral and alkaline conditions. Two functionally important subfamily-specific residues, Gluβ482 and Aspβ484, were found. Ionization of these residues at alkaline pH promoted the collapse of a buried network of stabilizing interactions that consequently disrupted the functional protein conformation. The subfamily-specific position Aspβ484 was selected as a hotspot for mutation to engineer enzyme variant tolerant to alkaline medium. The corresponding Dβ484N mutant was produced and showed 9-fold increase in stability at alkaline conditions. Bioinformatic analysis of subfamily-specific positions can be further explored to study mechanisms of protein inactivation and to design more stable variants for the engineering of homologous Ntn-hydrolases with improved catalytic properties.

Computational Design of a pH Stable Enzyme: Understanding Molecular Mechanism of Penicillin Acylase's Adaptation to Alkaline Conditions

PubMed Central

Suplatov, Dmitry; Panin, Nikolay; Kirilin, Evgeny; Shcherbakova, Tatyana; Kudryavtsev, Pavel; Švedas, Vytas

2014-01-01

Protein stability provides advantageous development of novel properties and can be crucial in affording tolerance to mutations that introduce functionally preferential phenotypes. Consequently, understanding the determining factors for protein stability is important for the study of structure-function relationship and design of novel protein functions. Thermal stability has been extensively studied in connection with practical application of biocatalysts. However, little work has been done to explore the mechanism of pH-dependent inactivation. In this study, bioinformatic analysis of the Ntn-hydrolase superfamily was performed to identify functionally important subfamily-specific positions in protein structures. Furthermore, the involvement of these positions in pH-induced inactivation was studied. The conformational mobility of penicillin acylase in Escherichia coli was analyzed through molecular modeling in neutral and alkaline conditions. Two functionally important subfamily-specific residues, Gluβ482 and Aspβ484, were found. Ionization of these residues at alkaline pH promoted the collapse of a buried network of stabilizing interactions that consequently disrupted the functional protein conformation. The subfamily-specific position Aspβ484 was selected as a hotspot for mutation to engineer enzyme variant tolerant to alkaline medium. The corresponding Dβ484N mutant was produced and showed 9-fold increase in stability at alkaline conditions. Bioinformatic analysis of subfamily-specific positions can be further explored to study mechanisms of protein inactivation and to design more stable variants for the engineering of homologous Ntn-hydrolases with improved catalytic properties. PMID:24959852

Cations Form Sequence Selective Motifs within DNA Grooves via a Combination of Cation-Pi and Ion-Dipole/Hydrogen Bond Interactions

PubMed Central

Stewart, Mikaela; Dunlap, Tori; Dourlain, Elizabeth; Grant, Bryce; McFail-Isom, Lori

2013-01-01

The fine conformational subtleties of DNA structure modulate many fundamental cellular processes including gene activation/repression, cellular division, and DNA repair. Most of these cellular processes rely on the conformational heterogeneity of specific DNA sequences. Factors including those structural characteristics inherent in the particular base sequence as well as those induced through interaction with solvent components combine to produce fine DNA structural variation including helical flexibility and conformation. Cation-pi interactions between solvent cations or their first hydration shell waters and the faces of DNA bases form sequence selectively and contribute to DNA structural heterogeneity. In this paper, we detect and characterize the binding patterns found in cation-pi interactions between solvent cations and DNA bases in a set of high resolution x-ray crystal structures. Specifically, we found that monovalent cations (Tl+) and the polarized first hydration shell waters of divalent cations (Mg2+, Ca2+) form cation-pi interactions with DNA bases stabilizing unstacked conformations. When these cation-pi interactions are combined with electrostatic interactions a pattern of specific binding motifs is formed within the grooves. PMID:23940752

Cations form sequence selective motifs within DNA grooves via a combination of cation-pi and ion-dipole/hydrogen bond interactions.

PubMed

Stewart, Mikaela; Dunlap, Tori; Dourlain, Elizabeth; Grant, Bryce; McFail-Isom, Lori

2013-01-01

The fine conformational subtleties of DNA structure modulate many fundamental cellular processes including gene activation/repression, cellular division, and DNA repair. Most of these cellular processes rely on the conformational heterogeneity of specific DNA sequences. Factors including those structural characteristics inherent in the particular base sequence as well as those induced through interaction with solvent components combine to produce fine DNA structural variation including helical flexibility and conformation. Cation-pi interactions between solvent cations or their first hydration shell waters and the faces of DNA bases form sequence selectively and contribute to DNA structural heterogeneity. In this paper, we detect and characterize the binding patterns found in cation-pi interactions between solvent cations and DNA bases in a set of high resolution x-ray crystal structures. Specifically, we found that monovalent cations (Tl⁺) and the polarized first hydration shell waters of divalent cations (Mg²⁺, Ca²⁺) form cation-pi interactions with DNA bases stabilizing unstacked conformations. When these cation-pi interactions are combined with electrostatic interactions a pattern of specific binding motifs is formed within the grooves.

[The study of complex-formation of DNA with the antimicrobial drug decamethoxine].

PubMed

Sorokin, V A; Blagoĭ, Iu P; Valeev, V A; Gladchenko, G O; Sukhodub, L F; Volianskiĭ, Iu L

1990-01-01

The interaction of effective antibacterial drug decametoxyn with natural DNA was studied by UV-spectroscopy. Decametoxyn shows a specificity to nucleotides: it decreases the cooperativity of melting and the thermal stability of DNA parts enriched by AT pairs. The characteristics of the helix-coil transition on the DNA parts enriched by GC-pairs are invariable. Interaction with AT-pairs results in their partial or complete melting at room temperature, followed by intermolecule aggregation. Interacting with phosphates decametoxyn manifests itself not as a dication but as two single-charged ions.

UV-Visible and Infrared Methods for Investigating Lipid-Rhodopsin Membrane Interactions

PubMed Central

Brown, Michael F.

2017-01-01

Summary Experimental UV-visible and Fourier transform infrared (FTIR) spectroscopic methods are described for characterizing lipid-protein interactions for the example of rhodopsin in a membrane bilayer environment. The combined use of FTIR and UV-visible difference spectroscopy monitors the structural and functional changes during rhodopsin activation. Such studies investigate how membrane lipids stabilize the various rhodopsin photoproducts, analogous to mutating the protein. Interpretation of the results entails a non-specific flexible surface model for explaining the role of membrane lipid-protein interactions in biological functions. PMID:22976026

Exploring the membrane fusion mechanism through force-induced disassembly of HIV-1 six-helix bundle

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

Gao, Kai; Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101; University of Chinese Academy of Sciences, Beijing 100049

Enveloped virus, such as HIV-1, employs membrane fusion mechanism to invade into host cell. HIV-1 gp41 ectodomain uses six-helix bundle configuration to accomplish this process. Using molecular dynamic simulations, we confirmed the stability of this six-helix bundle by showing high occupancy of hydrogen bonds and hydrophobic interactions. Key residues and interactions important for the bundle integration were characterized by force-induced unfolding simulations of six-helix bundle, exhibiting the collapse order of these groups of interactions. Moreover, our results in some way concerted with a previous theory that the formation of coiled-coil choose a route which involved cooperative interactions between the N-terminalmore » and C-terminal helix. -- Highlights: •Unfolding of HIV-1 gp41 six-helix bundle is studied by molecular dynamics simulations. •Specific interactions responsible for the stability of HIV-1 envelope post-fusion conformation were identified. •The gp41 six-helix bundle transition inducing membrane fusion might be a cooperative process of the three subunits.« less

The roles of amensalistic and commensalistic interactions in large ecological network stability

PubMed Central

Mougi, Akihiko

2016-01-01

Ecological communities comprise diverse species and their interactions. Notably, ecological and evolutionary studies have revealed that reciprocal interactions such as predator–prey, competition, and mutualism, are key drivers of community dynamics. However, there is an argument that many species interactions are asymmetric, where one species unilaterally affects another species (amensalism or commensalism). This raises the unanswered question of what is the role of unilateral interactions in community dynamics. Here I use a theoretical approach to demonstrate that unilateral interactions greatly enhance community stability. The results suggested that amensalism and commensalism were more stabilizing than symmetrical interactions, such as competition and mutualism, but they were less stabilizing than an asymmetric antagonistic interaction. A mix of unilateral interactions increased stability. Furthermore, in communities with all interaction types, unilateral interactions tended to increase stability. This study suggests that unilateral interactions play a major role in maintaining communities, underlining the need to further investigate their roles in ecosystem dynamics. PMID:27406267

Kinetic stabilization of Bacillus licheniformis alpha-amylase through introduction of hydrophobic residues at the surface.

PubMed

Machius, Mischa; Declerck, Nathalie; Huber, Robert; Wiegand, Georg

2003-03-28

It is generally assumed that in proteins hydrophobic residues are not favorable at solvent-exposed sites, and that amino acid substitutions on the surface have little effect on protein thermostability. Contrary to these assumptions, we have identified hyperthermostable variants of Bacillus licheniformis alpha-amylase (BLA) that result from the incorporation of hydrophobic residues at the surface. Under highly destabilizing conditions, a variant combining five stabilizing mutations unfolds 32 times more slowly and at a temperature 13 degrees C higher than the wild-type. Crystal structure analysis at 1.7 A resolution suggests that stabilization is achieved through (a) extension of the concept of increased hydrophobic packing, usually applied to cavities, to surface indentations, (b) introduction of favorable aromatic-aromatic interactions on the surface, (c) specific stabilization of intrinsic metal binding sites, and (d) stabilization of a beta-sheet by introducing a residue with high beta-sheet forming propensity. All mutated residues are involved in forming complex, cooperative interaction networks that extend from the interior of the protein to its surface and which may therefore constitute "weak points" where BLA unfolding is initiated. This might explain the unexpectedly large effect induced by some of the substitutions on the kinetic stability of BLA. Our study shows that substantial protein stabilization can be achieved by stabilizing surface positions that participate in underlying cooperatively formed substructures. At such positions, even the apparently thermodynamically unfavorable introduction of hydrophobic residues should be explored.

Balancing the stability and the catalytic specificities of OP hydrolases with enhanced V-agent activities.

PubMed

Reeves, T E; Wales, M E; Grimsley, J K; Li, P; Cerasoli, D M; Wild, J R

2008-06-01

Rational site-directed mutagenesis and biophysical analyses have been used to explore the thermodynamic stability and catalytic capabilities of organophosphorus hydrolase (OPH) and its genetically modified variants. There are clear trade-offs in the stability of modifications that enhance catalytic activities. For example, the H254R/H257L variant has higher turnover numbers for the chemical warfare agents VX (144 versus 14 s(-1) for the native enzyme (wild type) and VR (Russian VX, 465 versus 12 s(-1) for wild type). These increases are accompanied by a loss in stability in which the total Gibb's free energy for unfolding is 19.6 kcal/mol, which is 5.7 kcal/mol less than that of the wild-type enzyme. X-ray crystallographic studies support biophysical data that suggest amino acid residues near the active site contribute to the chemical and thermal stability through hydrophobic and cation-pi interactions. The cation-pi interactions appear to contribute an additional 7 kcal/mol to the overall global stability of the enzyme. Using rational design, it has been possible to make amino acid changes in this region that restored the stability, yet maintained effective V-agent activities, with turnover numbers of 68 and 36 s(-1) for VX and VR, respectively. This study describes the first rationally designed, stability/activity balance for an OPH enzyme with a legitimate V-agent activity, and its crystal structure.

Characterization of Molecular Determinants of the Conformational Stability of Macrophage Migration Inhibitory Factor: Leucine 46 Hydrophobic Pocket

PubMed Central

El-Turk, Farah; Fauvet, Bruno; Ashrafi, Amer; Ouertatani-Sakouhi, Hajer; Cho, Min-Kyu; Neri, Marilisa; Cascella, Michele; Rothlisberger, Ursula; Pojer, Florence; Zweckstetter, Markus; Lashuel, Hilal

2012-01-01

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF’s trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state. PMID:23028743

Characterization of molecular determinants of the conformational stability of macrophage migration inhibitory factor: leucine 46 hydrophobic pocket.

PubMed

El-Turk, Farah; Fauvet, Bruno; Ashrafi, Amer; Ouertatani-Sakouhi, Hajer; Cho, Min-Kyu; Neri, Marilisa; Cascella, Michele; Rothlisberger, Ursula; Pojer, Florence; Zweckstetter, Markus; Lashuel, Hilal

2012-01-01

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF's trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state.

Identification and Characterization of Noncovalent Interactions That Drive Binding and Specificity in DD-Peptidases and β-Lactamases.

PubMed

Hargis, Jacqueline C; Vankayala, Sai Lakshmana; White, Justin K; Woodcock, H Lee

2014-02-11

Bacterial resistance to standard (i.e., β-lactam-based) antibiotics has become a global pandemic. Simultaneously, research into the underlying causes of resistance has slowed substantially, although its importance is universally recognized. Key to unraveling critical details is characterization of the noncovalent interactions that govern binding and specificity (DD-peptidases, antibiotic targets, versus β-lactamases, the evolutionarily derived enzymes that play a major role in resistance) and ultimately resistance as a whole. Herein, we describe a detailed investigation that elicits new chemical insights into these underlying intermolecular interactions. Benzylpenicillin and a novel β-lactam peptidomimetic complexed to the Stremptomyces R61 peptidase are examined using an arsenal of computational techniques: MD simulations, QM/MM calculations, charge perturbation analysis, QM/MM orbital analysis, bioinformatics, flexible receptor/flexible ligand docking, and computational ADME predictions. Several key molecular level interactions are identified that not only shed light onto fundamental resistance mechanisms, but also offer explanations for observed specificity. Specifically, an extended π-π network is elucidated that suggests antibacterial resistance has evolved, in part, due to stabilizing aromatic interactions. Additionally, interactions between the protein and peptidomimetic substrate are identified and characterized. Of particular interest is a water-mediated salt bridge between Asp217 and the positively charged N-terminus of the peptidomimetic, revealing an interaction that may significantly contribute to β-lactam specificity. Finally, interaction information is used to suggest modifications to current β-lactam compounds that should both improve binding and specificity in DD-peptidases and their physiochemical properties.

TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.

PubMed

Lin, Jiangguo; Countryman, Preston; Buncher, Noah; Kaur, Parminder; E, Longjiang; Zhang, Yiyun; Gibson, Greg; You, Changjiang; Watkins, Simon C; Piehler, Jacob; Opresko, Patricia L; Kad, Neil M; Wang, Hong

2014-02-01

Human telomeres are maintained by the shelterin protein complex in which TRF1 and TRF2 bind directly to duplex telomeric DNA. How these proteins find telomeric sequences among a genome of billions of base pairs and how they find protein partners to form the shelterin complex remains uncertain. Using single-molecule fluorescence imaging of quantum dot-labeled TRF1 and TRF2, we study how these proteins locate TTAGGG repeats on DNA tightropes. By virtue of its basic domain TRF2 performs an extensive 1D search on nontelomeric DNA, whereas TRF1's 1D search is limited. Unlike the stable and static associations observed for other proteins at specific binding sites, TRF proteins possess reduced binding stability marked by transient binding (∼ 9-17 s) and slow 1D diffusion on specific telomeric regions. These slow diffusion constants yield activation energy barriers to sliding ∼ 2.8-3.6 κ(B)T greater than those for nontelomeric DNA. We propose that the TRF proteins use 1D sliding to find protein partners and assemble the shelterin complex, which in turn stabilizes the interaction with specific telomeric DNA. This 'tag-team proofreading' represents a more general mechanism to ensure a specific set of proteins interact with each other on long repetitive specific DNA sequences without requiring external energy sources.

Interaction strength combinations and the overfishing of a marine food web.

PubMed

Bascompte, Jordi; Melián, Carlos J; Sala, Enric

2005-04-12

The stability of ecological communities largely depends on the strength of interactions between predators and their prey. Here we show that these interaction strengths are structured nonrandomly in a large Caribbean marine food web. Specifically, the cooccurrence of strong interactions on two consecutive levels of food chains occurs less frequently than expected by chance. Even when they occur, these strongly interacting chains are accompanied by strong omnivory more often than expected by chance. By using a food web model, we show that these interaction strength combinations reduce the likelihood of trophic cascades after the overfishing of top predators. However, fishing selectively removes predators that are overrepresented in strongly interacting chains. Hence, the potential for strong community-wide effects remains a threat.

Interaction strength combinations and the overfishing of a marine food web

PubMed Central

Bascompte, Jordi; Melián, Carlos J.; Sala, Enric

2005-01-01

The stability of ecological communities largely depends on the strength of interactions between predators and their prey. Here we show that these interaction strengths are structured nonrandomly in a large Caribbean marine food web. Specifically, the cooccurrence of strong interactions on two consecutive levels of food chains occurs less frequently than expected by chance. Even when they occur, these strongly interacting chains are accompanied by strong omnivory more often than expected by chance. By using a food web model, we show that these interaction strength combinations reduce the likelihood of trophic cascades after the overfishing of top predators. However, fishing selectively removes predators that are overrepresented in strongly interacting chains. Hence, the potential for strong community-wide effects remains a threat. PMID:15802468

Novel interactions of ankyrins-G at the costameres: The muscle-specific Obscurin/Titin-Binding-related Domain (OTBD) binds plectin and filamin C

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

Maiweilidan, Yimingjiang; Klauza, Izabela; Kordeli, Ekaterini, E-mail: ekaterini.kordeli@inserm.fr

2011-04-01

Ankyrins, the adapters of the spectrin skeleton, are involved in local accumulation and stabilization of integral proteins to the appropriate membrane domains. In striated muscle, tissue-dependent alternative splicing generates unique Ank3 gene products (ankyrins-G); they share the Obscurin/Titin-Binding-related Domain (OTBD), a muscle-specific insert of the C-terminal domain which is highly conserved among ankyrin genes, and binds obscurin and titin to Ank1 gene products. We previously proposed that OTBD sequences constitute a novel domain of protein-protein interactions which confers ankyrins with specific cellular functions in muscle. Here we searched for muscle proteins binding to ankyrin-G OTBD by yeast two hybrid assay,more » and we found plectin and filamin C, two organizing elements of the cytoskeleton with essential roles in myogenesis, muscle cell cytoarchitecture, and muscle disease. The three proteins coimmunoprecipitate from skeletal muscle extracts and colocalize at costameres in adult muscle fibers. During in vitro myogenesis, muscle ankyrins-G are first expressed in postmitotic myocytes undergoing fusion to myotubes. In western blots of subcellular fractions from C2C12 cells, the majority of muscle ankyrins-G appear associated with membrane compartments. Occasional but not extensive co-localization at nascent costameres suggested that ankyrin-G interactions with plectin and filamin C are not involved in costamere assembly; they would rather reinforce stability and/or modulate molecular interactions in sarcolemma microdomains by establishing novel links between muscle-specific ankyrins-G and the two costameric dystrophin-associated glycoprotein and integrin-based protein complexes. These results report the first protein-protein interactions involving the ankyrin-G OTBD domain and support the hypothesis that OTBD sequences confer ankyrins with a gain of function in vertebrates, bringing further consolidation and resilience of the linkage between sarcomeres and sarcolemma.« less

SLP-2 interacts with prohibitins in the mitochondrial inner membrane and contributes to their stability.

PubMed

Da Cruz, Sandrine; Parone, Philippe A; Gonzalo, Philippe; Bienvenut, Willy V; Tondera, Daniel; Jourdain, Alexis; Quadroni, Manfredo; Martinou, Jean-Claude

2008-05-01

Stomatin is a member of a large family of proteins including prohibitins, HflK/C, flotillins, mechanoreceptors and plant defense proteins, that are thought to play a role in protein turnover. Using different proteomic approaches, we and others have identified SLP-2, a member of the stomatin gene family, as a component of the mitochondria. In this study, we show that SLP-2 is strongly associated with the mitochondrial inner membrane and that it interacts with prohibitins. Depleting HeLa cells of SLP-2 lead to increased proteolysis of prohibitins and of subunits of the respiratory chain complexes I and IV. Further supporting the role of SLP-2 in regulating the stability of specific mitochondrial proteins, we found that SLP-2 is up-regulated under conditions of mitochondrial stress leading to increased protein turnover. These data indicate that SLP-2 plays a role in regulating the stability of mitochondrial proteins including prohibitins and subunits of respiratory chain complexes.

Mechanisms of monoclonal antibody stabilization and release from silk biomaterials

PubMed Central

Guziewicz, Nicholas A.; Massetti, Andrew J.; Perez-Ramirez, Bernardo J.; Kaplan, David L.

2013-01-01

The availability of stabilization and sustained delivery systems for antibody therapeutics remains a major clinical challenge, despite the growing development of antibodies for a wide range of therapeutic applications due to their specificity and efficacy. A mechanistic understanding of protein-matrix interactions is critical for the development of such systems and is currently lacking as a mode to guide the field. We report mechanistic insight to address this need by using well-defined matrices based on silk gels, in combination with a monoclonal antibody. Variables including antibody loading, matrix density, charge interactions, hydrophobicity and water access were assessed to clarify mechanisms involved in the release of antibody from the biomaterial matrix. The results indicate that antibody release is primarily governed by hydrophobic interactions and hydration resistance, which are controlled by silk matrix chemistry, peptide domain distribution and protein density. Secondary ionic repulsions are also critical in antibody stabilization and release. Matrix modification by free methionine incorporation was found to be an effective strategy for mitigating encapsulation induced antibody oxidation. Additionally, these studies highlight a characterization approach to improve the understanding and development of other protein sustained delivery systems, with broad applicability to the rapidly developing monoclonal antibody field. PMID:23859659

Selection of peptides interfering with protein-protein interaction.

PubMed

Gaida, Annette; Hagemann, Urs B; Mattay, Dinah; Räuber, Christina; Müller, Kristian M; Arndt, Katja M

2009-01-01

Cell physiology depends on a fine-tuned network of protein-protein interactions, and misguided interactions are often associated with various diseases. Consequently, peptides, which are able to specifically interfere with such adventitious interactions, are of high interest for analytical as well as medical purposes. One of the most abundant protein interaction domains is the coiled-coil motif, and thus provides a premier target. Coiled coils, which consist of two or more alpha-helices wrapped around each other, have one of the simplest interaction interfaces, yet they are able to confer highly specific homo- and heterotypic interactions involved in virtually any cellular process. While there are several ways to generate interfering peptides, the combination of library design with a powerful selection system seems to be one of the most effective and promising approaches. This chapter guides through all steps of such a process, starting with library options and cloning, detailing suitable selection techniques and ending with purification for further down-stream characterization. Such generated peptides will function as versatile tools to interfere with the natural function of their targets thereby illuminating their down-stream signaling and, in general, promoting understanding of factors leading to specificity and stability in protein-protein interactions. Furthermore, peptides interfering with medically relevant proteins might become important diagnostics and therapeutics.

Role of tyrosine 33 residue for the stabilization of the tetrameric structure of human cytidine deaminase.

PubMed

Micozzi, Daniela; Pucciarelli, Stefania; Carpi, Francesco M; Costanzi, Stefano; De Sanctis, Giampiero; Polzonetti, Valeria; Natalini, Paolo; Santarelli, Ivano F; Vita, Alberto; Vincenzetti, Silvia

2010-11-01

In the present work the effect of a mutation on tyrosine 33 residue (Y33G) of human cytidine deaminase (CDA) was investigated with regard to protein solubility and specific activity. Osmolytes and CDA ligands were used to increase the yield and the specific activity of the protein. The mutant enzyme was purified and subjected to a kinetic characterization and to stability studies. These investigations reinforced the hypothesis that in human CDA the side chain of Y33 is involved in intersubunit interactions with four glutamate residues (E108) forming a double latch that connects each of the two pairs of monomers of the tetrameric CDA. Copyright © 2010 Elsevier B.V. All rights reserved.

Differences in Pornography Use Among Couples: Associations with Satisfaction, Stability, and Relationship Processes.

PubMed

Willoughby, Brian J; Carroll, Jason S; Busby, Dean M; Brown, Cameron C

2016-01-01

The present study utilized a sample of 1755 adult couples in heterosexual romantic relationships to examine how different patterns of pornography use between romantic partners may be associated with relationship outcomes. While pornography use has been generally associated with some negative and some positive couple outcomes, no study has yet explored how differences between partners may uniquely be associated with relationship well-being. Results suggested that greater discrepancies between partners in pornography use were related to less relationship satisfaction, less stability, less positive communication, and more relational aggression. Mediation analyses suggested that greater pornography use discrepancies were primarily associated with elevated levels of male relational aggression, lower female sexual desire, and less positive communication for both partners which then predicted lower relational satisfaction and stability for both partners. Results generally suggest that discrepancies in pornography use at the couple level are related to negative couple outcomes. Specifically, pornography differences may alter specific couple interaction processes which, in turn, may influence relationship satisfaction and stability. Implications for scholars and clinicians interested in how pornography use is associated with couple process are discussed.

Double row loop-coil configuration for high-speed electrodynamic maglev suspension, guidance, propulsion and guideway directional switching

DOEpatents

He, Jianliang; Rote, Donald M.

1996-01-01

A stabilization and propulsion system comprising a series of loop-coils arranged in parallel rows wherein two rows combine to form one of two magnetic rails. Levitation and lateral stability are provided when the induced field in the magnetic rails interacts with the superconducting magnets mounted on the magnetic levitation vehicle. The loop-coils forming the magnetic rails have specified dimensions and a specified number of turns and by constructing differently these specifications, for one rail with respect to the other, the angle of tilt of the vehicle can be controlled during directional switching. Propulsion is provided by the interaction of a traveling magnetic wave associated with the coils forming the rails and the super conducting magnets on the vehicle.

Double row loop-coil configuration for high-speed electrodynamic maglev suspension, guidance, propulsion and guideway directional switching

DOEpatents

He, J.; Rote, D.M.

1996-05-21

A stabilization and propulsion system are disclosed comprising a series of loop-coils arranged in parallel rows wherein two rows combine to form one of two magnetic rails. Levitation and lateral stability are provided when the induced field in the magnetic rails interacts with the superconducting magnets mounted on the magnetic levitation vehicle. The loop-coils forming the magnetic rails have specified dimensions and a specified number of turns and by constructing differently these specifications, for one rail with respect to the other, the angle of tilt of the vehicle can be controlled during directional switching. Propulsion is provided by the interaction of a traveling magnetic wave associated with the coils forming the rails and the superconducting magnets on the vehicle. 12 figs.

A direct interaction between leucine-rich repeat kinase 2 and specific β-tubulin isoforms regulates tubulin acetylation.

PubMed

Law, Bernard M H; Spain, Victoria A; Leinster, Veronica H L; Chia, Ruth; Beilina, Alexandra; Cho, Hyun J; Taymans, Jean-Marc; Urban, Mary K; Sancho, Rosa M; Blanca Ramírez, Marian; Biskup, Saskia; Baekelandt, Veerle; Cai, Huaibin; Cookson, Mark R; Berwick, Daniel C; Harvey, Kirsten

2014-01-10

Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of β-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease.

Community specificity: life and afterlife effects of genes.

PubMed

Whitham, Thomas G; Gehring, Catherine A; Lamit, Louis J; Wojtowicz, Todd; Evans, Luke M; Keith, Arthur R; Smith, David Solance

2012-05-01

Community-level genetic specificity results when individual genotypes or populations of the same species support different communities. Our review of the literature shows that genetic specificity exhibits both life and afterlife effects; it is a widespread phenomenon occurring in diverse taxonomic groups, aquatic to terrestrial ecosystems, and species-poor to species-rich systems. Such specificity affects species interactions, evolution, ecosystem processes and leads to community feedbacks on the performance of the individuals expressing the traits. Thus, genetic specificity by communities appears to be fundamentally important, suggesting that specificity is a major driver of the biodiversity and stability of the world's ecosystems. Copyright © 2012. Published by Elsevier Ltd.

I-mfa domain proteins specifically interact with HTLV-1 Tax and repress its transactivating functions.

PubMed

Kusano, Shuichi; Yoshimitsu, Makoto; Hachiman, Miho; Ikeda, Masanori

2015-12-01

The I-mfa domain proteins HIC (also known as MDFIC) and I-mfa (also known as MDFI) are candidate tumor suppressor genes that are involved in cellular and viral transcriptional regulation. Here, we show that HIC and I-mfa directly interact with human T-cell leukemia virus type-1 (HTLV-1) Tax protein in vitro. In addition, HIC and I-mfa repress Tax-dependent transactivation of an HTLV-1 long terminal repeat (LTR) reporter construct in COS-1, Jurkat and high-Tax-producing HTLV-1-infected T cells. HIC also interacts with Tax through its I-mfa domain in vivo and represses Tax-dependent transactivation of HTLV-1 LTR and NF-κB reporter constructs in an interaction-dependent manner. Furthermore, we show that HIC decreases the nuclear distribution and stimulates the proteasomal degradation of Tax. These data reveal that HIC specifically interacts with HTLV-1 Tax and negatively regulates Tax transactivational activity by altering its subcellular distribution and stability. Copyright © 2015 Elsevier Inc. All rights reserved.

Stabilization of an immunoglobulin fold domain by an engineered disulfide bond at the buried hydrophobic region.

PubMed

Hagihara, Yoshihisa; Mine, Shouhei; Uegaki, Koichi

2007-12-14

We report for the first time the stabilization of an immunoglobulin fold domain by an engineered disulfide bond. In the llama single-domain antibody, which has human chorionic gonadotropin as its specific antigen, Ala49 and Ile70 are buried in the structure. A mutant with an artificial disulfide bond at this position showed a 10 degrees C higher midpoint temperature of thermal unfolding than that without the extra disulfide bond. The modified domains exhibited an antigen binding affinity comparable with that of the wild-type domain. Ala49 and Ile70 are conserved in camel and llama single-domain antibody frameworks. Therefore, domains against different antigens are expected to be stabilized by the engineered disulfide bond examined here. In addition to the effect of the loop constraints in the unfolded state, thermodynamic analysis indicated that internal interaction and hydration also control the stability of domains with disulfide bonds. The change in physical properties resulting from mutation often causes unpredictable and destabilizing effects on these interactions. The introduction of a hydrophobic cystine into the hydrophobic region maintains the hydrophobicity of the protein and is expected to minimize the unfavorable mutational effects.

A glycolytic metabolon in Saccharomyces cerevisiae is stabilized by F-actin.

PubMed

Araiza-Olivera, Daniela; Chiquete-Felix, Natalia; Rosas-Lemus, Mónica; Sampedro, José G; Peña, Antonio; Mujica, Adela; Uribe-Carvajal, Salvador

2013-08-01

In the Saccharomyces cerevisiae glycolytic pathway, 11 enzymes catalyze the stepwise conversion of glucose to two molecules of ethanol plus two CO₂ molecules. In the highly crowded cytoplasm, this pathway would be very inefficient if it were dependent on substrate/enzyme diffusion. Therefore, the existence of a multi-enzymatic glycolytic complex has been suggested. This complex probably uses the cytoskeleton to stabilize the interaction of the various enzymes. Here, the role of filamentous actin (F-actin) in stabilization of a putative glycolytic metabolon is reported. Experiments were performed in isolated enzyme/actin mixtures, cytoplasmic extracts and permeabilized yeast cells. Polymerization of actin was promoted using phalloidin or inhibited using cytochalasin D or latrunculin. The polymeric filamentous F-actin, but not the monomeric globular G-actin, stabilized both the interaction of isolated glycolytic pathway enzyme mixtures and the whole fermentation pathway, leading to higher fermentation activity. The associated complexes were resistant against inhibition as a result of viscosity (promoted by the disaccharide trehalose) or inactivation (using specific enzyme antibodies). In S. cerevisiae, a glycolytic metabolon appear to assemble in association with F-actin. In this complex, fermentation activity is enhanced and enzymes are partially protected against inhibition by trehalose or by antibodies. © 2013 FEBS.

An Innovative Structural Mode Selection Methodology: Application for the X-33 Launch Vehicle Finite Element Model

NASA Technical Reports Server (NTRS)

Hidalgo, Homero, Jr.

2000-01-01

An innovative methodology for determining structural target mode selection and mode selection based on a specific criterion is presented. An effective approach to single out modes which interact with specific locations on a structure has been developed for the X-33 Launch Vehicle Finite Element Model (FEM). We presented Root-Sum-Square (RSS) displacement method computes resultant modal displacement for each mode at selected degrees of freedom (DOF) and sorts to locate modes with highest values. This method was used to determine modes, which most influenced specific locations/points on the X-33 flight vehicle such as avionics control components, aero-surface control actuators, propellant valve and engine points for use in flight control stability analysis and for flight POGO stability analysis. Additionally, the modal RSS method allows for primary or global target vehicle modes to also be identified in an accurate and efficient manner.

Protein modeling and molecular dynamics simulation of the two novel surfactant proteins SP-G and SP-H.

PubMed

Rausch, Felix; Schicht, Martin; Bräuer, Lars; Paulsen, Friedrich; Brandt, Wolfgang

2014-11-01

Surfactant proteins are well known from the human lung where they are responsible for the stability and flexibility of the pulmonary surfactant system. They are able to influence the surface tension of the gas-liquid interface specifically by directly interacting with single lipids. This work describes the generation of reliable protein structure models to support the experimental characterization of two novel putative surfactant proteins called SP-G and SP-H. The obtained protein models were complemented by predicted posttranslational modifications and placed in a lipid model system mimicking the pulmonary surface. Molecular dynamics simulations of these protein-lipid systems showed the stability of the protein models and the formation of interactions between protein surface and lipid head groups on an atomic scale. Thereby, interaction interface and strength seem to be dependent on orientation and posttranslational modification of the protein. The here presented modeling was fundamental for experimental localization studies and the simulations showed that SP-G and SP-H are theoretically able to interact with lipid systems and thus are members of the surfactant protein family.

Using atomic force microscopy and surface plasmon resonance to detect specific interactions between ricin and anti-ricin aptamers

USDA-ARS?s Scientific Manuscript database

Nucleic acid aptamers have been widely used as binding reagents for the label free detections of biomolecules. Compare to antibodies, aptamers have demonstrated advantages such as easy synthesis, low cost, and better stability. Therefore, aptamers can be integrated into various detection platforms ...

Effect of Sustained Maternal Responsivity on Later Vocabulary Development in Children with Fragile X Syndrome

ERIC Educational Resources Information Center

Brady, Nancy; Warren, Steven F.; Fleming, Kandace; Keller, Juliana; Sterling, Audra

2014-01-01

Purpose: This research explored whether sustained maternal responsivity (a parent-child interaction style characterized by warmth, nurturance, and stability as well as specific behaviors, such as contingent positive responses to child initiations) was a significant variable predicting vocabulary development of children with fragile X syndrome…

SR proteins in Vertical Integration of Gene Expression from Transcription to RNA Processing to Translation

PubMed Central

Zhong, Xiang-Yang; Wang, Pingping; Han, Joonhee; Rosenfeld, Michael G.; Fu, Xiang-Dong

2009-01-01

Summary SR proteins have been studied extensively as a family of RNA binding proteins that participate in both constitutive and regulated pre-mRNA splicing in mammalian cells. However, SR proteins were first discovered as factors that interact with transcriptionally active chromatin. Recent studies have now uncovered properties that connect these once apparently disparate functions, showing that a subset of SR proteins seem to bind directly to the histone 3 tail, play an active role in transcriptional elongation, and co-localize with genes that are engaged in specific intra- and inter-chromosome interactions for coordinated regulation of gene expression in the nucleus. These transcription-related activities are also coupled with a further expansion of putative functions of specific SR protein family members in RNA metabolism downstream of mRNA splicing, from RNA export to stability control to translation. These findings therefore highlight the broader roles of SR proteins in vertical integration of gene expression and provide mechanistic insights into their contributions to genome stability and proper cell cycle progression in higher eukaryotic cells. PMID:19595711

SR proteins in vertical integration of gene expression from transcription to RNA processing to translation.

PubMed

Zhong, Xiang-Yang; Wang, Pingping; Han, Joonhee; Rosenfeld, Michael G; Fu, Xiang-Dong

2009-07-10

SR proteins have been studied extensively as a family of RNA-binding proteins that participate in both constitutive and regulated pre-mRNA splicing in mammalian cells. However, SR proteins were first discovered as factors that interact with transcriptionally active chromatin. Recent studies have now uncovered properties that connect these once apparently disparate functions, showing that a subset of SR proteins seem to bind directly to the histone 3 tail, play an active role in transcriptional elongation, and colocalize with genes that are engaged in specific intra- and interchromosome interactions for coordinated regulation of gene expression in the nucleus. These transcription-related activities are also coupled with a further expansion of putative functions of specific SR protein family members in RNA metabolism downstream of mRNA splicing, from RNA export to stability control to translation. These findings, therefore, highlight the broader roles of SR proteins in vertical integration of gene expression and provide mechanistic insights into their contributions to genome stability and proper cell-cycle progression in higher eukaryotic cells.

Optimization-based controller design for rotorcraft

NASA Technical Reports Server (NTRS)

Tsing, N.-K.; Fan, M. K. H.; Barlow, J.; Tits, A. L.; Tischler, M. B.

1993-01-01

An optimization-based methodology for linear control system design is outlined by considering the design of a controller for a UH-60 rotorcraft in hover. A wide range of design specifications is taken into account: internal stability, decoupling between longitudinal and lateral motions, handling qualities, and rejection of windgusts. These specifications are investigated while taking into account physical limitations in the swashplate displacements and rates of displacement. The methodology crucially relies on user-machine interaction for tradeoff exploration.

Chromatic patchy particles: Effects of specific interactions on liquid structure

DOE PAGES

Vasilyev, Oleg A.; Tkachenko, Alexei V.; Klumov, Boris A.

2015-07-13

We study the structural and thermodynamic properties of patchy particle liquids, with a special focus on the role of “color,” i.e., specific interactions between individual patches. A possible experimental realization of such “chromatic” interactions is by decorating the particle patches with single-stranded DNA linkers. The complementarity of the linkers can promote selective bond formation between predetermined pairs of patches. By using MD simulations, we compare the local connectivity, the bond orientation order, and other structural properties of the aggregates formed by the “colored” and “colorless” systems. The analysis is done for spherical particles with two different patch arrangements (tetrahedral andmore » cubic). It is found that the aggregated (liquid) phase of the “colorless” patchy particles is better connected, denser and typically has stronger local order than the corresponding “colored” one. This, in turn, makes the colored liquid less stable thermodynamically. Specifically, we predict that in a typical case the chromatic interactions should increase the relative stability of the crystalline phase with respect to the disordered liquid, thus expanding its region in the phase diagram.« less

Stratifin regulates stabilization of receptor tyrosine kinases via interaction with ubiquitin-specific protease 8 in lung adenocarcinoma.

PubMed

Kim, Yunjung; Shiba-Ishii, Aya; Nakagawa, Tomoki; Iemura, Shun-Ichiro; Natsume, Tohru; Nakano, Noriyuki; Matsuoka, Ryota; Sakashita, Shingo; Lee, SangJoon; Kawaguchi, Atsushi; Sato, Yukio; Noguchi, Masayuki

2018-06-07

Previously we have reported that stratifin (SFN, 14-3-3 sigma) acts as a novel oncogene, accelerating the tumor initiation and progression of lung adenocarcinoma. Here, pull-down assay and LC-MS/MS analysis revealed that ubiquitin-specific protease 8 (USP8) specifically bound to SFN in lung adenocarcinoma cells. Both USP8 and SFN showed higher expression in human lung adenocarcinoma than in normal lung tissue, and USP8 expression was significantly correlated with SFN expression. Expression of SFN, but not of USP8, was associated with histological subtype, pathological stage, and poor prognosis. USP8 stabilizes receptor tyrosine kinases (RTKs) such as EGFR and MET by deubiquitination, contributing to the proliferative activity of many human cancers including non-small cell lung cancer. In vitro, USP8 binds to SFN and they co-localize at the early endosomes in lung adenocarcinoma cells. Moreover, USP8 or SFN knockdown leads to downregulation of tumor cellular proliferation and upregulation of apoptosis, p-EGFR or p-MET, which are related to the degradation pathway, and accumulation of ubiquitinated RTKs, leading to lysosomal degradation. Additionally, mutant USP8, which is unable to bind to SFN, reduces the expression of RTKs and p-STAT3. We also found that interaction with SFN is critical for USP8 to exert its autodeubiquitination function and avoid dephosphorylation by PP1. Our findings demonstrate that SFN enhances RTK stabilization through abnormal USP8 regulation in lung adenocarcinoma, suggesting that SFN could be a more suitable therapeutic target for lung adenocarcinoma than USP8.

Ready to rumble: how team personality composition and task conflict interact to improve performance.

PubMed

Bradley, Bret H; Klotz, Anthony C; Postlethwaite, Bennett E; Brown, Kenneth G

2013-03-01

Although prior work has proposed a number of conditions under which task conflict in teams may improve performance, composition variables have been left unexplored. Given the effects of personality traits on team processes and outcomes demonstrated in prior work, investigating whether specific personality compositions influence the effect of task conflict on team performance is critical to researchers' understanding of conflict in teams. Our results indicate that team-level averages of both openness to experience and emotional stability function as moderators of the relationship between task conflict and team performance. Specifically, task conflict had a positive impact on performance in teams with high levels of openness or emotional stability; in contrast, task conflict had a negative impact on performance in teams with low levels of openness or emotional stability. Thus, when task conflict emerges, teams composed of members who are open minded or emotionally stable are best able to leverage conflict to improve performance. Implications for theory and practice are discussed.

Neto-Mediated Intracellular Interactions Shape Postsynaptic Composition at the Drosophila Neuromuscular Junction

PubMed Central

Ramos, Cathy I.; Igiesuorobo, Oghomwen; Wang, Qi; Serpe, Mihaela

2015-01-01

The molecular mechanisms controlling the subunit composition of glutamate receptors are crucial for the formation of neural circuits and for the long-term plasticity underlying learning and memory. Here we use the Drosophila neuromuscular junction (NMJ) to examine how specific receptor subtypes are recruited and stabilized at synaptic locations. In flies, clustering of ionotropic glutamate receptors (iGluRs) requires Neto (Neuropillin and Tolloid-like), a highly conserved auxiliary subunit that is essential for NMJ assembly and development. Drosophila neto encodes two isoforms, Neto-α and Neto-β, with common extracellular parts and distinct cytoplasmic domains. Mutations that specifically eliminate Neto-β or its intracellular domain were generated. When Neto-β is missing or is truncated, the larval NMJs show profound changes in the subtype composition of iGluRs due to reduced synaptic accumulation of the GluRIIA subunit. Furthermore, neto-β mutant NMJs fail to accumulate p21-activated kinase (PAK), a critical postsynaptic component implicated in the synaptic stabilization of GluRIIA. Muscle expression of either Neto-α or Neto-β rescued the synaptic transmission at neto null NMJs, indicating that Neto conserved domains mediate iGluRs clustering. However, only Neto-β restored PAK synaptic accumulation at neto null NMJs. Thus, Neto engages in intracellular interactions that regulate the iGluR subtype composition by preferentially recruiting and/or stabilizing selective receptor subtypes. PMID:25905467

Human Sirtuin 2 Localization, Transient Interactions, and Impact on the Proteome Point to Its Role in Intracellular Trafficking.

PubMed

Budayeva, Hanna G; Cristea, Ileana M

2016-10-01

Human sirtuin 2 (SIRT2) is an NAD + -dependent deacetylase that primarily functions in the cytoplasm, where it can regulate α-tubulin acetylation levels. SIRT2 is linked to cancer progression, neurodegeneration, and infection with bacteria or viruses. However, the current knowledge about its interactions and the means through which it exerts its functions has remained limited. Here, we aimed to gain a better understanding of its cellular functions by characterizing SIRT2 subcellular localization, the identity and relative stability of its protein interactions, and its impact on the proteome of primary human fibroblasts. To assess the relative stability of SIRT2 interactions, we used immunoaffinity purification in conjunction with both label-free and metabolic labeling quantitative mass spectrometry. In addition to the expected associations with cytoskeleton proteins, including its known substrate TUBA1A, our results reveal that SIRT2 specifically interacts with proteins functioning in membrane trafficking, secretory processes, and transcriptional regulation. By quantifying their relative stability, we found most interactions to be transient, indicating a dynamic SIRT2 environment. We discover that SIRT2 localizes to the ER-Golgi intermediate compartment (ERGIC), and that this recruitment requires an intact ER-Golgi trafficking pathway. Further expanding these findings, we used microscopy and interaction assays to establish the interaction and coregulation of SIRT2 with liprin-β1 scaffolding protein (PPFiBP1), a protein with roles in focal adhesions disassembly. As SIRT2 functions may be accomplished via interactions, enzymatic activity, and transcriptional regulation, we next assessed the impact of SIRT2 levels on the cellular proteome. SIRT2 knockdown led to changes in the levels of proteins functioning in membrane trafficking, including some of its interaction partners. Altogether, our study expands the knowledge of SIRT2 cytoplasmic functions to define a previously unrecognized involvement in intracellular trafficking pathways, which may contribute to its roles in cellular homeostasis and human diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Human Sirtuin 2 Localization, Transient Interactions, and Impact on the Proteome Point to Its Role in Intracellular Trafficking*

PubMed Central

Budayeva, Hanna G.; Cristea, Ileana M.

2016-01-01

Human sirtuin 2 (SIRT2) is an NAD+-dependent deacetylase that primarily functions in the cytoplasm, where it can regulate α-tubulin acetylation levels. SIRT2 is linked to cancer progression, neurodegeneration, and infection with bacteria or viruses. However, the current knowledge about its interactions and the means through which it exerts its functions has remained limited. Here, we aimed to gain a better understanding of its cellular functions by characterizing SIRT2 subcellular localization, the identity and relative stability of its protein interactions, and its impact on the proteome of primary human fibroblasts. To assess the relative stability of SIRT2 interactions, we used immunoaffinity purification in conjunction with both label-free and metabolic labeling quantitative mass spectrometry. In addition to the expected associations with cytoskeleton proteins, including its known substrate TUBA1A, our results reveal that SIRT2 specifically interacts with proteins functioning in membrane trafficking, secretory processes, and transcriptional regulation. By quantifying their relative stability, we found most interactions to be transient, indicating a dynamic SIRT2 environment. We discover that SIRT2 localizes to the ER-Golgi intermediate compartment (ERGIC), and that this recruitment requires an intact ER-Golgi trafficking pathway. Further expanding these findings, we used microscopy and interaction assays to establish the interaction and coregulation of SIRT2 with liprin-β1 scaffolding protein (PPFiBP1), a protein with roles in focal adhesions disassembly. As SIRT2 functions may be accomplished via interactions, enzymatic activity, and transcriptional regulation, we next assessed the impact of SIRT2 levels on the cellular proteome. SIRT2 knockdown led to changes in the levels of proteins functioning in membrane trafficking, including some of its interaction partners. Altogether, our study expands the knowledge of SIRT2 cytoplasmic functions to define a previously unrecognized involvement in intracellular trafficking pathways, which may contribute to its roles in cellular homeostasis and human diseases. PMID:27503897

A dynamic model for generating actuator specifications for small arms barrel active stabilization

NASA Astrophysics Data System (ADS)

Pathak, Anupam; Brei, Diann; Luntz, Jonathan; Lavigna, Chris

2006-03-01

Due to stresses encountered in combat, it is known that soldier marksmanship noticeably decreases regardless of prior training. Active stabilization systems in small arms have potential to address this problem to increase soldier survivability and mission effectiveness. The key to success is proper actuator design, but this is highly dependent on proper specification which is challenging due to the human/weapon interaction. This paper presents a generic analytical dynamic model which is capable of defining the necessary actuation specifications for a wide range of small arms platforms. The model is unique because it captures the human interface--shoulder and arm--that introduces the jitter disturbance in addition to the geometry, inertial properties and active stabilization stiffness of the small arms platform. Because no data to date is available for actual shooter-induced disturbance in field conditions, a method is given using the model to back-solve from measured shooting range variability data the disturbance amplitude information relative to the input source (arm or shoulder). As examples of the applicability of the model to various small arms systems, two different weapon systems were investigated: the M24 sniper weapon and the M16 assault rifle. In both cases, model based simulations provided valuable insight into impact on the actuation specifications (force, displacement, phase, frequency) due to the interplay of the human-weapon-active stabilization interface including the effect of shooter-disturbance frequency, disturbance location (shoulder vs. arm), and system parameters (stiffness, barrel rotation).

The Regulatory Capacity of Bivalent Genes—A Theoretical Approach

PubMed Central

Thalheim, Torsten; Herberg, Maria; Loeffler, Markus; Galle, Joerg

2017-01-01

Bivalent genes are frequently associated with developmental and lineage specification processes. Resolving their bivalency enables fast changes in their expression, which potentially can trigger cell fate decisions. Here, we provide a theoretical model of bivalency that allows for predictions on the occurrence, stability and regulatory capacity of this prominent modification state. We suggest that bivalency enables balanced gene expression heterogeneity that constitutes a prerequisite of robust lineage priming in somatic stem cells. Moreover, we demonstrate that interactions between the histone and DNA methylation machineries together with the proliferation activity control the stability of the bivalent state and can turn it into an unmodified state. We suggest that deregulation of these interactions underlies cell transformation processes as associated with acute myeloid leukemia (AML) and provide a model of AML blast formation following deregulation of the Ten-eleven Translocation (TET) pathway. PMID:28513551

Investigating the association between social interactions and personality states dynamics

PubMed Central

Finnerty, Ailbhe N.; Staiano, Jacopo; Teso, Stefano; Passerini, Andrea; Pianesi, Fabio; Lepri, Bruno

2017-01-01

The recent personality psychology literature has coined the name of personality states to refer to states having the same behavioural, affective and cognitive content (described by adjectives) as the corresponding trait, but for a shorter duration. The variability in personality states may be the reaction to specific characteristics of situations. The aim of our study is to investigate whether specific situational factors, that is, different configurations of face-to-face interactions, are predictors of variability of personality states in a work environment. The obtained results provide evidence that within-person variability in personality is associated with variation in face-to-face interactions. Interestingly, the effects differ by type and level of the personality states: adaptation effects for Agreeableness and Emotional Stability, whereby the personality states of an individual trigger similar states in other people interacting with them and complementarity effects for Openness to Experience, whereby the personality states of an individual trigger opposite states in other people interacting with them. Overall, these findings encourage further research to characterize face-to-face and social interactions in terms of their relevance to personality states. PMID:28989732

Investigating the association between social interactions and personality states dynamics.

PubMed

Gundogdu, Didem; Finnerty, Ailbhe N; Staiano, Jacopo; Teso, Stefano; Passerini, Andrea; Pianesi, Fabio; Lepri, Bruno

2017-09-01

The recent personality psychology literature has coined the name of personality states to refer to states having the same behavioural, affective and cognitive content (described by adjectives) as the corresponding trait, but for a shorter duration. The variability in personality states may be the reaction to specific characteristics of situations. The aim of our study is to investigate whether specific situational factors, that is, different configurations of face-to-face interactions, are predictors of variability of personality states in a work environment. The obtained results provide evidence that within-person variability in personality is associated with variation in face-to-face interactions. Interestingly, the effects differ by type and level of the personality states: adaptation effects for Agreeableness and Emotional Stability, whereby the personality states of an individual trigger similar states in other people interacting with them and complementarity effects for Openness to Experience, whereby the personality states of an individual trigger opposite states in other people interacting with them. Overall, these findings encourage further research to characterize face-to-face and social interactions in terms of their relevance to personality states.

Assessing the utility of gene co-expression stability in combination with correlation in the analysis of protein-protein interaction networks

PubMed Central

2011-01-01

Background Gene co-expression, in the form of a correlation coefficient, has been valuable in the analysis, classification and prediction of protein-protein interactions. However, it is susceptible to bias from a few samples having a large effect on the correlation coefficient. Gene co-expression stability is a means of quantifying this bias, with high stability indicating robust, unbiased co-expression correlation coefficients. We assess the utility of gene co-expression stability as an additional measure to support the co-expression correlation in the analysis of protein-protein interaction networks. Results We studied the patterns of co-expression correlation and stability in interacting proteins with respect to their interaction promiscuity, levels of intrinsic disorder, and essentiality or disease-relatedness. Co-expression stability, along with co-expression correlation, acts as a better classifier of hub proteins in interaction networks, than co-expression correlation alone, enabling the identification of a class of hubs that are functionally distinct from the widely accepted transient (date) and obligate (party) hubs. Proteins with high levels of intrinsic disorder have low co-expression correlation and high stability with their interaction partners suggesting their involvement in transient interactions, except for a small group that have high co-expression correlation and are typically subunits of stable complexes. Similar behavior was seen for disease-related and essential genes. Interacting proteins that are both disordered have higher co-expression stability than ordered protein pairs. Using co-expression correlation and stability, we found that transient interactions are more likely to occur between an ordered and a disordered protein while obligate interactions primarily occur between proteins that are either both ordered, or disordered. Conclusions We observe that co-expression stability shows distinct patterns in structurally and functionally different groups of proteins and interactions. We conclude that it is a useful and important measure to be used in concert with gene co-expression correlation for further insights into the characteristics of proteins in the context of their interaction network. PMID:22369639

Revisiting Speech Rate and Utterance Length Manipulations in Stuttering Speakers

ERIC Educational Resources Information Center

Blomgren, Michael; Goberman, Alexander M.

2008-01-01

The goal of this study was to evaluate stuttering frequency across a multidimensional (2 x 2) hierarchy of speech performance tasks. Specifically, this study examined the interaction between changes in length of utterance and levels of speech rate stability. Forty-four adult male speakers participated in the study (22 stuttering speakers and 22…

Flexible DNA bending in HU–DNA cocrystal structures

PubMed Central

Swinger, Kerren K.; Lemberg, Kathryn M.; Zhang, Ying; Rice, Phoebe A.

2003-01-01

HU and IHF are members of a family of prokaryotic proteins that interact with the DNA minor groove in a sequence-specific (IHF) or non-specific (HU) manner to induce and/or stabilize DNA bending. HU plays architectural roles in replication initiation, transcription regulation and site-specific recombination, and is associated with bacterial nucleoids. Cocrystal structures of Anabaena HU bound to DNA (1P71, 1P78, 1P51) reveal that while underlying proline intercalation and asymmetric charge neutralization mechanisms of DNA bending are similar for IHF and HU, HU stabilizes different DNA bend angles (∼105–140°). The two bend angles within a single HU complex are not coplanar, and the resulting dihedral angle is consistent with negative supercoiling. Comparison of HU–DNA and IHF–DNA structures suggests that sharper bending is correlated with longer DNA binding sites and smaller dihedral angles. An HU-induced bend may be better modeled as a hinge, not a rigid bend. The ability to induce or stabilize varying bend angles is consistent with HU’s role as an architectural cofactor in many different systems that may require differing geometries. PMID:12853489

Distinct reaction pathway promoted by non-divalent-metal cations in a tertiary stabilized hammerhead ribozyme

PubMed Central

Roychowdhury-Saha, Manami; Burke, Donald H.

2007-01-01

Divalent ion sensitivity of hammerhead ribozymes is significantly reduced when the RNA structure includes appropriate tertiary stabilization. Therefore, we investigated the activity of the tertiary stabilized “RzB” hammerhead ribozyme in several nondivalent ions. Ribozyme RzB is active in spermidine and Na+ alone, although the cleavage rates are reduced by more than 1,000-fold relative to the rates observed in Mg2+ and in transition metal ions. The trivalent cobalt hexammine (CoHex) ion is often used as an exchange-inert analog of hydrated magnesium ion. Trans-cleavage rates exceeded 8 min−1 in 20 mM CoHex, which promoted cleavage through outersphere interactions. The stimulation of catalysis afforded by the tertiary structural interactions within RzB does not require Mg2+, unlike other extended hammerhead ribozymes. Site-specific interaction with at least one Mg2+ ion is suggested by CoHex competition experiments. In the presence of a constant, low concentration of Mg2+, low concentrations of CoHex decreased the rate by two to three orders of magnitude relative to the rate in Mg2+ alone. Cleavage rates increased as CoHex concentrations were raised further, but the final fraction cleaved was lower than what was observed in CoHex or Mg2+ alone. These observations suggest that Mg2+ and CoHex compete for binding and that they cause misfolded structures when they are together. The results of this study support the existence of an alternate catalytic mechanism used by nondivalent ions (especially CoHex) that is distinct from the one promoted by divalent metal ions, and they imply that divalent metals influence catalysis through a specific nonstructural role. PMID:17456566

Combined Photoemission Spectroscopy and Electrochemical Study of a Mixture of (Oxy)carbides as Potential Innovative Supports and Electrocatalysts.

PubMed

Calvillo, Laura; Valero-Vidal, Carlos; Agnoli, Stefano; Sezen, Hikmet; Rüdiger, Celine; Kunze-Liebhäuser, Julia; Granozzi, Gaetano

2016-08-03

Active and stable non-noble metal materials, able to substitute Pt as catalyst or to reduce the Pt amount, are vitally important for the extended commercialization of energy conversion technologies, such as fuel cells and electrolyzers. Here, we report a fundamental study of nonstoichiometric tungsten carbide (WxC) and its interaction with titanium oxycarbide (TiOxCy) under electrochemical working conditions. In particular, the electrochemical activity and stability of the WxC/TiOxCy system toward the ethanol electrooxidation reaction (EOR) and hydrogen evolution reaction (HER) are investigated. The chemical changes caused by the applied potential are established by combining photoemission spectroscopy and electrochemistry. WxC is not active toward the ethanol electrooxidation reaction at room temperature but it is highly stable under these conditions thanks to the formation of a passive thin film on the surface, consisting mainly of WO2 and W2O5, which prevents the full oxidation of WxC. In addition, WxC is able to adsorb ethanol, forming ethoxy groups on the surface, which constitutes the first step for the ethanol oxidation. The interaction between WxC and TiOxCy plays an important role in the electrochemical stability of WxC since specific orientations of the substrate are able to stabilize WxC and prevent its corrosion. The beneficial interaction with the substrate and the specific surface chemistry makes tungsten carbide a good electrocatalyst support or cocatalyst for direct ethanol fuel cells. However, WxC is active toward the HER and chemically stable under hydrogen reduction conditions, since no changes in the chemical composition or dissolution of the film are observed. This makes tungsten carbide a good candidate as electrocatalyst support or cocatalyst for the electrochemical production of hydrogen.

Spectroscopic investigations of novel pharmaceuticals: Stability and resonant interaction with laser beam

NASA Astrophysics Data System (ADS)

Smarandache, Adriana; Boni, Mihai; Andrei, Ionut Relu; Handzlik, Jadwiga; Kiec-Kononowicz, Katarzyna; Staicu, Angela; Pascu, Mihail-Lucian

2017-09-01

This paper presents data about photophysics of two novel thio-hydantoins that exhibit promising pharmaceutical properties in multidrug resistance control. Time stability studies are necessary to establish the proper use of these compounds in different applications. As for their administration as drugs, it is imperative to know their shelf life, as well as storage conditions. At the same time, laser induced modified properties of the two new compounds are valuable to further investigate their specific interactions with other materials, including biological targets. The two new thio-hydantoins under generic names SZ-2 and SZ-7 were prepared as solutions in dimethyl sulfoxide at different concentrations, as well as in deionised water. For the stability assay they were kept in various light/temperature conditions up to 60 days. The stability was estimates based on UV-vis absorption measurements. The samples in bulk shape were exposed different time intervals to laser radiation emitted at 266 nm as the fourth harmonic of a Nd:YAG laser. The resonant interaction of the studied compounds with laser beams was analysed through spectroscopic methods UV-vis and FTIR absorption, as well as laser induced fluorescence spectroscopy. As for stability assay, only solutions kept in dark at 4 °C have preserved the absorption characteristics, considering the cumulated measuring errors, less than one week. The vibrational changes that occur in their FTIR and modified fluorescence spectra upon laser beam exposure are also discussed. A result of the experimental analysis is that modifications are induced in molecular structures of the investigated compounds by resonant interaction with laser radiation. This fact evidences that the molecules are photoreactive and their characteristics might be shaped through controlled laser radiation exposure using appropriate protocols. This conclusion opens many opportunities both in the biomedical field, but also in other industrial activities involving the use of hydantoins.

Transfer of dipolar gas through the discrete localized mode.

PubMed

Bai, Xiao-Dong; Zhang, Ai-Xia; Xue, Ju-Kui

2013-12-01

By considering the discrete nonlinear Schrödinger model with dipole-dipole interactions for dipolar condensate, the existence, the types, the stability, and the dynamics of the localized modes in a nonlinear lattice are discussed. It is found that the contact interaction and the dipole-dipole interactions play important roles in determining the existence, the type, and the stability of the localized modes. Because of the coupled effects of the contact interaction and the dipole-dipole interactions, rich localized modes and their stability nature can exist: when the contact interaction is larger and the dipole-dipole interactions is smaller, a discrete bright breather occurs. In this case, while the on-site interaction can stabilize the discrete breather, the dipole-dipole interactions will destabilize the discrete breather; when both the contact interaction and the dipole-dipole interactions are larger, a discrete kink appears. In this case, both the on-site interaction and the dipole-dipole interactions can stabilize the discrete kink, but the discrete kink is more unstable than the ordinary discrete breather. The predicted results provide a deep insight into the dynamics of blocking, filtering, and transfer of the norm in nonlinear lattices for dipolar condensates.

Non-catalytic Roles for XPG with BRCA1 and BRCA2 in Homologous Recombination and Genome Stability

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

Trego, Kelly S.; Groesser, Torsten; Davalos, Albert R.

XPG is a structure-specific endonuclease required for nucleotide excision repair, and incision-defective XPG mutations cause the skin cancer-prone syndrome xeroderma pigmentosum. Truncating mutations instead cause the neurodevelopmental progeroid disorder Cockayne syndrome, but little is known about how XPG loss results in this devastating disease. In this paper, we identify XPG as a partner of BRCA1 and BRCA2 in maintaining genomic stability through homologous recombination (HRR). XPG depletion causes DNA double-strand breaks, chromosomal abnormalities, cell-cycle delays, defective HRR, inability to overcome replication fork stalling, and replication stress. XPG directly interacts with BRCA2, RAD51, and PALB2, and XPG depletion reduces their chromatinmore » binding and subsequent RAD51 foci formation. Upstream in HRR, XPG interacts directly with BRCA1. Its depletion causes BRCA1 hyper-phosphorylation and persistent chromatin binding. Finally, these unexpected findings establish XPG as an HRR protein with important roles in genome stability and suggest how XPG defects produce severe clinical consequences including cancer and accelerated aging.« less

Non-catalytic Roles for XPG with BRCA1 and BRCA2 in Homologous Recombination and Genome Stability

DOE PAGES

Trego, Kelly S.; Groesser, Torsten; Davalos, Albert R.; ...

2016-01-28

XPG is a structure-specific endonuclease required for nucleotide excision repair, and incision-defective XPG mutations cause the skin cancer-prone syndrome xeroderma pigmentosum. Truncating mutations instead cause the neurodevelopmental progeroid disorder Cockayne syndrome, but little is known about how XPG loss results in this devastating disease. In this paper, we identify XPG as a partner of BRCA1 and BRCA2 in maintaining genomic stability through homologous recombination (HRR). XPG depletion causes DNA double-strand breaks, chromosomal abnormalities, cell-cycle delays, defective HRR, inability to overcome replication fork stalling, and replication stress. XPG directly interacts with BRCA2, RAD51, and PALB2, and XPG depletion reduces their chromatinmore » binding and subsequent RAD51 foci formation. Upstream in HRR, XPG interacts directly with BRCA1. Its depletion causes BRCA1 hyper-phosphorylation and persistent chromatin binding. Finally, these unexpected findings establish XPG as an HRR protein with important roles in genome stability and suggest how XPG defects produce severe clinical consequences including cancer and accelerated aging.« less

Drastic stabilization of parallel DNA hybridizations by a polylysine comb-type copolymer with hydrophilic graft chain.

PubMed

Miyoshi, Daisuke; Ueda, Yu-Mi; Shimada, Naohiko; Nakano, Shu-Ichi; Sugimoto, Naoki; Maruyama, Atsushi

2014-09-01

Electrostatic interactions play a major role in protein-DNA interactions. As a model system of a cationic protein, herein we focused on a comb-type copolymer of a polycation backbone and dextran side chains, poly(L-lysine)-graft-dextran (PLL-g-Dex), which has been reported to form soluble interpolyelectrolyte complexes with DNA strands. We investigated the effects of PLL-g-Dex on the conformation and thermodynamics of DNA oligonucleotides forming various secondary structures. Thermodynamic analysis of the DNA structures showed that the parallel conformations involved in both DNA duplexes and triplexes were significantly and specifically stabilized by PLL-g-Dex. On the basis of thermodynamic parameters, it was further possible to design DNA switches that undergo structural transition responding to PLL-g-Dex from an antiparallel duplex to a parallel triplex even with mismatches in the third strand hybridization. These results suggest that polycationic molecules are able to induce structural polymorphism of DNA oligonucleotides, because of the conformation-selective stabilization effects. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme

DOE PAGES

Lee, Hui-Ting; Kilburn, D.; Behrouzi, R.; ...

2014-12-05

The native structure of the Azoarcus group I ribozyme is stabilized by the cooperative formation of tertiary interactions between double helical domains. Thus, even single mutations that break this network of tertiary interactions reduce ribozyme activity in physiological Mg2+ concentrations. Here, we report that molecular crowding comparable to that in the cell compensates for destabilizing mutations in the Azoarcus ribozyme. Small angle X-ray scattering, native polyacrylamide gel electrophoresis and activity assays were used to compare folding free energies in dilute and crowded solutions containing 18% PEG1000. Crowder molecules allowed the wild-type and mutant ribozymes to fold at similarly low Mg2+more » concentrations and stabilized the active structure of the mutant ribozymes under physiological conditions. This compensation helps explains why ribozyme mutations are often less deleterious in the cell than in the test tube. Nevertheless, crowding did not rescue the high fraction of folded but less active structures formed by double and triple mutants. We conclude that crowding broadens the fitness landscape by stabilizing compact RNA structures without improving the specificity of self-assembly.« less

Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme

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

Lee, Hui-Ting; Kilburn, D.; Behrouzi, R.

The native structure of the Azoarcus group I ribozyme is stabilized by the cooperative formation of tertiary interactions between double helical domains. Thus, even single mutations that break this network of tertiary interactions reduce ribozyme activity in physiological Mg2+ concentrations. Here, we report that molecular crowding comparable to that in the cell compensates for destabilizing mutations in the Azoarcus ribozyme. Small angle X-ray scattering, native polyacrylamide gel electrophoresis and activity assays were used to compare folding free energies in dilute and crowded solutions containing 18% PEG1000. Crowder molecules allowed the wild-type and mutant ribozymes to fold at similarly low Mg2+more » concentrations and stabilized the active structure of the mutant ribozymes under physiological conditions. This compensation helps explains why ribozyme mutations are often less deleterious in the cell than in the test tube. Nevertheless, crowding did not rescue the high fraction of folded but less active structures formed by double and triple mutants. We conclude that crowding broadens the fitness landscape by stabilizing compact RNA structures without improving the specificity of self-assembly.« less

De novo self-assembling collagen heterotrimers using explicit positive and negative design.

PubMed

Xu, Fei; Zhang, Lei; Koder, Ronald L; Nanda, Vikas

2010-03-23

We sought to computationally design model collagen peptides that specifically associate as heterotrimers. Computational design has been successfully applied to the creation of new protein folds and functions. Despite the high abundance of collagen and its key role in numerous biological processes, fibrous proteins have received little attention as computational design targets. Collagens are composed of three polypeptide chains that wind into triple helices. We developed a discrete computational model to design heterotrimer-forming collagen-like peptides. Stability and specificity of oligomerization were concurrently targeted using a combined positive and negative design approach. The sequences of three 30-residue peptides, A, B, and C, were optimized to favor charge-pair interactions in an ABC heterotrimer, while disfavoring the 26 competing oligomers (i.e., AAA, ABB, BCA). Peptides were synthesized and characterized for thermal stability and triple-helical structure by circular dichroism and NMR. A unique A:B:C-type species was not achieved. Negative design was partially successful, with only A + B and B + C competing mixtures formed. Analysis of computed versus experimental stabilities helps to clarify the role of electrostatics and secondary-structure propensities determining collagen stability and to provide important insight into how subsequent designs can be improved.

Terminal Duplex Stability and Nucleotide Identity Differentially Control siRNA Loading and Activity in RNA Interference

PubMed Central

Angart, Phillip A.; Carlson, Rebecca J.; Adu-Berchie, Kwasi

2016-01-01

Efficient short interfering RNA (siRNA)-mediated gene silencing requires selection of a sequence that is complementary to the intended target and possesses sequence and structural features that encourage favorable functional interactions with the RNA interference (RNAi) pathway proteins. In this study, we investigated how terminal sequence and structural characteristics of siRNAs contribute to siRNA strand loading and silencing activity and how these characteristics ultimately result in a functionally asymmetric duplex in cultured HeLa cells. Our results reiterate that the most important characteristic in determining siRNA activity is the 5′ terminal nucleotide identity. Our findings further suggest that siRNA loading is controlled principally by the hybridization stability of the 5′ terminus (Nucleotides: 1–2) of each siRNA strand, independent of the opposing terminus. Postloading, RNA-induced silencing complex (RISC)–specific activity was found to be improved by lower hybridization stability in the 5′ terminus (Nucleotides: 3–4) of the loaded siRNA strand and greater hybridization stability toward the 3′ terminus (Nucleotides: 17–18). Concomitantly, specific recognition of the 5′ terminal nucleotide sequence by human Argonaute 2 (Ago2) improves RISC half-life. These findings indicate that careful selection of siRNA sequences can maximize both the loading and the specific activity of the intended guide strand. PMID:27399870

The molecular mechanism of nuclear transport revealed by atomic-scale measurements

PubMed Central

Hough, Loren E; Dutta, Kaushik; Sparks, Samuel; Temel, Deniz B; Kamal, Alia; Tetenbaum-Novatt, Jaclyn; Rout, Michael P; Cowburn, David

2015-01-01

Nuclear pore complexes (NPCs) form a selective filter that allows the rapid passage of transport factors (TFs) and their cargoes across the nuclear envelope, while blocking the passage of other macromolecules. Intrinsically disordered proteins (IDPs) containing phenylalanyl-glycyl (FG)-rich repeats line the pore and interact with TFs. However, the reason that transport can be both fast and specific remains undetermined, through lack of atomic-scale information on the behavior of FGs and their interaction with TFs. We used nuclear magnetic resonance spectroscopy to address these issues. We show that FG repeats are highly dynamic IDPs, stabilized by the cellular environment. Fast transport of TFs is supported because the rapid motion of FG motifs allows them to exchange on and off TFs extremely quickly through transient interactions. Because TFs uniquely carry multiple pockets for FG repeats, only they can form the many frequent interactions needed for specific passage between FG repeats to cross the NPC. DOI: http://dx.doi.org/10.7554/eLife.10027.001 PMID:26371551

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells.

PubMed

Kresoja-Rakic, Jelena; Felley-Bosco, Emanuela

2018-04-25

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA structures and motifs. In this RNA-pulldown protocol, commercially synthesized RNA probes are labeled with a modified form of biotin, desthiobiotin, at the 3' terminus of the RNA strand, which reversibly binds to streptavidin and thus allows elution of proteins under more physiological conditions. The RNA-desthiobiotin is immobilized through interaction with streptavidin on magnetic beads, which are used to pull down proteins that specifically interact with the RNA of interest. Non-denatured and active proteins from the cytosolic fraction of mesothelioma cells are used as the source of proteins. The method described here can be applied to detect the interaction between known RNA binding proteins and a 25-nucleotide (nt) long RNA probe containing a sequence of interest. This is useful to complete the functional characterization of stabilizing or destabilizing elements present in RNA molecules achieved using a reporter vector assay.

Therapeutic peptides for cancer therapy. Part I - peptide inhibitors of signal transduction cascades.

PubMed

Bidwell, Gene L; Raucher, Drazen

2009-10-01

Therapeutic peptides have great potential as anticancer agents owing to their ease of rational design and target specificity. However, their utility in vivo is limited by low stability and poor tumor penetration. The authors review the development of peptide inhibitors with potential for cancer therapy. Peptides that inhibit signal transduction cascades are discussed. The authors searched Medline for articles concerning the development of therapeutic peptides and their delivery. Given our current knowledge of protein sequences, structures and interaction interfaces, therapeutic peptides that inhibit interactions of interest are easily designed. These peptides are advantageous because they are highly specific for the interaction of interest, and they are much more easily developed than small molecule inhibitors of the same interactions. The main hurdle to application of peptides for cancer therapy is their poor pharmacokinetic and biodistribution parameters. Therefore, successful development of peptide delivery vectors could potentially make possible the use of this new and very promising class of anticancer agents.

Crystal nucleation initiated by transient ion-surface interactions at aerosol interfaces

PubMed Central

Davis, Ryan D.; Tolbert, Margaret A.

2017-01-01

Particle collisions are a common occurrence in the atmosphere, but no empirical observations exist to fully predict the potential effects of these collisions on air quality and climate projections. The current consensus of heterogeneous crystal nucleation pathways relevant to the atmosphere dictates that collisions with amorphous particles have no effect on the crystallization relative humidity (RH) of aqueous inorganic aerosols because there is no stabilizing ion-surface interaction to facilitate the formation of crystal nuclei. In contrast to this view of heterogeneous nucleation, we report laboratory observations demonstrating that collisions with hydrophobic amorphous organic aerosols induced crystallization of aqueous inorganic microdroplets at high RH, the effect of which was correlated with destabilizing water-mediated ion-specific surface interactions. These same organic aerosols did not induce crystallization once internally mixed in the droplet, pointing toward a previously unconsidered transient ion-specific crystal nucleation pathway that can promote aerosol crystallization via particle collisions. PMID:28776032

Crystal nucleation initiated by transient ion-surface interactions at aerosol interfaces.

PubMed

Davis, Ryan D; Tolbert, Margaret A

2017-07-01

Particle collisions are a common occurrence in the atmosphere, but no empirical observations exist to fully predict the potential effects of these collisions on air quality and climate projections. The current consensus of heterogeneous crystal nucleation pathways relevant to the atmosphere dictates that collisions with amorphous particles have no effect on the crystallization relative humidity (RH) of aqueous inorganic aerosols because there is no stabilizing ion-surface interaction to facilitate the formation of crystal nuclei. In contrast to this view of heterogeneous nucleation, we report laboratory observations demonstrating that collisions with hydrophobic amorphous organic aerosols induced crystallization of aqueous inorganic microdroplets at high RH, the effect of which was correlated with destabilizing water-mediated ion-specific surface interactions. These same organic aerosols did not induce crystallization once internally mixed in the droplet, pointing toward a previously unconsidered transient ion-specific crystal nucleation pathway that can promote aerosol crystallization via particle collisions.

Asymmetrically localized proteins stabilize basal bodies against ciliary beating forces

PubMed Central

Galati, Domenico F.

2016-01-01

Basal bodies are radially symmetric, microtubule-rich structures that nucleate and anchor motile cilia. Ciliary beating produces asymmetric mechanical forces that are resisted by basal bodies. To resist these forces, distinct regions within the basal body ultrastructure and the microtubules themselves must be stable. However, the molecular components that stabilize basal bodies remain poorly defined. Here, we determine that Fop1 functionally interacts with the established basal body stability components Bld10 and Poc1. We find that Fop1 and microtubule glutamylation incorporate into basal bodies at distinct stages of assembly, culminating in their asymmetric enrichment at specific triplet microtubule regions that are predicted to experience the greatest mechanical force from ciliary beating. Both Fop1 and microtubule glutamylation are required to stabilize basal bodies against ciliary beating forces. Our studies reveal that microtubule glutamylation and Bld10, Poc1, and Fop1 stabilize basal bodies against the forces produced by ciliary beating via distinct yet interdependent mechanisms. PMID:27807131

USP22 acts as an oncogene by regulating the stability of cyclooxygenase-2 in non-small cell lung cancer

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

Xiao, Haibo; Tian, Yue; Yang, Yang

2015-05-08

The histone ubiquitin hydrolase ubiquitin-specific protease 22 (USP22) is an epigenetic modifier and an oncogene that is upregulated in many types of cancer. In non-small cell lung cancer (NSCLC), aberrant expression of USP22 is a predictor of poor survival, as is high expression of cyclooxygenase-2 (COX-2). Despite its oncogenic role, few substrates of USP22 have been identified and its mechanism of action in cancer remains unclear. Here, we identified COX-2 as a direct substrate of USP22 and showed that its levels are modulated by USP22 mediated deubiquitination. Silencing of USP22 downregulated COX-2, decreased its half-life, and inhibited lung carcinoma cellmore » proliferation by directly interacting with and modulating the stability and activity of COX-2 through the regulation of its ubiquitination status. The findings of the present study suggest a potential mechanism underlying the oncogenic role of USP22 mediated by the modulation of the stability and activity of COX-2. - Highlights: • USP22 interacts with COX-2. • USP22 deubiquitinates and stabilizes COX-2. • USP22 is required for COX-2-mediated upregulation of prostaglandin E2.« less

Stability of local secondary structure determines selectivity of viral RNA chaperones.

PubMed

Bravo, Jack P K; Borodavka, Alexander; Barth, Anders; Calabrese, Antonio N; Mojzes, Peter; Cockburn, Joseph J B; Lamb, Don C; Tuma, Roman

2018-05-18

To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA-RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus (RV) NSP2 and avian reovirus σNS. Here, we compared the abilities of NSP2 and σNS to mediate sequence-specific interactions between RV genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while σNS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric σNS oligomerizes into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA-RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.

Secondary structure encodes a cooperative tertiary folding funnel in the Azoarcus ribozyme

PubMed Central

Mustoe, Anthony M.; Al-Hashimi, Hashim M.; Brooks, Charles L.

2016-01-01

A requirement for specific RNA folding is that the free-energy landscape discriminate against non-native folds. While tertiary interactions are critical for stabilizing the native fold, they are relatively non-specific, suggesting additional mechanisms contribute to tertiary folding specificity. In this study, we use coarse-grained molecular dynamics simulations to explore how secondary structure shapes the tertiary free-energy landscape of the Azoarcus ribozyme. We show that steric and connectivity constraints posed by secondary structure strongly limit the accessible conformational space of the ribozyme, and that these so-called topological constraints in turn pose strong free-energy penalties on forming different tertiary contacts. Notably, native A-minor and base-triple interactions form with low conformational free energy, while non-native tetraloop/tetraloop–receptor interactions are penalized by high conformational free energies. Topological constraints also give rise to strong cooperativity between distal tertiary interactions, quantitatively matching prior experimental measurements. The specificity of the folding landscape is further enhanced as tertiary contacts place additional constraints on the conformational space, progressively funneling the molecule to the native state. These results indicate that secondary structure assists the ribozyme in navigating the otherwise rugged tertiary folding landscape, and further emphasize topological constraints as a key force in RNA folding. PMID:26481360

Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation

PubMed Central

Taylor, James A.; Pastrana, Cesar L.; Butterer, Annika; Pernstich, Christian; Gwynn, Emma J.; Sobott, Frank; Moreno-Herrero, Fernando; Dillingham, Mark S.

2015-01-01

The segregation of many bacterial chromosomes is dependent on the interactions of ParB proteins with centromere-like DNA sequences called parS that are located close to the origin of replication. In this work, we have investigated the binding of Bacillus subtilis ParB to DNA in vitro using a variety of biochemical and biophysical techniques. We observe tight and specific binding of a ParB homodimer to the parS sequence. Binding of ParB to non-specific DNA is more complex and displays apparent positive co-operativity that is associated with the formation of larger, poorly defined, nucleoprotein complexes. Experiments with magnetic tweezers demonstrate that non-specific binding leads to DNA condensation that is reversible by protein unbinding or force. The condensed DNA structure is not well ordered and we infer that it is formed by many looping interactions between neighbouring DNA segments. Consistent with this view, ParB is also able to stabilize writhe in single supercoiled DNA molecules and to bridge segments from two different DNA molecules in trans. The experiments provide no evidence for the promotion of non-specific DNA binding and/or condensation events by the presence of parS sequences. The implications of these observations for chromosome segregation are discussed. PMID:25572315

Observational signatures of neutron stars in low-mass X-ray binaries climbing a stability peak

NASA Astrophysics Data System (ADS)

Kantor, E. M.; Gusakov, M. E.; Chugunov, A. I.

2016-01-01

In the recent papers by Gusakov et al., a new scenario describing evolution of rapidly rotating neutron stars (NSs) in low-mass X-ray binaries was proposed. The scenario accounts for a resonant interaction of normal r-modes with superfluid inertial modes at some specific internal stellar temperatures (`resonance temperatures'). This interaction results in an enhanced damping of r-mode and appearance of the `stability peaks' in the temperature - spin frequency plane, which split the r-mode instability window in the vicinity of the resonance temperatures. The scenario suggests that the hot and rapidly rotating NSs spend most of their life climbing up these peaks and, in particular, are observed there at the moment. We analyse in detail possible observational signatures of this suggestion. In particular, we show that these objects may exhibit `anti-glitches' - sudden frequency jumps on a time-scale of hours-months.

Orbiting pairs of walking droplets: Dynamics and stability

NASA Astrophysics Data System (ADS)

Oza, Anand U.; Siéfert, Emmanuel; Harris, Daniel M.; Moláček, Jan; Bush, John W. M.

2017-05-01

A decade ago, Couder and Fort [Phys. Rev. Lett. 97, 154101 (2006)], 10.1103/PhysRevLett.97.154101 discovered that a millimetric droplet sustained on the surface of a vibrating fluid bath may self-propel through a resonant interaction with its own wave field. We here present the results of a combined experimental and theoretical investigation of the interactions of such walking droplets. Specifically, we delimit experimentally the different regimes for an orbiting pair of identical walkers and extend the theoretical model of Oza et al. [J. Fluid Mech. 737, 552 (2013)], 10.1017/jfm.2013.581 in order to rationalize our observations. A quantitative comparison between experiment and theory highlights the importance of spatial damping of the wave field. Our results also indicate that walkers adapt their impact phase according to the local wave height, an effect that stabilizes orbiting bound states.

PML IV/ARF interaction enhances p53 SUMO-1 conjugation, activation, and senescence

PubMed Central

Ivanschitz, Lisa; Takahashi, Yuki; Jollivet, Florence; Ayrault, Olivier; Le Bras, Morgane; de Thé, Hugues

2015-01-01

Promyelocytic leukemia protein (PML) nuclear bodies (NBs) recruit multiple partners, including p53 and many of its regulators. NBs are believed to facilitate several posttranslational modifications and are key regulators of senescence. PML, the organizer of NBs, is expressed as a number of splice variants that all efficiently recruit p53 partners. However, overexpression of only one of them, PML IV, triggers p53-driven senescence. Here, we show that PML IV specifically binds ARF, a key p53 regulator. Similar to ARF, PML IV enhances global SUMO-1 conjugation, particularly that of p53, resulting in p53 stabilization and activation. ARF interacts with and stabilizes the NB-associated UBC9 SUMO-conjugating enzyme, possibly explaining PML IV-enhanced SUMOylation. These results unexpectedly link two key tumor suppressors, highlighting their convergence for global control of SUMO conjugation, p53 activation, and senescence induction. PMID:26578773

PML IV/ARF interaction enhances p53 SUMO-1 conjugation, activation, and senescence.

PubMed

Ivanschitz, Lisa; Takahashi, Yuki; Jollivet, Florence; Ayrault, Olivier; Le Bras, Morgane; de Thé, Hugues

2015-11-17

Promyelocytic leukemia protein (PML) nuclear bodies (NBs) recruit multiple partners, including p53 and many of its regulators. NBs are believed to facilitate several posttranslational modifications and are key regulators of senescence. PML, the organizer of NBs, is expressed as a number of splice variants that all efficiently recruit p53 partners. However, overexpression of only one of them, PML IV, triggers p53-driven senescence. Here, we show that PML IV specifically binds ARF, a key p53 regulator. Similar to ARF, PML IV enhances global SUMO-1 conjugation, particularly that of p53, resulting in p53 stabilization and activation. ARF interacts with and stabilizes the NB-associated UBC9 SUMO-conjugating enzyme, possibly explaining PML IV-enhanced SUMOylation. These results unexpectedly link two key tumor suppressors, highlighting their convergence for global control of SUMO conjugation, p53 activation, and senescence induction.

Mathematical Models of Continuous Flow Electrophoresis

NASA Technical Reports Server (NTRS)

Saville, D. A.; Snyder, R. S.

1985-01-01

Development of high resolution continuous flow electrophoresis devices ultimately requires comprehensive understanding of the ways various phenomena and processes facilitate or hinder separation. A comprehensive model of the actual three dimensional flow, temperature and electric fields was developed to provide guidance in the design of electrophoresis chambers for specific tasks and means of interpreting test data on a given chamber. Part of the process of model development includes experimental and theoretical studies of hydrodynamic stability. This is necessary to understand the origin of mixing flows observed with wide gap gravitational effects. To insure that the model accurately reflects the flow field and particle motion requires extensive experimental work. Another part of the investigation is concerned with the behavior of concentrated sample suspensions with regard to sample stream stability particle-particle interactions which might affect separation in an electric field, especially at high field strengths. Mathematical models will be developed and tested to establish the roles of the various interactions.

Binding the diproton in stars: anthropic limits on the strength of gravity

NASA Astrophysics Data System (ADS)

Barnes, Luke A.

2015-12-01

We calculate the properties and investigate the stability of stars that burn via strong (and electromagnetic) interactions, and compare their properties with those that, as in our Universe, include a rate-limiting weak interaction. It has been suggested that, if the diproton were bound, stars would burn ~1018 times brighter and faster via strong interactions, resulting in a universe that would fail to support life. By considering the representative case of a star in our Universe with initially equal numbers of protons and deuterons, we find that stable, "strong-burning" stars adjust their central densities and temperatures to have familiar surface temperatures, luminosities and lifetimes. There is no "diproton disaster". In addition, strong-burning stars are stable in a much larger region of the parameter space of fundamental constants, specifically the strength of electromagnetism and gravity. The strongest anthropic bound on stars in such universes is not their stability, as is the case for stars limited by the weak interaction, but rather their lifetime. Regardless of the strength of electromagnetism, all stars burn out in mere millions of years unless the gravitational coupling constant is extremely small, αGlesssim 10-30.

Construction and Self-Assembly of Single-Chain Polymer Nanoparticles via Coordination Association and Electrostatic Repulsion in Water.

PubMed

Zhu, Zhengguang; Xu, Na; Yu, Qiuping; Guo, Lei; Cao, Hui; Lu, Xinhua; Cai, Yuanli

2015-08-01

Simultaneous coordination-association and electrostatic-repulsion interactions play critical roles in the construction and stabilization of enzymatic function metal centers in water media. These interactions are promising for construction and self-assembly of artificial aqueous polymer single-chain nanoparticles (SCNPs). Herein, the construction and self-assembly of dative-bonded aqueous SCNPs are reported via simultaneous coordination-association and electrostatic-repulsion interactions within single chains of histamine-based hydrophilic block copolymer. The electrostatic-repulsion interactions are tunable through adjusting the imidazolium/imidazole ratio in response to pH, and in situ Cu(II)-coordination leads to the intramolecular association and single-chain collapse in acidic water. SCNPs are stabilized by the electrostatic repulsion of dative-bonded block and steric shielding of nonionic water-soluble block, and have a huge specific surface area of function metal centers accessible to substrates in acidic water. Moreover, SCNPs can assemble into micelles, networks, and large particles programmably in response to the solution pH. These unique media-sensitive phase-transformation behaviors provide a general, facile, and versatile platform for the fabrication of enzyme-inspired smart aqueous catalysts. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of Type Three Secretion System Translocator Interactions with Phospholipid Membranes.

PubMed

Adam, Philip R; Barta, Michael L; Dickenson, Nicholas E

2017-01-01

In vitro characterization of type III secretion system (T3SS) translocator proteins has proven challenging due to complex purification schemes and their hydrophobic nature that often requires detergents to provide protein solubility and stability. Here, we provide experimental details for several techniques that overcome these hurdles, allowing for the direct characterization of the Shigella translocator protein IpaB with respect to phospholipid membrane interaction. The techniques specifically discussed in this chapter include membrane interaction/liposome flotation, liposome sensitive fluorescence quenching, and protein-mediated liposome disruption assays. These assays have provided valuable insight into the role of IpaB in T3SS-mediated phospholipid membrane interactions by Shigella and should readily extend to other members of this important class of proteins.

Structure-Function Based Molecular Relationships in Ewing's Sarcoma

PubMed Central

2015-01-01

Ewing's Sarcoma Oncogene (ews) on chromosome 22q12 is encoding a ubiquitously expressed RNA-binding protein (EWS) with unknown function that is target of tumor-specific chromosomal translocations in Ewing's sarcoma family of tumors. A model of transcription complex was proposed in which the heterodimer Rpb4/7 binds to EAD, connecting it to Core RNA Pol II. The DNA-binding domain, provided by EFP, is bound to the promoter. Rpb4/7 binds RNA, stabilizing the transcription complex. The complex Rpb4/7 can stabilize the preinitiation complexes by converting the conformation of RNA Pol II. EWS may change its conformation, so that NTD becomes accessible. Two different mechanisms of interaction between EWS and RNA Pol II are proposed: (I) an intermolecular EWS-EWS interaction between two molecules, pushing conformation from “closed” to “open” state, or (II) an intramolecular interaction inside the molecule of EWS, pushing conformation of the molecule from “closed” to “open” state. The modified forms of EWS may interact with Pol II subunits hsRpb5 and hsRpb7. The EWS and EFPs binding partners are described schematically in a model, an attempt to link the transcription with the splicing. The proposed model helps to understand the functional molecular interactions in cancer, to find new partners and ways to treat cancer. PMID:25688366

Protocol, pattern and paper: interactive stabilization of immunohistochemical knowledge.

PubMed

Nederbragt, Hubertus

2010-12-01

This paper analyzes the investigation of the distribution of the protein tenascin-C in canine mammary tumors. The method involved immunohistochemistry of tissue slices, performed by the application of an antibody to tenascin-C that specifically can be made visible for microscopic inspection. The first phase of the project is the making of the protocol, the second the deduction of a pattern of tenascin-C distribution in tumors and the third the writing of a paper. Each of the phases is analyzed separately, using the concept of resistance and accommodation. My purpose is to show that in each phase of the process of producing knowledge, the scientist meets resistances which force him to accommodate by changing his conceptual, technical and methodological approaches. In reverse, the details of the non-human agent (protocol, pattern or paper) have to be accommodated to the wishes and expectations of the scientist. Through this interaction a situation of stability of knowledge is reached at the end of each phase. In the protocol phase, resistance is found in the antibody and tissue slices. In the phase of pattern deduction the resistance is in the pathological diagnosis of the tumors and the expectations and hypothesis with which the scientist had entered the project; in the criteria to be used for assigning the slices to a tenascin-C pattern; and in the responses of colleagues and supervisor. In the paper-writing phase the interaction is between the scientist and the scientific community which should take on board the knowledge from the research project. When stabilization of knowledge is obtained in one of the phases, the agents of resistance turn into allies in the next phase, giving support to accommodating the resistances in this later phase. Second, the stabilization of knowledge of the protocol is further enhanced when stabilization of the pattern is achieved; in addition, knowledge of the pattern is more definite when it has become stabilized and closed knowledge within the science community. Copyright © 2010 Elsevier Ltd. All rights reserved.

A Project-Based Biochemistry Laboratory Promoting the Understanding and Uses of Fluorescence Spectroscopy in the Study of Biomolecular Structures and Interactions

ERIC Educational Resources Information Center

Briese, Nicholas; Jakubowsk, Henry V.

2007-01-01

A laboratory project for a first semester biochemistry course is described, which integrates the traditional classroom study of the structure and function of biomolecules with the laboratory study of these molecules using fluorescence spectroscopy. Students are assigned a specific question addressing the stability/function of lipids, proteins, or…

Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis

PubMed Central

Arunmanee, Wanatchaporn; Pathania, Monisha; Solovyova, Alexandra S.; Le Brun, Anton P.; Ridley, Helen; Baslé, Arnaud; van den Berg, Bert; Lakey, Jeremy H.

2016-01-01

The outer membrane (OM) of gram-negative bacteria is an unusual asymmetric bilayer with an external monolayer of lipopolysaccharide (LPS) and an inner layer of phospholipids. The LPS layer is rigid and stabilized by divalent cation cross-links between phosphate groups on the core oligosaccharide regions. This means that the OM is robust and highly impermeable to toxins and antibiotics. During their biogenesis, OM proteins (OMPs), which function as transporters and receptors, must integrate into this ordered monolayer while preserving its impermeability. Here we reveal the specific interactions between the trimeric porins of Enterobacteriaceae and LPS. Isolated porins form complexes with variable numbers of LPS molecules, which are stabilized by calcium ions. In earlier studies, two high-affinity sites were predicted to contain groups of positively charged side chains. Mutation of these residues led to the loss of LPS binding and, in one site, also prevented trimerization of the porin, explaining the previously observed effect of LPS mutants on porin folding. The high-resolution X-ray crystal structure of a trimeric porin–LPS complex not only helps to explain the mutagenesis results but also reveals more complex, subtle porin–LPS interactions and a bridging calcium ion. PMID:27493217

Site-Specific Dynamics of β-Sheet Peptides with (D) Pro-Gly Turns Probed by Laser-Excited Temperature-Jump Infrared Spectroscopy.

PubMed

Popp, Alexander; Scheerer, David; Chi, Heng; Keiderling, Timothy A; Hauser, Karin

2016-05-04

Turn residues and side-chain interactions play an important role for the folding of β-sheets. We investigated the conformational dynamics of a three-stranded β-sheet peptide ((D) P(D) P) and a two-stranded β-hairpin (WVYY-(D) P) by time-resolved temperature-jump (T-jump) infrared spectroscopy. Both peptide sequences contain (D) Pro-Gly residues that favor a tight β-turn. The three-stranded β-sheet (Ac-VFITS(D) PGKTYTEV(D) PGOKILQ-NH2 ) is stabilized by the turn sequences, whereas the β-hairpin (SWTVE(D) PGKYTYK-NH2 ) folding is assisted by both the turn sequence and hydrophobic cross-strand interactions. Relaxation times after the T-jump were monitored as a function of temperature and occur on a sub-microsecond time scale, (D) P(D) P being faster than WVYY-(D) P. The Xxx-(D) Pro tertiary amide provides a detectable IR band, allowing us to probe the dynamics site-specifically. The relative importance of the turn versus the intrastrand stability in β-sheet formation is discussed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Roles of specific membrane lipid domains in EGF receptor activation and cell adhesion molecule stabilization in a developing olfactory system.

PubMed

Gibson, Nicholas J; Tolbert, Leslie P; Oland, Lynne A

2009-09-29

Reciprocal interactions between glial cells and olfactory receptor neurons (ORNs) cause ORN axons entering the brain to sort, to fasciculate into bundles destined for specific glomeruli, and to form stable protoglomeruli in the developing olfactory system of an experimentally advantageous animal species, the moth Manduca sexta. Epidermal growth factor receptors (EGFRs) and the cell adhesion molecules (IgCAMs) neuroglian and fasciclin II are known to be important players in these processes. We report in situ and cell-culture studies that suggest a role for glycosphingolipid-rich membrane subdomains in neuron-glia interactions. Disruption of these subdomains by the use of methyl-beta-cyclodextrin results in loss of EGFR activation, depletion of fasciclin II in ORN axons, and loss of neuroglian stabilization in the membrane. At the cellular level, disruption leads to aberrant ORN axon trajectories, small antennal lobes, abnormal arrays of olfactory glomerul, and loss of normal glial cell migration. We propose that glycosphingolipid-rich membrane subdomains (possible membrane rafts or platforms) are essential for IgCAM-mediated EGFR activation and for anchoring of neuroglian to the cytoskeleton, both required for normal extension and sorting of ORN axons.

The coordinating role of IQGAP1 in the regulation of local, endosome-specific actin networks

PubMed Central

Samson, Edward B.; Tsao, David S.; Zimak, Jan; McLaughlin, R. Tyler; Trenton, Nicholaus J.; Mace, Emily M.; Orange, Jordan S.; Schweikhard, Volker

2017-01-01

ABSTRACT IQGAP1 is a large, multi-domain scaffold that helps orchestrate cell signaling and cytoskeletal mechanics by controlling interactions among a spectrum of receptors, signaling intermediates, and cytoskeletal proteins. While this coordination is known to impact cell morphology, motility, cell adhesion, and vesicular traffic, among other functions, the spatiotemporal properties and regulatory mechanisms of IQGAP1 have not been fully resolved. Herein, we describe a series of super-resolution and live-cell imaging analyses that identified a role for IQGAP1 in the regulation of an actin cytoskeletal shell surrounding a novel membranous compartment that localizes selectively to the basal cortex of polarized epithelial cells (MCF-10A). We also show that IQGAP1 appears to both stabilize the actin coating and constrain its growth. Loss of compartmental IQGAP1 initiates a disassembly mechanism involving rapid and unconstrained actin polymerization around the compartment and dispersal of its vesicle contents. Together, these findings suggest IQGAP1 achieves this control by harnessing both stabilizing and antagonistic interactions with actin. They also demonstrate the utility of these compartments for image-based investigations of the spatial and temporal dynamics of IQGAP1 within endosome-specific actin networks. PMID:28455356

WW Domains of the Yes-Kinase-Associated-Protein (YAP) Transcriptional Regulator Behave as Independent Units with Different Binding Preferences for PPxY Motif-Containing Ligands

PubMed Central

Iglesias-Bexiga, Manuel; Castillo, Francisco; Cobos, Eva S.; Oka, Tsutomu; Sudol, Marius; Luque, Irene

2015-01-01

YAP is a WW domain-containing effector of the Hippo tumor suppressor pathway, and the object of heightened interest as a potent oncogene and stemness factor. YAP has two major isoforms that differ in the number of WW domains they harbor. Elucidating the degree of co-operation between these WW domains is important for a full understanding of the molecular function of YAP. We present here a detailed biophysical study of the structural stability and binding properties of the two YAP WW domains aimed at investigating the relationship between both domains in terms of structural stability and partner recognition. We have carried out a calorimetric study of the structural stability of the two YAP WW domains, both isolated and in a tandem configuration, and their interaction with a set of functionally relevant ligands derived from PTCH1 and LATS kinases. We find that the two YAP WW domains behave as independent units with different binding preferences, suggesting that the presence of the second WW domain might contribute to modulate target recognition between the two YAP isoforms. Analysis of structural models and phage-display studies indicate that electrostatic interactions play a critical role in binding specificity. Together, these results are relevant to understand of YAP function and open the door to the design of highly specific ligands of interest to delineate the functional role of each WW domain in YAP signaling. PMID:25607641

Specificity determinants for the abscisic acid response element.

PubMed

Sarkar, Aditya Kumar; Lahiri, Ansuman

2013-01-01

Abscisic acid (ABA) response elements (ABREs) are a group of cis-acting DNA elements that have been identified from promoter analysis of many ABA-regulated genes in plants. We are interested in understanding the mechanism of binding specificity between ABREs and a class of bZIP transcription factors known as ABRE binding factors (ABFs). In this work, we have modeled the homodimeric structure of the bZIP domain of ABRE binding factor 1 from Arabidopsis thaliana (AtABF1) and studied its interaction with ACGT core motif-containing ABRE sequences. We have also examined the variation in the stability of the protein-DNA complex upon mutating ABRE sequences using the protein design algorithm FoldX. The high throughput free energy calculations successfully predicted the ability of ABF1 to bind to alternative core motifs like GCGT or AAGT and also rationalized the role of the flanking sequences in determining the specificity of the protein-DNA interaction.

Alpha particle effects in burning tokamak plasmas: overview and specific examples

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

Sigmar, D.J.

1986-07-01

Using the total power balance of an ignited tokamak plasma as a guideline, a range of alpha driven effects is surveyed regarding their impact on achieving and maintaining fusion burn. Specific examples of MHD and kinetic modes and multi species transport dynamics are discussed, including the possible interaction of these categories of effects. This power balance approach rather than a straightforward enumeration of possible effects serves to reveal their non-linear dependence and the ensuing fragility of our understanding of the approach to and maintenance of ignition. Specific examples are given of the interaction between ..cap alpha..-power driven sawtoothing and idealmore » MHD stability, and direct ..cap alpha..-effects on MHD modes including kinetic corrections. Anomalous ion heat transport and central impurity peaking mechanisms and anomalous and collisional ..cap alpha..-transport including the ambipolar electric field are discussed.« less

Specific phospholipid binding to Na,K-ATPase at two distinct sites.

PubMed

Habeck, Michael; Kapri-Pardes, Einat; Sharon, Michal; Karlish, Steven J D

2017-03-14

Membrane protein function can be affected by the physical state of the lipid bilayer and specific lipid-protein interactions. For Na,K-ATPase, bilayer properties can modulate pump activity, and, as observed in crystal structures, several lipids are bound within the transmembrane domain. Furthermore, Na,K-ATPase activity depends on phosphatidylserine (PS) and cholesterol, which stabilize the protein, and polyunsaturated phosphatidylcholine (PC) or phosphatidylethanolamine (PE), known to stimulate Na,K-ATPase activity. Based on lipid structural specificity and kinetic mechanisms, specific interactions of both PS and PC/PE have been inferred. Nevertheless, specific binding sites have not been identified definitively. We address this question with native mass spectrometry (MS) and site-directed mutagenesis. Native MS shows directly that one molecule each of 18:0/18:1 PS and 18:0/20:4 PC can bind specifically to purified human Na,K-ATPase (α 1 β 1 ). By replacing lysine residues at proposed phospholipid-binding sites with glutamines, the two sites have been identified. Mutations in the cytoplasmic αL8-9 loop destabilize the protein but do not affect Na,K-ATPase activity, whereas mutations in transmembrane helices (TM), αTM2 and αTM4, abolish the stimulation of activity by 18:0/20:4 PC but do not affect stability. When these data are linked to crystal structures, the underlying mechanism of PS and PC/PE effects emerges. PS (and cholesterol) bind between αTM 8, 9, 10, near the FXYD subunit, and maintain topological integrity of the labile C terminus of the α subunit (site A). PC/PE binds between αTM2, 4, 6, and 9 and accelerates the rate-limiting E 1 P-E 2 P conformational transition (site B). We discuss the potential physiological implications.

Zinc-Amyloid Interactions on a Millisecond Time-Scale Stabilize Non-Fibrillar Alzheimer Related Species

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

Noy,D.; Solomonov, I.; Sinkevich, O.

2008-01-01

The role of zinc, an essential element for normal brain function, in the pathology of Alzheimer's disease (AD) is poorly understood. On one hand, physiological and genetic evidence from transgenic mouse models supports its pathogenic role in promoting the deposition of the amyloid {beta}-protein (A{beta}) in senile plaques. On the other hand, levels of extracellular ('free') zinc in the brain, as inferred by the levels of zinc in cerebrospinal fluid, were found to be too low for inducing A{beta} aggregation. Remarkably, the release of transient high local concentrations of zinc during rapid synaptic events was reported. The role of suchmore » free zinc pulses in promoting A{beta} aggregation has never been established. Using a range of time-resolved structural and spectroscopic techniques, we found that zinc, when introduced in millisecond pulses of micromolar concentrations, immediately interacts with A{beta} 1-40 and promotes its aggregation. These interactions specifically stabilize non-fibrillar pathogenic related aggregate forms and prevent the formation of A{beta} fibrils (more benign species) presumably by interfering with the self-assembly process of A{beta}. These in vitro results strongly suggest a significant role for zinc pulses in A{beta} pathology. We further propose that by interfering with A{beta} self-assembly, which leads to insoluble, non-pathological fibrillar forms, zinc stabilizes transient, harmful amyloid forms. This report argues that zinc represents a class of molecular pathogens that effectively perturb the self-assembly of benign A{beta} fibrils, and stabilize harmful non-fibrillar forms.« less

Independent movement, dimerization and stability of tandem repeats of chicken brain alpha-spectrin

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

Kusunoki, H.; Minasov, G.; Macdonald, R.I.

Previous X-ray crystal structures have shown that linkers of five amino acid residues connecting pairs of chicken brain {alpha}-spectrin and human erythroid {beta}-spectrin repeats can undergo bending without losing their {alpha}-helical structure. To test whether bending at one linker can influence bending at an adjacent linker, the structures of two and three repeat fragments of chicken brain {alpha}-spectrin have been determined by X-ray crystallography. The structure of the three-repeat fragment clearly shows that bending at one linker can occur independently of bending at an adjacent linker. This observation increases the possible trajectories of modeled chains of spectrin repeats. Furthermore, themore » three-repeat molecule crystallized as an antiparallel dimer with a significantly smaller buried interfacial area than that of {alpha}-actinin, a spectrin-related molecule, but large enough and of a type indicating biological specificity. Comparison of the structures of the spectrin and {alpha}-actinin dimers supports weak association of the former, which could not be detected by analytical ultracentrifugation, versus strong association of the latter, which has been observed by others. To correlate features of the structure with solution properties and to test a previous model of stable spectrin and dystrophin repeats, the number of inter-helical interactions in each repeat of several spectrin structures were counted and compared to their thermal stabilities. Inter-helical interactions, but not all interactions, increased in parallel with measured thermal stabilities of each repeat and in agreement with the thermal stabilities of two and three repeats and also partial repeats of spectrin.« less

A Survey of Aspartate Phenylalanine and Glutamate Phenylalanine Interactions in the Protein Data Bank: Searching for Anion Pairs

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

Philip, Vivek M; Harris, Jason B; Adams, Rachel M

Protein structures are stabilized using noncovalent interactions. In addition to the traditional noncovalent interactions, newer types of interactions are thought to be present in proteins. One such interaction, an anion pair, in which the positively charged edge of an aromatic ring interacts with an anion, forming a favorable anion quadrupole interaction, has been previously proposed [Jackson, M. R., et al. (2007) J. Phys. Chem. B111, 8242 8249]. To study the role of anion interactions in stabilizing protein structure, we analyzed pairwise interactions between phenylalanine (Phe) and the anionic amino acids, aspartate (Asp) and glutamate (Glu). Particular emphasis was focused onmore » identification of Phe Asp or Glu pairs separated by less than 7 in the high-resolution, nonredundant Protein Data Bank. Simplifying Phe to benzene and Asp or Glu to formate molecules facilitated in silico analysis of the pairs. Kitaura Morokuma energy calculations were performed on roughly 19000 benzene formate pairs and the resulting energies analyzed as a function of distance and angle. Edgewise interactions typically produced strongly stabilizing interaction energies (2 to 7.3 kcal/mol), while interactions involving the ring face resulted in weakly stabilizing to repulsive interaction energies. The strongest, most stabilizing interactions were identified as preferentially occurring in buried residues. Anion pairs are found throughout protein structures, in helices as well as strands. Numerous pairs also had nearby cation interactions as well as potential stacking. While more than 1000 structures did not contain an anion pair, the 3134 remaining structures contained approximately 2.6 anion pairs per protein, suggesting it is a reasonably common motif that could contribute to the overall structural stability of a protein.« less

A Survey of Aspartate-Phenylalanine and Glutamate-Phenylalanine Interactions in the Protein Data Bank: Searching for Anion-pi Pairs

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

Philip, Vivek M; Harris, Jason B; Adams, Rachel M

Protein structures are stabilized using noncovalent interactions. In addition to the traditional noncovalent interactions, newer types of interactions are thought to be present in proteins. One such interaction, an anion-{pi} pair, in which the positively charged edge of an aromatic ring interacts with an anion, forming a favorable anion-quadrupole interaction, has been previously proposed [Jackson, M. R., et al. (2007) J. Phys. Chem. B111, 8242-8249]. To study the role of anion-{pi} interactions in stabilizing protein structure, we analyzed pairwise interactions between phenylalanine (Phe) and the anionic amino acids, aspartate (Asp) and glutamate (Glu). Particular emphasis was focused on identification ofmore » Phe-Asp or -Glu pairs separated by less than 7 {angstrom} in the high-resolution, nonredundant Protein Data Bank. Simplifying Phe to benzene and Asp or Glu to formate molecules facilitated in silico analysis of the pairs. Kitaura-Morokuma energy calculations were performed on roughly 19000 benzene-formate pairs and the resulting energies analyzed as a function of distance and angle. Edgewise interactions typically produced strongly stabilizing interaction energies (-2 to -7.3 kcal/mol), while interactions involving the ring face resulted in weakly stabilizing to repulsive interaction energies. The strongest, most stabilizing interactions were identified as preferentially occurring in buried residues. Anion-{pi} pairs are found throughout protein structures, in helices as well as {beta} strands. Numerous pairs also had nearby cation-{pi} interactions as well as potential {pi}-{pi} stacking. While more than 1000 structures did not contain an anion-{pi} pair, the 3134 remaining structures contained approximately 2.6 anion-{pi} pairs per protein, suggesting it is a reasonably common motif that could contribute to the overall structural stability of a protein.« less

A survey of aspartate-phenylalanine and glutamate-phenylalanine interactions in the protein data bank: searching for anion-π pairs.

PubMed

Philip, Vivek; Harris, Jason; Adams, Rachel; Nguyen, Don; Spiers, Jeremy; Baudry, Jerome; Howell, Elizabeth E; Hinde, Robert J

2011-04-12

Protein structures are stabilized using noncovalent interactions. In addition to the traditional noncovalent interactions, newer types of interactions are thought to be present in proteins. One such interaction, an anion-π pair, in which the positively charged edge of an aromatic ring interacts with an anion, forming a favorable anion-quadrupole interaction, has been previously proposed [Jackson, M. R., et al. (2007) J. Phys. Chem. B111, 8242-8249]. To study the role of anion-π interactions in stabilizing protein structure, we analyzed pairwise interactions between phenylalanine (Phe) and the anionic amino acids, aspartate (Asp) and glutamate (Glu). Particular emphasis was focused on identification of Phe-Asp or -Glu pairs separated by less than 7 Å in the high-resolution, nonredundant Protein Data Bank. Simplifying Phe to benzene and Asp or Glu to formate molecules facilitated in silico analysis of the pairs. Kitaura-Morokuma energy calculations were performed on roughly 19000 benzene-formate pairs and the resulting energies analyzed as a function of distance and angle. Edgewise interactions typically produced strongly stabilizing interaction energies (-2 to -7.3 kcal/mol), while interactions involving the ring face resulted in weakly stabilizing to repulsive interaction energies. The strongest, most stabilizing interactions were identified as preferentially occurring in buried residues. Anion-π pairs are found throughout protein structures, in helices as well as β strands. Numerous pairs also had nearby cation-π interactions as well as potential π-π stacking. While more than 1000 structures did not contain an anion-π pair, the 3134 remaining structures contained approximately 2.6 anion-π pairs per protein, suggesting it is a reasonably common motif that could contribute to the overall structural stability of a protein.

Dynamics in microbial communities: Unraveling mechanisms to identify principles

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

Konopka, Allan; Lindemann, Stephen R.; Fredrickson, Jim K.

2015-07-01

Diversity begets higher order properties such as functional stability and robustness in microbial communities, but principles that inform conceptual (and eventually predictive) models of community dynamics are lacking. Recent work has shown that selection as well as dispersal and drift shape communities, but the mechanistic bases for assembly of communities and the forces that maintain their function in the face of environmental perturbation are not well understood. Conceptually, some interactions among community members could generate endogenous dynamics in composition, even in the absence of environmental changes. These endogenous dynamics are further perturbed by exogenous forcing factors to produce a richermore » network of community interactions, and it is this “system” that is the basis for higher order community properties. Elucidation of principles that follow from this conceptual model requires identifying the mechanisms that (a) optimize diversity within a community and (b) impart community stability. The network of interactions between organisms can be an important element by providing a buffer against disturbance beyond the effect of functional redundancy, as alternative pathways with different combinations of microbes can be recruited to fulfill specific functions.« less

Interfacial Engineered Polyaniline/Sulfur-Doped TiO2 Nanotube Arrays for Ultralong Cycle Lifetime Fiber-Shaped, Solid-State Supercapacitors.

PubMed

Li, Chun; Wang, Zhuanpei; Li, Shengwen; Cheng, Jianli; Zhang, Yanning; Zhou, Jingwen; Yang, Dan; Tong, Dong-Ge; Wang, Bin

2018-05-30

Fiber-shaped supercapacitors (FSCs) have great promises in wearable electronics applications. However, the limited specific surface area and inadequate structural stability caused by the weak interfacial interactions of the electrodes result in relatively low specific capacitance and unsatisfactory cycle lifetime. Herein, solid-state FSCs with high energy density and ultralong cycle lifetime based on polyaniline (PANI)/sulfur-doped TiO 2 nanotube arrays (PANI/S-TiO 2 ) are fabricated by interfacial engineering. The experimental results and ab initio calculations reveal that S doping can effectively promote the conductivity of titania nanotubes and increase the binding energy of PANI anchored on the electrode surface, leading to a much stronger binding of PANI on the surface of the electrode and excellent electrode structure stability. As a result, the FSCs using the PANI/S-TiO 2 electrodes deliver a high specific capacitance of 91.9 mF cm -2 , a capacitance retention of 93.78% after 12 000 charge-discharge cycles, and an areal energy density of 3.2 μW h cm -2 . Meanwhile, the all-solid-state FSC device retains its excellent flexibility and stable electrochemical capacitance even after bending 150 cycles. The enhanced performances of FSCs could be attributed to the large surface area, reduced ion diffusion path, improved electrical conductivity, and engineered interfacial interaction of the rationally designed electrodes.

Application of nanotechnology in improving bioavailability and bioactivity of diet-derived phytochemicals

PubMed Central

Wang, Shu; Su, Rui; Nie, Shufang; Sun, Ming; Zhang, Jia; Wu, Dayong; Moustaid-Moussa, Naima

2013-01-01

Nanotechnology is an innovative approach that has potential applications in nutraceutical research. Phytochemicals have promising potential for maintaining and promoting health, as well as preventing and potentially treating some diseases. However, the generally low solubility, stability, bioavailability and target specificity, together with the side-effects seen when used at high levels, have limited their application. Indeed, nanoparticles can increase solubility and stability of phytochemicals, enhance their absorption, protect them from premature degradation in the body, and prolong their circulation time. Moreover, these nanoparticles exhibit high differential uptake efficiency in the target cells (or tissue) over normal cells (or tissue)through preventing them from prematurely interacting with the biological environment, enhanced permeation and retention effect in disease tissues, and improving their cellular uptake, resulting in decreased toxicity, In this review we outline the commonly used biocompatible and biodegradable nanoparticles including liposomes, emulsions, solid lipid nanoparticles, nanostructured lipid carriers, micelles and poly (lactic-co-glycolic acid) (PLGA) nanoparticles. We then summarize studies that have used these nanoparticles as carriers for EGCG, quercetin, resveratrol and curcuminadministration to enhance their aqueous solubility, stability, bioavailability, target specificity, and bioactivities. PMID:24406273

I-mfa domain proteins specifically interact with HTLV-1 Tax and repress its transactivating functions

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

Kusano, Shuichi, E-mail: skusano@m2.kufm.kagoshima-u.ac.jp; Yoshimitsu, Makoto; Hachiman, Miho

The I-mfa domain proteins HIC (also known as MDFIC) and I-mfa (also known as MDFI) are candidate tumor suppressor genes that are involved in cellular and viral transcriptional regulation. Here, we show that HIC and I-mfa directly interact with human T-cell leukemia virus type-1 (HTLV-1) Tax protein in vitro. In addition, HIC and I-mfa repress Tax-dependent transactivation of an HTLV-1 long terminal repeat (LTR) reporter construct in COS-1, Jurkat and high-Tax-producing HTLV-1-infected T cells. HIC also interacts with Tax through its I-mfa domain in vivo and represses Tax-dependent transactivation of HTLV-1 LTR and NF-κB reporter constructs in an interaction-dependent manner.more » Furthermore, we show that HIC decreases the nuclear distribution and stimulates the proteasomal degradation of Tax. These data reveal that HIC specifically interacts with HTLV-1 Tax and negatively regulates Tax transactivational activity by altering its subcellular distribution and stability. - Highlights: • I-mfa domain proteins, HIC and I-mfa, specifically interact with HTLV-1 Tax. • HIC and I-mfa repress the Tax-dependent transactivation of HTLV-1 LTR. • HIC represses the Tax-dependent transactivation of NF-κΒ. • HIC decreases the nuclear distribution of Tax. • HIC stimulates the proteasomal degradation of Tax.« less

Graphene sheets stabilized on genetically engineered M13 viral templates as conducting frameworks for hybrid energy-storage materials.

PubMed

Oh, Dahyun; Dang, Xiangnan; Yi, Hyunjung; Allen, Mark A; Xu, Kang; Lee, Yun Jung; Belcher, Angela M

2012-04-10

Utilization of the material-specific peptide-substrate interactions of M13 virus broadens colloidal stability window of graphene. The homogeneous distribution of graphene is maintained in weak acids and increased ionic strengths by complexing with virus. This graphene/virus conducting template is utilized in the synthesis of energy-storage materials to increase the conductivity of the composite electrode. Successful formation of the hybrid biological template is demonstrated by the mineralization of bismuth oxyfluoride as a cathode material for lithium-ion batteries, with increased loading and improved electronic conductivity. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures

PubMed Central

Schaffter, Samuel W; Green, Leopold N; Schneider, Joanna; Subramanian, Hari K K; Schulman, Rebecca

2018-01-01

Abstract The use of proteins that bind and catalyze reactions with DNA alongside DNA nanostructures has broadened the functionality of DNA devices. DNA binding proteins have been used to specifically pattern and tune structural properties of DNA nanostructures and polymerases have been employed to directly and indirectly drive structural changes in DNA structures and devices. Despite these advances, undesired and poorly understood interactions between DNA nanostructures and proteins that bind DNA continue to negatively affect the performance and stability of DNA devices used in conjunction with enzymes. A better understanding of these undesired interactions will enable the construction of robust DNA nanostructure-enzyme hybrid systems. Here, we investigate the undesired disassembly of DNA nanotubes in the presence of viral RNA polymerases (RNAPs) under conditions used for in vitro transcription. We show that nanotubes and individual nanotube monomers (tiles) are non-specifically transcribed by T7 RNAP, and that RNA transcripts produced during non-specific transcription disassemble the nanotubes. Disassembly requires a single-stranded overhang on the nanotube tiles where transcripts can bind and initiate disassembly through strand displacement, suggesting that single-stranded domains on other DNA nanostructures could cause unexpected interactions in the presence of viral RNA polymerases. PMID:29718412

T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures.

PubMed

Schaffter, Samuel W; Green, Leopold N; Schneider, Joanna; Subramanian, Hari K K; Schulman, Rebecca; Franco, Elisa

2018-06-01

The use of proteins that bind and catalyze reactions with DNA alongside DNA nanostructures has broadened the functionality of DNA devices. DNA binding proteins have been used to specifically pattern and tune structural properties of DNA nanostructures and polymerases have been employed to directly and indirectly drive structural changes in DNA structures and devices. Despite these advances, undesired and poorly understood interactions between DNA nanostructures and proteins that bind DNA continue to negatively affect the performance and stability of DNA devices used in conjunction with enzymes. A better understanding of these undesired interactions will enable the construction of robust DNA nanostructure-enzyme hybrid systems. Here, we investigate the undesired disassembly of DNA nanotubes in the presence of viral RNA polymerases (RNAPs) under conditions used for in vitro transcription. We show that nanotubes and individual nanotube monomers (tiles) are non-specifically transcribed by T7 RNAP, and that RNA transcripts produced during non-specific transcription disassemble the nanotubes. Disassembly requires a single-stranded overhang on the nanotube tiles where transcripts can bind and initiate disassembly through strand displacement, suggesting that single-stranded domains on other DNA nanostructures could cause unexpected interactions in the presence of viral RNA polymerases.

Redox control of protein-DNA interactions: from molecular mechanisms to significance in signal transduction, gene expression, and DNA replication.

PubMed

Shlomai, Joseph

2010-11-01

Protein-DNA interactions play a key role in the regulation of major cellular metabolic pathways, including gene expression, genome replication, and genomic stability. They are mediated through the interactions of regulatory proteins with their specific DNA-binding sites at promoters, enhancers, and replication origins in the genome. Redox signaling regulates these protein-DNA interactions using reactive oxygen species and reactive nitrogen species that interact with cysteine residues at target proteins and their regulators. This review describes the redox-mediated regulation of several master regulators of gene expression that control the induction and suppression of hundreds of genes in the genome, regulating multiple metabolic pathways, which are involved in cell growth, development, differentiation, and survival, as well as in the function of the immune system and cellular response to intracellular and extracellular stimuli. It also discusses the role of redox signaling in protein-DNA interactions that regulate DNA replication. Specificity of redox regulation is discussed, as well as the mechanisms providing several levels of redox-mediated regulation, from direct control of DNA-binding domains through the indirect control, mediated by release of negative regulators, regulation of redox-sensitive protein kinases, intracellular trafficking, and chromatin remodeling.

Molecular insights into the stabilization of protein-protein interactions with small molecule: The FKBP12-rapamycin-FRB case study

NASA Astrophysics Data System (ADS)

Chaurasia, Shilpi; Pieraccini, Stefano; De Gonda, Riccardo; Conti, Simone; Sironi, Maurizio

2013-11-01

Targetting protein-protein interactions is a challenging task in drug discovery process. Despite the challenges, several studies provided evidences for the development of small molecules modulating protein-protein interactions. Here we consider a typical case of protein-protein interaction stabilization: the complex between FKBP12 and FRB with rapamycin. We have analyzed the stability of the complex and characterized its interactions at the atomic level by performing free energy calculations and computational alanine scanning. It is shown that rapamycin stabilizes the complex by acting as a bridge between the two proteins; and the complex is stable only in the presence of rapamycin.

Specific effects of surface carboxyl groups on anionic polystyrene particles in their interactions with mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Jiang, Xiue; Musyanovych, Anna; Röcker, Carlheinz; Landfester, Katharina; Mailänder, Volker; Nienhaus, G. Ulrich

2011-05-01

Nanoparticle uptake by living cells is governed by chemical interactions between functional groups on the nanoparticle as well as the receptors on cell surfaces. Here we have investigated the uptake of anionic polystyrene (PS) nanoparticles of ~100 nm diameter by mesenchymal stem cells (MSCs) using spinning-disk confocal optical microscopy combined with a quantitative analysis of the fluorescence images. Two types of anionic PS nanoparticles with essentially identical sizes and ζ-potentials were employed in this study, carboxyl-functionalized nanoparticles (CPS) and plain PS nanoparticles, both coated with anionic detergent for stabilization. CPS nanoparticles were observed to internalize more rapidly and accumulate to a much higher level than plain PS nanoparticles. The relative importance of different uptake mechanisms for the two types of nanoparticles was investigated by using specific inhibitors. CPS nanoparticles were internalized mainly via the clathrin-mediated mechanism, whereas plain PS nanoparticles mainly utilized the macropinocytosis pathway. The pronounced difference in the internalization behavior of CPS and plain PS nanoparticles points to a specific interaction of the carboxyl group with receptors on the cell surface.

Structural mechanism underlying capsaicin binding and activation of TRPV1 ion channel

PubMed Central

Cheng, Wei; Yang, Wei; Yu, Peilin; Song, Zhenzhen; Yarov-Yarovoy, Vladimir; Zheng, Jie

2015-01-01

Capsaicin bestows spiciness by activating TRPV1 channel with exquisite potency and selectivity. Capsaicin-bound channel structure was previously resolved by cryo-EM at 4.2-to-4.5 Å resolution, however important details required for mechanistic understandings are unavailable: capsaicin was registered as a small electron density, reflecting neither its chemical structure nor specific ligand-channel interactions. We obtained the missing atomic-level details by iterative computation, which were confirmed by systematic site-specific functional tests. We observed that the bound capsaicin takes “tail-up, head-down” configurations. The vanillyl and amide groups form specific interactions to anchor its bound position, while the aliphatic tail may sample a range of conformations, making it invisible in cryo-EM images. Capsaicin stabilizes the open state by “pull-and-contact” interactions between the vanillyl group and the S4-S5 linker. Our study provided a structural mechanism for the agonistic function of capsaicin and its analogs, and demonstrated an effective approach to obtain atomic level information from cryo-EM structures. PMID:26053297

Fabrication of an electrochemical sensor based on spiropyran for sensitive and selective detection of fluoride ion.

PubMed

Tao, Jia; Zhao, Peng; Li, Yinhui; Zhao, Wenjie; Xiao, Yue; Yang, Ronghua

2016-04-28

In the past decades, numerous electrochemical sensors based on exogenous electroactive substance have been reported. Due to non-specific interaction between the redox mediator and the target, the instability caused by false signal may not be avoided. To address this issue, in this paper, a new electrochemical sensor based on spiropyran skeleton, namely SPOSi, was designed for specific electrochemical response to fluoride ions (F(-)). The breakage of Si-O induced by F(-) based on the specific nucleophilic substitution reaction between F(-) and silica would directly produce a hydroquinone structure for electrochemical signal generation. To improve the sensitivity, SPOSi probe was assembled on the single-walled carbon nanotubes (SWCNTs) modified glassy carbon electrode (GCE) through the π-π conjugating interaction. This electrode was successfully applied to monitor F(-) with a detection limit of 8.3 × 10(-8) M. Compared with the conventional F(-) ion selected electrode (ISE) which utilized noncovalent interaction, this method displays higher stability and a comparable sensitivity in the urine samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Phospho-selective mechanisms of arrestin conformations and functions revealed by unnatural amino acid incorporation and 19F-NMR

PubMed Central

Yang, Fan; Yu, Xiao; Liu, Chuan; Qu, Chang-Xiu; Gong, Zheng; Liu, Hong-Da; Li, Fa-Hui; Wang, Hong-Mei; He, Dong-Fang; Yi, Fan; Song, Chen; Tian, Chang-Lin; Xiao, Kun-Hong; Wang, Jiang-Yun; Sun, Jin-Peng

2015-01-01

Specific arrestin conformations are coupled to distinct downstream effectors, which underlie the functions of many G-protein-coupled receptors (GPCRs). Here, using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance (19F-NMR) spectroscopy, we demonstrate that distinct receptor phospho-barcodes are translated to specific β-arrestin-1 conformations and direct selective signalling. With its phosphate-binding concave surface, β-arrestin-1 ‘reads' the message in the receptor phospho-C-tails and distinct phospho-interaction patterns are revealed by 19F-NMR. Whereas all functional phosphopeptides interact with a common phosphate binding site and induce the movements of finger and middle loops, different phospho-interaction patterns induce distinct structural states of β-arrestin-1 that are coupled to distinct arrestin functions. Only clathrin recognizes and stabilizes GRK2-specific β-arrestin-1 conformations. The identified receptor-phospho-selective mechanism for arrestin conformation and the spacing of the multiple phosphate-binding sites in the arrestin enable arrestin to recognize plethora phosphorylation states of numerous GPCRs, contributing to the functional diversity of receptors. PMID:26347956

Complementarity of stability patches at the interfaces of protein complexes: Implication for the structural organization of energetic hot spots.

PubMed

Kuttner, Yosef Y; Engel, Stanislav

2018-02-01

A rational design of protein complexes with defined functionalities and of drugs aimed at disrupting protein-protein interactions requires fundamental understanding of the mechanisms underlying the formation of specific protein complexes. Efforts to develop efficient small-molecule or protein-based binders often exploit energetic hot spots on protein surfaces, namely, the interfacial residues that provide most of the binding free energy in the complex. The molecular basis underlying the unusually high energy contribution of the hot spots remains obscure, and its elucidation would facilitate the design of interface-targeted drugs. To study the nature of the energetic hot spots, we analyzed the backbone dynamic properties of contact surfaces in several protein complexes. We demonstrate that, in most complexes, the backbone dynamic landscapes of interacting surfaces form complementary "stability patches," in which static areas from the opposing surfaces superimpose, and that these areas are predominantly located near the geometric center of the interface. We propose that a diminished enthalpy-entropy compensation effect augments the degree to which residues positioned within the complementary stability patches contribute to complex affinity, thereby giving rise to the energetic hot spots. These findings offer new insights into the nature of energetic hot spots and the role that backbone dynamics play in facilitating intermolecular recognition. Mapping the interfacial stability patches may provide guidance for protein engineering approaches aimed at improving the stability of protein complexes and could facilitate the design of ligands that target complex interfaces. © 2017 Wiley Periodicals, Inc.

DAP12 impacts trafficking and surface stability of killer immunoglobulin-like receptors on natural killer cells

PubMed Central

Mulrooney, Tiernan J.; Posch, Phillip E.; Hurley, Carolyn Katovich

2013-01-01

KIR aid in the regulation of NK cell activity. In this study, the effect of the interaction between the KIR2DS and their adapter, DAP12, was investigated beyond the previously defined signaling function. Flow cytometry analysis showed enhanced KIR2DS surface expression on NKL cells when cotransfected with DAP12. Conversely, KIR2DS4 surface expression on primary cells was decreased when the cells were treated with DAP12-specific siRNA. Treatment of the KIR2DS and DAP12-transfected cells with CHX or BFA repressed KIR2DS surface expression, revealing a role for DAP12 in trafficking newly synthesized KIR to the cell surface. Immunoprecipitation of DAP12 revealed an interaction of DAP12 with an immature isoform of KIR2DS, indicating that the interaction likely initiates within the ER. An internalization assay demonstrated a significant impact of DAP12 on KIR2DS surface stability. Confocal microscopy showed that internalized KIR2DS molecules are recruited to lysosomal compartments independent of DAP12 expression. Our results suggest that in vivo conditions that adversely affect DAP12 expression will indirectly reduce surface expression and stability of KIR2DS. These effects could significantly impact ligand recognition and strength of signaling through KIR2DS molecules. PMID:23715743

Thioamide Substitution Selectively Modulates Proteolysis and Receptor Activity of Therapeutic Peptide Hormones.

PubMed

Chen, Xing; Mietlicki-Baase, Elizabeth G; Barrett, Taylor M; McGrath, Lauren E; Koch-Laskowski, Kieran; Ferrie, John J; Hayes, Matthew R; Petersson, E James

2017-11-22

Peptide hormones are attractive as injectable therapeutics and imaging agents, but they often require extensive modification by mutagenesis and/or chemical synthesis to prevent rapid in vivo degradation. Alternatively, the single-atom, O-to-S modification of peptide backbone thioamidation has the potential to selectively perturb interactions with proteases while preserving interactions with other proteins, such as target receptors. Here, we use the validated diabetes therapeutic, glucagon-like peptide-1 (GLP-1), and the target of clinical investigation, gastric inhibitory polypeptide (GIP), as proof-of-principle peptides to demonstrate the value of thioamide substitution. In GLP-1 and GIP, a single thioamide near the scissile bond renders these peptides up to 750-fold more stable than the corresponding oxopeptides toward cleavage by dipeptidyl peptidase 4, the principal regulator of their in vivo stability. These stabilized analogues are nearly equipotent with their parent peptide in cyclic AMP activation assays, but the GLP-1 thiopeptides have much lower β-arrestin potency, making them novel agonists with altered signaling bias. Initial tests show that a thioamide GLP-1 analogue is biologically active in rats, with an in vivo potency for glycemic control surpassing that of native GLP-1. Taken together, these experiments demonstrate the potential for thioamides to modulate specific protein interactions to increase proteolytic stability or tune activation of different signaling pathways.

Salt-bridge networks within globular and disordered proteins: characterizing trends for designable interactions.

PubMed

Basu, Sankar; Mukharjee, Debasish

2017-07-01

There has been considerable debate about the contribution of salt bridges to the stabilization of protein folds, in spite of their participation in crucial protein functions. Salt bridges appear to contribute to the activity-stability trade-off within proteins by bringing high-entropy charged amino acids into close contacts during the course of their functions. The current study analyzes the modes of association of salt bridges (in terms of networks) within globular proteins and at protein-protein interfaces. While the most common and trivial type of salt bridge is the isolated salt bridge, bifurcated salt bridge appears to be a distinct salt-bridge motif having a special topology and geometry. Bifurcated salt bridges are found ubiquitously in proteins and interprotein complexes. Interesting and attractive examples presenting different modes of interaction are highlighted. Bifurcated salt bridges appear to function as molecular clips that are used to stitch together large surface contours at interacting protein interfaces. The present work also emphasizes the key role of salt-bridge-mediated interactions in the partial folding of proteins containing long stretches of disordered regions. Salt-bridge-mediated interactions seem to be pivotal to the promotion of "disorder-to-order" transitions in small disordered protein fragments and their stabilization upon binding. The results obtained in this work should help to guide efforts to elucidate the modus operandi of these partially disordered proteins, and to conceptualize how these proteins manage to maintain the required amount of disorder even in their bound forms. This work could also potentially facilitate explorations of geometrically specific designable salt bridges through the characterization of composite salt-bridge networks. Graphical abstract ᅟ.

Intermolecular orbital interaction in π systems

NASA Astrophysics Data System (ADS)

Zhao, Rundong; Zhang, Rui-Qin

2018-04-01

Intermolecular interactions, in regard to which people tend to emphasise the noncovalent van der Waals (vdW) forces when conducting investigations throughout chemistry, can influence the structure, stability and function of molecules and materials. Despite the ubiquitous nature of vdW interactions, a simplified electrostatic model has been popularly adopted to explain common intermolecular interactions, especially those existing in π-involved systems. However, this classical model has come under fire in revealing specific issues such as substituent effects, due to its roughness; and it has been followed in past decades by sundry explanations which sometimes bring in nebulous descriptions. In this account, we try to summarise and present a unified model for describing and analysing the binding mechanism of such systems from the viewpoint of energy decomposition. We also emphasise a commonly ignored factor - orbital interaction, pointing out that the noncovalent intermolecular orbital interactions actually exhibit similar bonding and antibonding phenomena as those in covalent bonds.

Interaction of size-selected gold nanoclusters with dopamine

NASA Astrophysics Data System (ADS)

Montone, Georgia R.; Hermann, Eric; Kandalam, Anil K.

2016-12-01

We present density functional theory based results on the interaction of size-selected gold nanoclusters, Au10 and Au20, with dopamine molecule. The gold clusters interact strongly with the nitrogen site of dopamine, thereby forming stable gold-dopamine complexes. Our calculations further show that there is no site specificity on the planar Au10 cluster with all the edge gold atoms equally preferred. On the other hand, in the pyramidal Au20 cluster, the vertex metal atom is the most active site. As the size increased from Au10 to Au20, the interaction strength has shown a declining trend. The effect of aqueous environment on the interaction strengths were also studied by solvation model. It is found that the presence of solvent water stabilizes the interaction between the metal cluster and dopamine molecule, even though for Au10 cluster the energy ordering of the isomers changed from that of the gas-phase.

Interaction of Human Enteric Viruses with Microbial Compounds: Implication for Virus Persistence and Disinfection Treatments.

PubMed

Waldman, Prunelle; Meseguer, Alba; Lucas, Françoise; Moulin, Laurent; Wurtzer, Sébastien

2017-12-05

Although the interaction between phages and bacteria has already been well described, it only recently emerged that human viruses also interact with bacteria in the mammalian gut. We studied whether this interaction could occur in tap water and thus confer enteric viruses protection against temperature and the classical disinfection treatments used in drinking water production. We demonstrated that the addition of lipopolysaccharide or peptidoglycan of bacterial origin to enterovirus provides thermal protection through stabilization of the viral capsid. This interaction plays a role when viruses are exposed to disinfection that targets the capsid, but less so when the virus genome is directly targeted. The interaction seems to be serotype-specific, suggesting that the capsid protein sequence could be important. The protection is linked to a direct association between viral particles and bacterial compounds as observed by microscopy. These results show that bacterial compounds present in the environment can affect virus inactivation.

Role of the noise on the transient dynamics of an ecosystem of interacting species

NASA Astrophysics Data System (ADS)

Spagnolo, B.; La Barbera, A.

2002-11-01

We analyze the transient dynamics of an ecosystem described by generalized Lotka-Volterra equations in the presence of a multiplicative noise and a random interaction parameter between the species. We consider specifically three cases: (i) two competing species, (ii) three interacting species (one predator-two preys), (iii) n-interacting species. The interaction parameter in case (i) is a stochastic process which obeys a stochastic differential equation. We find noise delayed extinction of one of two species, which is akin to the noise-enhanced stability phenomenon. Other two noise-induced effects found are temporal oscillations and spatial patterns of the two competing species. In case (ii) the noise induces correlated spatial patterns of the predator and of the two preys concentrations. Finally, in case (iii) we find the asymptotic behavior of the time average of the ith population when the ecosystem is composed of a great number of interacting species.

Cation specific binding with protein surface charges

PubMed Central

Hess, Berk; van der Vegt, Nico F. A.

2009-01-01

Biological organization depends on a sensitive balance of noncovalent interactions, in particular also those involving interactions between ions. Ion-pairing is qualitatively described by the law of “matching water affinities.” This law predicts that cations and anions (with equal valence) form stable contact ion pairs if their sizes match. We show that this simple physical model fails to describe the interaction of cations with (molecular) anions of weak carboxylic acids, which are present on the surfaces of many intra- and extracellular proteins. We performed molecular simulations with quantitatively accurate models and observed that the order K+ < Na+ < Li+ of increasing binding affinity with carboxylate ions is caused by a stronger preference for forming weak solvent-shared ion pairs. The relative insignificance of contact pair interactions with protein surfaces indicates that thermodynamic stability and interactions between proteins in alkali salt solutions is governed by interactions mediated through hydration water molecules. PMID:19666545

Genotype x environment interaction and growth stability of several elm clones resistant to Dutch elm disease

Treesearch

Alberto Santini; Francesco Pecori; Alessia L. Pepori; Luisa Ghelardini

2012-01-01

The elm breeding program carried out in Italy at the Institute of Plant Protection - Consiglio Nazionale delle Ricercje (CNR) during the last 40 years aimed to develop Dutch elm disease (DED)-resistant elm selections specific to the Mediterranean environment. The need for genotypes adapted to Mediterranean conditions was evident from the poor performance of the Dutch...

Moderator Effects of Working Memory on the Stability of ADHD Symptoms by Dopamine Receptor Gene Polymorphisms during Development

ERIC Educational Resources Information Center

Trampush, Joey W.; Jacobs, Michelle M.; Hurd, Yasmin L.; Newcorn, Jeffrey H.; Halperin, Jeffrey M.

2014-01-01

We tested the hypothesis that dopamine D1 and D2 receptor gene (DRD1 and DRD2, respectively) polymorphisms and the development of working memory skills can interact to influence symptom change over 10 years in children with attention-deficit/hyperactivity disorder (ADHD). Specifically, we examined whether improvements in working memory maintenance…

Influence of evolution on the stability of ecological communities.

PubMed

Loeuille, Nicolas

2010-12-01

In randomly assembled communities, diversity is known to have a destabilizing effect. Evolution may affect this result, but our theoretical knowledge of its role is mostly limited to models of small food webs. In the present article, I introduce evolution in a two-species Lotka-Volterra model in which I vary the interaction type and the cost constraining evolution. Regardless of the cost type, evolution tends to stabilize the dynamics more often in trophic interactions than for mutualism or competition. I then use simulations to study the effect of evolution in larger communities that contain all interaction types. Results suggest that evolution usually stabilizes the dynamics. This stabilizing effect is stronger when evolution affects trophic interactions, but happens for all interaction types. Stabilization decreases with diversity and evolution becomes destabilizing in very diverse communities. This suggests that evolution may not counteract the destabilizing effect of diversity observed in random communities. © 2010 Blackwell Publishing Ltd/CNRS.

Physical and Chemical Toeholds for Exoplanet Bioastronomy

NASA Technical Reports Server (NTRS)

Hoehler, Tori

2013-01-01

If a search for exoplanet life were mounted today, the likely focus would be to detect oxygen (or ozone) in the atmosphere of a water-bearing rocky planet orbiting roughly 1AU from a G-type star. This appropriately conservative and practical default is necessary in large part because biological input on the question of where and how to look for life has progressed little beyond a purely empirical reliance on the example of terrestrial biology. However, fundamental physical and chemical considerations may impose significant yet universal constraints on biological potential. The liquid water + oxygen paradigm will be considered as an example, with a focus on the question, is liquid water a prerequisite for life? . Life requires a solvent to mediate interactions among biological molecules. A key class of these interactions is molecular recognition with high specificity, which is essential for high fidelity catalysis and (especially) information processing. For example, to correctly reproduce a string consisting of 600,000 units of information (e.g., 600 kilobases, equivalent to the genome of the smallest free living terrestrial organisms) with a 90% success rate requires specificity greater than 10(exp 7):1 for the target molecule vs. incorrect alternatives. Such specificity requires (i) that the correct molecular association is energetically stabilized by at least 40 kJ/mol relative to alternatives, and (ii) that the system is able to sample among possible states (alternative molecular associations) rapidly enough to allow the system to fall under thermodynamic control and express the energetic stabilization. We argue that electrostatic interactions are required to confer the necessary energetic stabilization vs. a large library of molecular alternatives, and that a solvent with polarity and dielectric properties comparable to water is required for the system to sample among possible states and express thermodynamic control. Electrostatic associations can be made in non-polar solvents, but the resulting complexes are too stable to be "unmade" with sufficient frequency to confer thermodynamic control on the system. Such considerations suggest that water, or a solvent with properties very like water, is necessary to support high-fidelity information processing a feature that must be common to all biology and can therefore be considered a critical prerequisite for life.

Fetal bovine serum influences the stability and bioactivity of resveratrol analogues: A polyphenol-protein interaction approach.

PubMed

Tang, Fen; Xie, Yixi; Cao, Hui; Yang, Hua; Chen, Xiaoqing; Xiao, Jianbo

2017-03-15

Fetal bovine serum (FBS) is a universal growth supplement of cell and tissue culture media. Herein, the influences of FBS on the stability and antioxidant activity of 21 resveratrol analogues were investigated using a polyphenol-protein interaction approach. The structure-stability relationships of resveratrol analogues in FBS showed a clear decrease in the stability of hydroxylated resveratrol analogues in the order: resorcinol-type>pyrogallol-type>catechol-type. The glycosylation and methoxylation of resveratrol analogues enhanced their stability. A linear relationship between the stability of resveratrol analogues in FBS and the affinity of resveratrol analogues-FBS interaction was found. The oxidation process is not the only factor governing the stability of resveratrol analogues in FBS. These results facilitated the insightful investigation of the role of polyphenol-protein interactions in serum, thereby providing some fundamental clues for future clinical research and pharmacological studies on natural small molecules. Copyright © 2016 Elsevier Ltd. All rights reserved.

Rational modification of protein stability by targeting surface sites leads to complicated results

PubMed Central

Xiao, Shifeng; Patsalo, Vadim; Shan, Bing; Bi, Yuan; Green, David F.; Raleigh, Daniel P.

2013-01-01

The rational modification of protein stability is an important goal of protein design. Protein surface electrostatic interactions are not evolutionarily optimized for stability and are an attractive target for the rational redesign of proteins. We show that surface charge mutants can exert stabilizing effects in distinct and unanticipated ways, including ones that are not predicted by existing methods, even when only solvent-exposed sites are targeted. Individual mutation of three solvent-exposed lysines in the villin headpiece subdomain significantly stabilizes the protein, but the mechanism of stabilization is very different in each case. One mutation destabilizes native-state electrostatic interactions but has a larger destabilizing effect on the denatured state, a second removes the desolvation penalty paid by the charged residue, whereas the third introduces unanticipated native-state interactions but does not alter electrostatics. Our results show that even seemingly intuitive mutations can exert their effects through unforeseen and complex interactions. PMID:23798426

Direct observation of individual particle armored bubble interaction, stability, and coalescence dynamics.

PubMed

Tan, Sin-Ying; Ata, Seher; Wanless, Erica J

2013-07-18

The interactions between two individual particle-stabilized bubbles were investigated, in the absence of surfactant, using a combination of coalescence rig and high-speed video camera. This combination allows the visualization of bubble coalescence dynamics which provide information on bubble stability. Experimental data suggested that bubble stability is enhanced by both the adsorption of particles at the interface as indicated by the long induction time and the increase in damping coefficient at high surface coverage. The interaction between an armored bubble and a bare bubble (asymmetric interaction) can be destabilized through the addition of a small amount of salt, which suggested that electrostatic interactions play a significant role in bubble stability. Interestingly, the DLVO theory cannot be used to describe the bubble stability in the case of a symmetric interaction as coalescence was inhibited at 0.1 M KCl in both the absence and presence of particles at the interfaces. Furthermore, bubbles can also be destabilized by increasing the particle hydrophobicity. This behavior is due to thinner liquid films between bubbles and an increase in film drainage rate. The fraction of particles detached from the bubble surface after film rupture was found to be very similar within the range of solution ionic strength, surface coverage, and particle hydrophobicity studied. This lack of dependence implies that the kinetic energy generated by the coalescing bubbles is larger than the attachment energy of the particles and dominates the detachment process. This study illuminates the stability behavior of individual particle-stabilized bubbles and has potential impact on processes which involve their interaction.

Sequences Flanking the Gephyrin-Binding Site of GlyRβ Tune Receptor Stabilization at Synapses

PubMed Central

Grünewald, Nora; Salvatico, Charlotte; Kress, Vanessa

2018-01-01

Abstract The efficacy of synaptic transmission is determined by the number of neurotransmitter receptors at synapses. Their recruitment depends upon the availability of postsynaptic scaffolding molecules that interact with specific binding sequences of the receptor. At inhibitory synapses, gephyrin is the major scaffold protein that mediates the accumulation of heteromeric glycine receptors (GlyRs) via the cytoplasmic loop in the β-subunit (β-loop). This binding involves high- and low-affinity interactions, but the molecular mechanism of this bimodal binding and its implication in GlyR stabilization at synapses remain unknown. We have approached this question using a combination of quantitative biochemical tools and high-density single molecule tracking in cultured rat spinal cord neurons. The high-affinity binding site could be identified and was shown to rely on the formation of a 310-helix C-terminal to the β-loop core gephyrin-binding motif. This site plays a structural role in shaping the core motif and represents the major contributor to the synaptic confinement of GlyRs by gephyrin. The N-terminal flanking sequence promotes lower affinity interactions by occupying newly identified binding sites on gephyrin. Despite its low affinity, this binding site plays a modulatory role in tuning the mobility of the receptor. Together, the GlyR β-loop sequences flanking the core-binding site differentially regulate the affinity of the receptor for gephyrin and its trapping at synapses. Our experimental approach thus bridges the gap between thermodynamic aspects of receptor-scaffold interactions and functional receptor stabilization at synapses in living cells. PMID:29464196

Structural insight into the role of Gln293Met mutation on the Peloruside A/Laulimalide association with αβ-tubulin from molecular dynamics simulations, binding free energy calculations and weak interactions analysis.

PubMed

Zúñiga, Matías A; Alderete, Joel B; Jaña, Gonzalo A; Jiménez, Verónica A

2017-07-01

Peloruside A (PLA) and Laulimalide (LAU) are novel microtubule-stabilizing agents with promising properties against different cancer types. These ligands share a non-taxoid binding site at the outer surface of β-tubulin and promote microtubule stabilization by bridging two adjacent αβ-tubulin dimers from parallel protofilaments. Recent site-directed mutagenesis experiments confirmed the existence of a unique β-tubulin site mutation (Gln293Met) that specifically increased the activity of PLA and caused resistance to LAU, without affecting the stability of microtubules in the absence of the ligands. In this work, fully atomistic molecular dynamics simulations were carried out to examine the PLA and LAU association with native and mutated αβ-tubulin in the search for structural and energetic evidence to explain the role of Gln293Met mutation on determining the activity of these ligands. Our results revealed that Gln293Met mutation induced the loss of relevant LAU-tubulin contacts but exerted negligible changes in the interaction networks responsible for PLA-tubulin association. Binding free energy calculations (MM/GBSA and MM/PBSA), and weak interaction analysis (aNCI) predicted an increased affinity for PLA, and a weakened association for LAU after mutation, thus suggesting that Gln293Met mutation exerts its action by a modulation of drug-tubulin interactions. These results are valuable to increase understanding about PLA and LAU activity and to assist the future design of novel agents targeting the PLA/LAU binding pocket.

Structural insight into the role of Gln293Met mutation on the Peloruside A/Laulimalide association with αβ-tubulin from molecular dynamics simulations, binding free energy calculations and weak interactions analysis

NASA Astrophysics Data System (ADS)

Zúñiga, Matías A.; Alderete, Joel B.; Jaña, Gonzalo A.; Jiménez, Verónica A.

2017-07-01

Peloruside A (PLA) and Laulimalide (LAU) are novel microtubule-stabilizing agents with promising properties against different cancer types. These ligands share a non-taxoid binding site at the outer surface of β-tubulin and promote microtubule stabilization by bridging two adjacent αβ-tubulin dimers from parallel protofilaments. Recent site-directed mutagenesis experiments confirmed the existence of a unique β-tubulin site mutation (Gln293Met) that specifically increased the activity of PLA and caused resistance to LAU, without affecting the stability of microtubules in the absence of the ligands. In this work, fully atomistic molecular dynamics simulations were carried out to examine the PLA and LAU association with native and mutated αβ-tubulin in the search for structural and energetic evidence to explain the role of Gln293Met mutation on determining the activity of these ligands. Our results revealed that Gln293Met mutation induced the loss of relevant LAU-tubulin contacts but exerted negligible changes in the interaction networks responsible for PLA-tubulin association. Binding free energy calculations (MM/GBSA and MM/PBSA), and weak interaction analysis (aNCI) predicted an increased affinity for PLA, and a weakened association for LAU after mutation, thus suggesting that Gln293Met mutation exerts its action by a modulation of drug-tubulin interactions. These results are valuable to increase understanding about PLA and LAU activity and to assist the future design of novel agents targeting the PLA/LAU binding pocket.

Interdomain Contacts Control Native State Switching of RfaH on a Dual-Funneled Landscape

PubMed Central

Ramírez-Sarmiento, César A.; Noel, Jeffrey K.; Valenzuela, Sandro L.; Artsimovitch, Irina

2015-01-01

RfaH is a virulence factor from Escherichia coli whose C-terminal domain (CTD) undergoes a dramatic α-to-β conformational transformation. The CTD in its α-helical fold is stabilized by interactions with the N-terminal domain (NTD), masking an RNA polymerase binding site until a specific recruitment site is encountered. Domain dissociation is triggered upon binding to DNA, allowing the NTD to interact with RNA polymerase to facilitate transcription while the CTD refolds into the β-barrel conformation that interacts with the ribosome to activate translation. However, structural details of this transformation process in the context of the full protein remain to be elucidated. Here, we explore the mechanism of the α-to-β conformational transition of RfaH in the full-length protein using a dual-basin structure-based model. Our simulations capture several features described experimentally, such as the requirement of disruption of interdomain contacts to trigger the α-to-β transformation, confirms the roles of previously indicated residues E48 and R138, and suggests a new important role for F130, in the stability of the interdomain interaction. These native basins are connected through an intermediate state that builds up upon binding to the NTD and shares features from both folds, in agreement with previous in silico studies of the isolated CTD. We also examine the effect of RNA polymerase binding on the stabilization of the β fold. Our study shows that native-biased models are appropriate for interrogating the detailed mechanisms of structural rearrangements during the dramatic transformation process of RfaH. PMID:26230837

Disruption of Rhodopsin Dimerization with Synthetic Peptides Targeting an Interaction Interface*

PubMed Central

Jastrzebska, Beata; Chen, Yuanyuan; Orban, Tivadar; Jin, Hui; Hofmann, Lukas; Palczewski, Krzysztof

2015-01-01

Although homo- and heterodimerizations of G protein-coupled receptors (GPCRs) are well documented, GPCR monomers are able to assemble in different ways, thus causing variations in the interactive interface between receptor monomers among different GPCRs. Moreover, the functional consequences of this phenomenon, which remain to be clarified, could be specific for different GPCRs. Synthetic peptides derived from transmembrane (TM) domains can interact with a full-length GPCR, blocking dimer formation and affecting its function. Here we used peptides corresponding to TM helices of bovine rhodopsin (Rho) to investigate the Rho dimer interface and functional consequences of its disruption. Incubation of Rho with TM1, TM2, TM4, and TM5 peptides in rod outer segment (ROS) membranes shifted the resulting detergent-solubilized protein migration through a gel filtration column toward smaller molecular masses with a reduced propensity for dimer formation in a cross-linking reaction. Binding of these TM peptides to Rho was characterized by both mass spectrometry and a label-free assay from which dissociation constants were calculated. A BRET (bioluminescence resonance energy transfer) assay revealed that the physical interaction between Rho molecules expressed in membranes of living cells was blocked by the same four TM peptides identified in our in vitro experiments. Although disruption of the Rho dimer/oligomer had no effect on the rates of G protein activation, binding of Gt to the activated receptor stabilized the dimer. However, TM peptide-induced disruption of dimer/oligomer decreased receptor stability, suggesting that Rho supramolecular organization could be essential for ROS stabilization and receptor trafficking. PMID:26330551

Monocyte-endothelial adhesion in chronic rheumatoid arthritis. In situ detection of selectin and integrin-dependent interactions.

PubMed Central

Grober, J S; Bowen, B L; Ebling, H; Athey, B; Thompson, C B; Fox, D A; Stoolman, L M

1993-01-01

Blood monocytes are the principal reservoir for tissue macrophages in rheumatoid synovitis. Receptor-mediated adhesive interactions between circulating cells and the synovial venules initiate recruitment. These interactions have been studied primarily in cultured endothelial cells. Thus the functional activities of specific adhesion receptors, such as the endothelial selectins and the leukocytic integrins, have not been evaluated directly in diseased tissues. We therefore examined monocyte-microvascular interactions in rheumatoid synovitis by modifying the Stamper-Woodruff frozen section binding assay initially developed to study lymphocyte homing. Specific binding of monocytes to venules lined by low or high endothelium occurred at concentrations as low as 5 x 10(5) cells/ml. mAbs specific for P-selectin (CD62, GMP-140/PADGEM) blocked adhesion by > 90% in all synovitis specimens examined. In contrast, P-selectin-mediated adhesion to the microvasculature was either lower or absent in frozen sections of normal foreskin and placenta. mAbs specific for E-selectin (ELAM-1) blocked 20-50% of monocyte attachment in several RA synovial specimens but had no effect in others. mAbs specific for LFA-1, Mo1/Mac 1, the integrin beta 2-chain, and L-selectin individually inhibited 30-40% of adhesion. An mAb specific for the integrin beta 1-chain inhibited the attachment of elutriated monocytes up to 20%. We conclude that P-selectin associated with the synovial microvasculature initiates shear-resistant adhesion of monocytes in the Stamper-Woodruff assay and stabilizes bonds formed by other selectins and the integrins. Thus the frozen section binding assay permits direct evaluation of leukocyte-microvascular adhesive interactions in inflamed tissues and suggests a prominent role for P-selectin in monocyte recruitment in vivo. Images PMID:7685772

Directional, stabilizing, and disruptive trait selection as alternative mechanisms for plant community assembly.

PubMed

Rolhauser, Andrés G; Pucheta, Eduardo

2017-03-01

How plant functional traits (e.g., seed mass) drive species abundance within communities remains an unsolved question. Borrowing concepts from natural selection theory, we propose that trait-abundance relationships can generally correspond to one of three modes of trait selection: directional (a rectilinear relationship, where species at one end of a trait axis are most abundant), stabilizing (an n-shaped relationship), and disruptive (a u-shaped relationship). Stabilizing selection (i.e., the functional convergence of abundant species) would result from positive density-dependent interactions (e.g., facilitation) or due to generalized trade-offs in resource acquisition/use, while disruptive selection (i.e., the divergence of abundant species) would result from negative density-dependent interactions (e.g., competition) or due to environmental heterogeneity. These selection modes can be interpreted as proxies for community-level trait-fitness functions, which establish the degree to which traits are truly "functional". We searched for selection modes in a desert annual-plant community in Argentina (which was divided into winter and summer guilds) to test the hypothesis that the relative importance of disruptive mechanisms (competition, disturbances) decreases with the increase of abiotic stress, a stabilizing agent. Average density was analyzed as a function of eight traits generally linked to resource acquisition and competitive ability (maximum plant height, leaf size, specific leaf area, specific root length), resource retention and stress tolerance (leaf dissection, leaf dry matter content, specific root volume), and regeneration (seed mass) using multiple quadratic-regression models. Trait selection was stabilizing and/or directional when the environment was harshest (winter) and disruptive and/or directional when conditions were milder (summer). Selection patterns differed between guilds for two important traits: plant height and seed mass. These results suggest that abiotic stress may drive within-community functional convergence independently of the trait considered, opposing the view that some traits may be inherently convergent while others divergent. Our quadratic model-based approach provides standardized metrics of both linear and nonlinear selection that may allow simple comparisons among communities subjected to contrasting environmental conditions. These concepts, rooted in natural selection theory, may clarify the functional link between traits and species abundance, and thus help untangle the contributions of deterministic and stochastic processes on community assembly. © 2017 by the Ecological Society of America.

Enhanced assembly and colloidal stabilization of primate erythroparvovirus 1 virus-like particles for improved surface engineering.

PubMed

Sánchez-Rodríguez, Sandra Paola; Morán-García, Areli del Carmen; Bolonduro, Olurotimi; Dordick, Jonathan S; Bustos-Jaimes, Ismael

2016-04-15

Virus-like particles (VLPs) are the product of the self-assembly, either in vivo or in vitro, of structural components of viral capsids. These particles are excellent scaffolds for surface display of biomolecules that can be used in vaccine development and tissue-specific drug delivery. Surface engineering of VLPs requires structural stability and chemical reactivity. Herein, we report the enhanced assembly, colloidal stabilization and fluorescent labeling of primate erythroparvovirus 1 (PE1V), generally referred to as parvovirus B19. In vitro assembly of the VP2 protein of PE1V produces VLPs, which are prone to flocculate and hence undergo limited chemical modification by thiol-specific reagents like the fluorogenic monobromobimane (mBBr). We determined that the addition of 0.2M l-arginine during the assembly process produced an increased yield of soluble VLPs with good dispersion stability. Fluorescent labeling of VLPs suspended in phosphate buffered saline (PBS) added with 0.2M l-Arg was achieved in significantly shorter times than the flocculated VLPs assembled in only PBS buffer. Finally, to demonstrate the potential application of this approach, mBBr-labeled VLPs were successfully used to tag human hepatoma HepG2 cells. This new method for assembly and labeling PE1V VLPs eases its applications and provides insights on the manipulation of this biomaterial for further developments. Application of virus-derived biomaterials sometimes requires surface modification for diverse purposes, including enhanced cell-specific interaction, the inclusion of luminescent probes for bioimaging, or the incorporation of catalytic properties for the production of enzyme nanocarriers. In this research, we reported for the first time the colloidal stabilization of the primate erythroparvovirus 1 (PE1V) virus-like particles (VLPs). Also, we report the chemical modification of the natural Cys residues located on the surface of these VLPs with a fluorescent probe, as well as its application for tagging hepatoma cells in vitro. Keeping in mind that PE1V is a human pathogen, virus-host interactions already exist in human cells, and they can be exploited for therapeutic and research aims. This study will impact on the speed in which the scientific community will be able to manipulate PE1V VLPs for diverse purposes. Additionally, this study may provide insights on the colloidal properties of these VLPs as well as in the effect of different protein additives used for protein stabilization. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Effects of external stress field on the charge stability of nitrogen vacancy centers in diamond

NASA Astrophysics Data System (ADS)

Yao, Miao-Miao; Zhu, Tian-Yuan; Shu, Da-Jun

2017-07-01

The interaction of the atom-like defects in semiconductors with external fields provides an avenue to quantum information processing and nanoscale sensors. Meanwhile, external fields may induce instability of the desired charge state of the defects. It is essential to understand how the charge state of a defect is affected by external fields that introduced in diverse applications. In this letter, we explore the stability of the negatively charged state (NV-) and the neutral state (NV0) of the nitrogen vacancy (NV) center in diamond under stress by first-principles calculations. We find that the relative stability of NV- to NV0 is always reduced by the stress if the NV center is free to relax its orientation. Once the NV center has formed and retains its orientation, however, the relative stability of NV- can be always enhanced by compressive stress along its trigonal symmetry axis. We believe that the results are not only significant for control of the charge stability of NV center but also enlightening for applications based on specific charge states of other kinds of defects in the stress field.

Three-dimensional reconstructions come to life--interactive 3D PDF animations in functional morphology.

PubMed

van de Kamp, Thomas; dos Santos Rolo, Tomy; Vagovič, Patrik; Baumbach, Tilo; Riedel, Alexander

2014-01-01

Digital surface mesh models based on segmented datasets have become an integral part of studies on animal anatomy and functional morphology; usually, they are published as static images, movies or as interactive PDF files. We demonstrate the use of animated 3D models embedded in PDF documents, which combine the advantages of both movie and interactivity, based on the example of preserved Trigonopterus weevils. The method is particularly suitable to simulate joints with largely deterministic movements due to precise form closure. We illustrate the function of an individual screw-and-nut type hip joint and proceed to the complex movements of the entire insect attaining a defence position. This posture is achieved by a specific cascade of movements: Head and legs interlock mutually and with specific features of thorax and the first abdominal ventrite, presumably to increase the mechanical stability of the beetle and to maintain the defence position with minimal muscle activity. The deterministic interaction of accurately fitting body parts follows a defined sequence, which resembles a piece of engineering.

Three-Dimensional Reconstructions Come to Life – Interactive 3D PDF Animations in Functional Morphology

PubMed Central

van de Kamp, Thomas; dos Santos Rolo, Tomy; Vagovič, Patrik; Baumbach, Tilo; Riedel, Alexander

2014-01-01

Digital surface mesh models based on segmented datasets have become an integral part of studies on animal anatomy and functional morphology; usually, they are published as static images, movies or as interactive PDF files. We demonstrate the use of animated 3D models embedded in PDF documents, which combine the advantages of both movie and interactivity, based on the example of preserved Trigonopterus weevils. The method is particularly suitable to simulate joints with largely deterministic movements due to precise form closure. We illustrate the function of an individual screw-and-nut type hip joint and proceed to the complex movements of the entire insect attaining a defence position. This posture is achieved by a specific cascade of movements: Head and legs interlock mutually and with specific features of thorax and the first abdominal ventrite, presumably to increase the mechanical stability of the beetle and to maintain the defence position with minimal muscle activity. The deterministic interaction of accurately fitting body parts follows a defined sequence, which resembles a piece of engineering. PMID:25029366

RNA degradation and models for post-transcriptional gene-silencing.

PubMed

Meins, F

2000-06-01

Post-transcriptional gene silencing (PTGS) is a form of stable but potentially reversible epigenetic modification, which frequently occurs in transgenic plants. The interaction in trans of genes with similar transcribed sequences results in sequence-specific degradation of RNAs derived from the genes involved. Highly expressed single-copy loci, transcribed inverted repeats, and poorly transcribed complex loci can act as sources of signals that trigger PTGS. In some cases, mobile, sequence-specific silencing signals can move from cell to cell or even over long distances in the plant. Several current models hold that silencing signals are 'aberrant' RNAs (aRNA), which differ in some way from normal mRNAs. The most likely candidates are small antisense RNAs (asRNA) and double-stranded RNAs (dsRNA). Direct evidence that these or other aRNAs found in silent tissues can induce PTGS is still lacking. Most current models assume that silencing signals interact with target RNAs in a sequence-specific fashion. This results in degradation, usually in the cytoplasm, by exonucleolytic as well as endonucleolytic pathways, which are not necessarily PTGS-specific. Biochemical-switch models hold that the silent state is maintained by a positive auto-regulatory loop. One possibility is that concentrations of hypothetical silencing signals above a critical threshold trigger their own production by self-replication, by degradation of target RNAs, or by a combination of both mechanisms. These models can account for the stability, reversibility and multiplicity of silent states; the strong influence of transcription rate of target genes on the incidence and stability of silencing, and the amplification and systemic propagation of motile silencing signals.

On the Importance of Polar Interactions for Complexes Containing Intrinsically Disordered Proteins

PubMed Central

Wong, Eric T. C.; Na, Dokyun; Gsponer, Jörg

2013-01-01

There is a growing recognition for the importance of proteins with large intrinsically disordered (ID) segments in cell signaling and regulation. ID segments in these proteins often harbor regions that mediate molecular recognition. Coupled folding and binding of the recognition regions has been proposed to confer high specificity to interactions involving ID segments. However, researchers recently questioned the origin of the interaction specificity of ID proteins because of the overrepresentation of hydrophobic residues in their interaction interfaces. Here, we focused on the role of polar and charged residues in interactions mediated by ID segments. Making use of the extended nature of most ID segments when in complex with globular proteins, we first identified large numbers of complexes between globular proteins and ID segments by using radius-of-gyration-based selection criteria. Consistent with previous studies, we found the interfaces of these complexes to be enriched in hydrophobic residues, and that these residues contribute significantly to the stability of the interaction interface. However, our analyses also show that polar interactions play a larger role in these complexes than in structured protein complexes. Computational alanine scanning and salt-bridge analysis indicate that interfaces in ID complexes are highly complementary with respect to electrostatics, more so than interfaces of globular proteins. Follow-up calculations of the electrostatic contributions to the free energy of binding uncovered significantly stronger Coulombic interactions in complexes harbouring ID segments than in structured protein complexes. However, they are counter-balanced by even higher polar-desolvation penalties. We propose that polar interactions are a key contributing factor to the observed high specificity of ID segment-mediated interactions. PMID:23990768

Substituent Effects on the Self-Assembly/Coassembly and Hydrogelation of Phenylalanine Derivatives.

PubMed

Liyanage, Wathsala; Nilsson, Bradley L

2016-01-26

Supramolecular hydrogels derived from the self-assembly of organic molecules have been exploited for applications ranging from drug delivery to tissue engineering. The relationship between the structure of the assembly motif and the emergent properties of the resulting materials is often poorly understood, impeding rational approaches for the creation of next-generation materials. Aromatic π-π interactions play a significant role in the self-assembly of many supramolecular hydrogelators, but the exact nature of these interactions lacks definition. Conventional models that describe π-π interactions rely on quadrupolar electrostatic interactions between neighboring aryl groups in the π-system. However, recent experimental and computational studies reveal the potential importance of local dipolar interactions between elements of neighboring aromatic rings in stabilizing π-π interactions. Herein, we examine the nature of π-π interactions in the self- and coassembly of Fmoc-Phe-derived hydrogelators by systematically varying the electron-donating or electron-withdrawing nature of the side chain benzyl substituents and correlating these effects to the emergent assembly and gelation properties of the systems. These studies indicate a significant role for stabilizing dipolar interactions between neighboring benzyl groups in the assembled materials. Additional evidence for specific dipolar interactions is provided by high-resolution crystal structures obtained from dynamic transition of gel fibrils to crystals for several of the self-assembled/coassembled Fmoc-Phe derivatives. In addition to electronic effects, steric properties also have a significant effect on the interaction between neighboring benzyl groups in these assembled systems. These findings provide significant insight into the structure-function relationship for Fmoc-Phe-derived hydrogelators and give cues for the design of next-generation materials with desired emergent properties.

Orientation-dependent hydration structures at yttria-stabilized cubic zirconia surfaces

DOE PAGES

Hou, Binyang; Kim, Seunghyun; Kim, Taeho; ...

2016-11-30

Water interaction with surfaces is very important and plays key roles in many natural and technological processes. Because the experimental challenges that arise when studying the interaction water with specific crystalline surfaces, most studies on metal oxides have focused on powder samples, which averaged the interaction over different crystalline surfaces. As a result, studies on the crystal orientation-dependent interaction of water with metal oxides are rarely available in the literature. In this work, water adsorption at 8 mol % yttria-stabilized cubic single crystal zirconia (100) and (111) surfaces was studied in terms of interfacial hydration structures using high resolution X-raymore » reflectivity measurements. The interfacial electron density profiles derived from the structure factor analysis of the measured data show the existence of multiple layers of adsorbed water with additional peculiar metal adsorption near the oxide surfaces.Surface relaxation, depletion, and interaction between the adsorbed layers and bulk water are found to vary greatly between the two surfaces and are also different when compared to the previously studied (110) surface. The fractional ratio between chemisorbed and physisorbed water species were also quantitatively estimated, which turned out to vary dramatically from surface to surface. Finally, the result gives us a unique opportunity to reconsider the simplified 2:1 relation between chemisorption and physisorption, originally proposed by Morimoto et al. based on the adsorption isotherms of water on powder metal oxide samples.« less

Linking Findings in Microfluidics to Membrane Emulsification Process Design: The Importance of Wettability and Component Interactions with Interfaces

PubMed Central

Schroën, Karin; Ferrando, Montse; de Lamo-Castellví, Silvia; Sahin, Sami; Güell, Carme

2016-01-01

In microfluidics and other microstructured devices, wettability changes, as a result of component interactions with the solid wall, can have dramatic effects. In emulsion separation and emulsification applications, the desired behavior can even be completely lost. Wettability changes also occur in one phase systems, but the effect is much more far-reaching when using two-phase systems. For microfluidic emulsification devices, this can be elegantly demonstrated and quantified for EDGE (Edge-base Droplet GEneration) devices that have a specific behavior that allows us to distinguish between surfactant and liquid interactions with the solid surface. Based on these findings, design rules can be defined for emulsification with any micro-structured emulsification device, such as direct and premix membrane emulsification. In general, it can be concluded that mostly surface interactions increase the contact angle toward 90°, either through the surfactant, or the oil that is used. This leads to poor process stability, and very limited pressure ranges at which small droplets can be made in microfluidic systems, and cross-flow membrane emulsification. In a limited number of cases, surface interactions can also lead to lower contact angles, thereby increasing the operational stability. This paper concludes with a guideline that can be used to come to the appropriate combination of membrane construction material (or any micro-structured device), surfactants and liquids, in combination with process conditions. PMID:27187484

Structure-Energy Relationships of Halogen Bonds in Proteins.

PubMed

Scholfield, Matthew R; Ford, Melissa Coates; Carlsson, Anna-Carin C; Butta, Hawera; Mehl, Ryan A; Ho, P Shing

2017-06-06

The structures and stabilities of proteins are defined by a series of weak noncovalent electrostatic, van der Waals, and hydrogen bond (HB) interactions. In this study, we have designed and engineered halogen bonds (XBs) site-specifically to study their structure-energy relationship in a model protein, T4 lysozyme. The evidence for XBs is the displacement of the aromatic side chain toward an oxygen acceptor, at distances that are equal to or less than the sums of their respective van der Waals radii, when the hydroxyl substituent of the wild-type tyrosine is replaced by a halogen. In addition, thermal melting studies show that the iodine XB rescues the stabilization energy from an otherwise destabilizing substitution (at an equivalent noninteracting site), indicating that the interaction is also present in solution. Quantum chemical calculations show that the XB complements an HB at this site and that solvent structure must also be considered in trying to design molecular interactions such as XBs into biological systems. A bromine substitution also shows displacement of the side chain, but the distances and geometries do not indicate formation of an XB. Thus, we have dissected the contributions from various noncovalent interactions of halogens introduced into proteins, to drive the application of XBs, particularly in biomolecular design.

Characterization of Monobody Scaffold Interactions with Ligand via Force Spectroscopy and Steered Molecular Dynamics

NASA Astrophysics Data System (ADS)

Cheung, Luthur Siu-Lun; Shea, Daniel J.; Nicholes, Nathan; Date, Amol; Ostermeier, Marc; Konstantopoulos, Konstantinos

2015-02-01

Monobodies are antibody alternatives derived from fibronectin that are thermodynamically stable, small in size, and can be produced in bacterial systems. Monobodies have been engineered to bind a wide variety of target proteins with high affinity and specificity. Using alanine-scanning mutagenesis simulations, we identified two scaffold residues that are critical to the binding interaction between the monobody YS1 and its ligand, maltose-binding protein (MBP). Steered molecular dynamics (SMD) simulations predicted that the E47A and R33A mutations in the YS1 scaffold substantially destabilize the YS1-MBP interface by reducing the bond rupture force and the lifetime of single hydrogen bonds. SMD simulations further indicated that the R33A mutation weakens the hydrogen binding between all scaffold residues and MBP and not just between R33 and MBP. We validated the simulation data and characterized the effects of mutations on YS1-MBP binding by using single-molecule force spectroscopy and surface plasmon resonance. We propose that interfacial stability resulting from R33 of YS1 stacking with R344 of MBP synergistically stabilizes both its own bond and the interacting scaffold residues of YS1. Our integrated approach improves our understanding of the monobody scaffold interactions with a target, thus providing guidance for the improved engineering of monobodies.

Parabolized Stability Equations analysis of nonlinear interactions with forced eigenmodes to control subsonic jet instabilities

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

Itasse, Maxime, E-mail: Maxime.Itasse@onera.fr; Brazier, Jean-Philippe, E-mail: Jean-Philippe.Brazier@onera.fr; Léon, Olivier, E-mail: Olivier.Leon@onera.fr

2015-08-15

Nonlinear evolution of disturbances in an axisymmetric, high subsonic, high Reynolds number hot jet with forced eigenmodes is studied using the Parabolized Stability Equations (PSE) approach to understand how modes interact with one another. Both frequency and azimuthal harmonic interactions are analyzed by setting up one or two modes at higher initial amplitudes and various phases. While single mode excitation leads to harmonic growth and jet noise amplification, controlling the evolution of a specific mode has been made possible by forcing two modes (m{sub 1}, n{sub 1}), (m{sub 2}, n{sub 2}), such that the difference in azimuth and in frequencymore » matches the desired “target” mode (m{sub 1} − m{sub 2}, n{sub 1} − n{sub 2}). A careful setup of the initial amplitudes and phases of the forced modes, defined as the “killer” modes, has allowed the minimizing of the initially dominant instability in the near pressure field, as well as its estimated radiated noise with a 15 dB loss. Although an increase of the overall sound pressure has been found in the range of azimuth and frequency analyzed, the present paper reveals the possibility to make the initially dominant instability ineffective acoustically using nonlinear interactions with forced eigenmodes.« less

Binding the diproton in stars: anthropic limits on the strength of gravity

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

Barnes, Luke A., E-mail: L.Barnes@physics.usyd.edu.au

2015-12-01

We calculate the properties and investigate the stability of stars that burn via strong (and electromagnetic) interactions, and compare their properties with those that, as in our Universe, include a rate-limiting weak interaction. It has been suggested that, if the diproton were bound, stars would burn ∼10{sup 18} times brighter and faster via strong interactions, resulting in a universe that would fail to support life. By considering the representative case of a star in our Universe with initially equal numbers of protons and deuterons, we find that stable, 'strong-burning' stars adjust their central densities and temperatures to have familiar surfacemore » temperatures, luminosities and lifetimes. There is no 'diproton disaster'. In addition, strong-burning stars are stable in a much larger region of the parameter space of fundamental constants, specifically the strength of electromagnetism and gravity. The strongest anthropic bound on stars in such universes is not their stability, as is the case for stars limited by the weak interaction, but rather their lifetime. Regardless of the strength of electromagnetism, all stars burn out in mere millions of years unless the gravitational coupling constant is extremely small, α{sub G}∼< 10{sup −30}.« less

Temperature- and pH-dependent effect of lactate on in vitro redox stability of red meat myoglobins.

PubMed

Nair, M N; Suman, S P; Li, S; Ramanathan, R; Mancini, R A

2014-01-01

Our objective was to evaluate the influence of lactate on in vitro redox stability and thermostability of beef, horse, pork, and sheep myoglobins. Lactate (200 mM) had no effect (P>0.05) on redox stability at physiological (pH7.4, 37°C) and meat (pH 5.6, 4°C) conditions. However, lactate increased (P<0.05) metmyoglobin formation at a condition simulating stressed live skeletal muscle (pH 6.5, 37°C). The redox stability of myoglobins at stressed live skeletal muscle and meat conditions was species-specific (P<0.05). Myoglobin thermostability at 71°C was lower (P<0.05) in the presence of lactate compared with controls and was influenced (P<0.05) by species. The results of the present study indicate that the effects of lactate on myoglobin are temperature and pH dependent. The observed lack of influence of lactate on myoglobin redox stability at meat condition suggests that the color stability of lactate-enhanced fresh meat is not due to direct interactions between the ingredient and the heme protein. © 2013.

Stabilization of protein-protein interactions in drug discovery.

PubMed

Andrei, Sebastian A; Sijbesma, Eline; Hann, Michael; Davis, Jeremy; O'Mahony, Gavin; Perry, Matthew W D; Karawajczyk, Anna; Eickhoff, Jan; Brunsveld, Luc; Doveston, Richard G; Milroy, Lech-Gustav; Ottmann, Christian

2017-09-01

PPIs are involved in every disease and specific modulation of these PPIs with small molecules would significantly improve our prospects of developing therapeutic agents. Both industry and academia have engaged in the identification and use of PPI inhibitors. However in comparison, the opposite strategy of employing small-molecule stabilizers of PPIs is underrepresented in drug discovery. Areas covered: PPI stabilization has not been exploited in a systematic manner. Rather, this concept validated by a number of therapeutically used natural products like rapamycin and paclitaxel has been shown retrospectively to be the basis of the activity of synthetic molecules originating from drug discovery projects among them lenalidomide and tafamidis. Here, the authors cover the growing number of synthetic small-molecule PPI stabilizers to advocate for a stronger consideration of this as a drug discovery approach. Expert opinion: Both the natural products and the growing number of synthetic molecules show that PPI stabilization is a viable strategy for drug discovery. There is certainly a significant challenge to adapt compound libraries, screening techniques and downstream methodologies to identify, characterize and optimize PPI stabilizers, but the examples of molecules reviewed here in our opinion justify these efforts.

[Thermostabilization of glutamin(asparagin)ase from Pseudomonas aurantica BKMB-548].

PubMed

Kabanova, E A; Lebedeva, Z I; Berezov, T T

1985-01-01

In studies on kinetics of thermoinactivation of glutaminase (asparaginase) from Ps. arantiaca BKMB-548 at 50 degrees and pH 7.0 in presence or in absence of L-glutamate the enzyme inactivation was found to obey the first order equation. Both the glutaminase and asparaginase activities decreased at a similar rate. L-Glutamate stabilized the enzyme due to direct interaction with its molecule. Stability of the complex formed was evaluated quantitatively. L-Glutamate reacted apparently with a specific site on the surface of the enzyme molecule; Kdiss was 0.42 +/- 0.03 mM at pH 7.0 and 50 degrees. No cooperative effect was found. L-Aspartate protected the enzyme completely; stabilizing effects of L-cysteine, L-serine and glycine were similar to the effect of L-glutamate (94%, 84%, 83% and 82%, respectively). At the same time, glutarate, succinate, alpha-ketobutyrate, alpha-ketoglutarate, gamma-aminobutyrate and N-benzoyl glutamate did not exhibit the stabilization effect. The data obtained suggest that the high stabilizing effect might exhibit only the substances containing simultaneously free alpha-NH2 and alpha-COOH groups in a molecule, whereas presence of COOH groups at beta--or gamma-carbon atoms was not essential for the stabilizing effect.

Rotating non-Boussinesq Rayleigh-Benard convection

NASA Astrophysics Data System (ADS)

Moroz, Vadim Vladimir

This thesis makes quantitative predictions about the formation and stability of hexagonal and roll patterns in convecting system unbounded in horizontal direction. Starting from the Navier-Stokes, heat and continuity equations, the convection problem is then reduced to normal form equations using equivariant bifurcation theory. The relative stabilities of patterns lying on a hexagonal lattice in Fourier space are then determined using appropriate amplitude equations, with coefficients obtained via asymptotic expansion of the governing partial differential equations, with the conducting state being the base state, and the control parameter and the non-Boussinesq effects being small. The software package Mathematica was used to calculate amplitude coefficients of the appropriate coupled Ginzburg-Landau equations for the rigid-rigid and free-free case. A Galerkin code (initial version of which was written by W. Pesch et al.) is used to determine pattern stability further from onset and for strongly non-Boussinesq fluids. Specific predictions about the stability of hexagon and roll patterns for realistic experimental conditions are made. The dependence of the stability of the convective patterns on the Rayleigh number, planform wavenumber and the rotation rate is studied. Long- and shortwave instabilities, both steady and oscillatory, are identified. For small Prandtl numbers oscillatory sideband instabilities are found already very close to onset. A resonant mode interaction in hexagonal patterns arising in non-Boussinesq Rayleigh-Benard convection is studied using symmetry group methods. The lowest-order coupling terms for interacting patterns are identified. A bifurcation analysis of the resulting system of equations shows that the bifurcation is transcritical. Stability properties of resulting patterns are discussed. It is found that for some fluid properties the traditional hexagon convection solution does not exist. Analytical results are supported by numerical solutions of the convection equations using the Galerkin procedure and a Floquet analysis.

Nature and magnitude of aromatic base stacking in DNA and RNA: Quantum chemistry, molecular mechanics, and experiment.

PubMed

Sponer, Jiří; Sponer, Judit E; Mládek, Arnošt; Jurečka, Petr; Banáš, Pavel; Otyepka, Michal

2013-12-01

Base stacking is a major interaction shaping up and stabilizing nucleic acids. During the last decades, base stacking has been extensively studied by experimental and theoretical methods. Advanced quantum-chemical calculations clarified that base stacking is a common interaction, which in the first approximation can be described as combination of the three most basic contributions to molecular interactions, namely, electrostatic interaction, London dispersion attraction and short-range repulsion. There is not any specific π-π energy term associated with the delocalized π electrons of the aromatic rings that cannot be described by the mentioned contributions. The base stacking can be rather reasonably approximated by simple molecular simulation methods based on well-calibrated common force fields although the force fields do not include nonadditivity of stacking, anisotropy of dispersion interactions, and some other effects. However, description of stacking association in condensed phase and understanding of the stacking role in biomolecules remain a difficult problem, as the net base stacking forces always act in a complex and context-specific environment. Moreover, the stacking forces are balanced with many other energy contributions. Differences in definition of stacking in experimental and theoretical studies are explained. Copyright © 2013 Wiley Periodicals, Inc.

Differentially-Expressed Pseudogenes in HIV-1 Infection.

PubMed

Gupta, Aditi; Brown, C Titus; Zheng, Yong-Hui; Adami, Christoph

2015-09-29

Not all pseudogenes are transcriptionally silent as previously thought. Pseudogene transcripts, although not translated, contribute to the non-coding RNA pool of the cell that regulates the expression of other genes. Pseudogene transcripts can also directly compete with the parent gene transcripts for mRNA stability and other cell factors, modulating their expression levels. Tissue-specific and cancer-specific differential expression of these "functional" pseudogenes has been reported. To ascertain potential pseudogene:gene interactions in HIV-1 infection, we analyzed transcriptomes from infected and uninfected T-cells and found that 21 pseudogenes are differentially expressed in HIV-1 infection. This is interesting because parent genes of one-third of these differentially-expressed pseudogenes are implicated in HIV-1 life cycle, and parent genes of half of these pseudogenes are involved in different viral infections. Our bioinformatics analysis identifies candidate pseudogene:gene interactions that may be of significance in HIV-1 infection. Experimental validation of these interactions would establish that retroviruses exploit this newly-discovered layer of host gene expression regulation for their own benefit.

Probing intermolecular interactions in a diethylcarbamazine citrate salt by fast MAS 1H solid-state NMR spectroscopy and GIPAW calculations.

PubMed

Venâncio, Tiago; Oliveira, Lyege Magalhaes; Ellena, Javier; Boechat, Nubia; Brown, Steven P

2017-10-01

Fast magic-angle spinning (MAS) NMR is used to probe intermolecular interactions in a diethylcarbamazine salt, that is widely used as a treatment against adult worms of Wuchereria bancrofti which cause a common disease in tropical countries named filariasis. Specifically, a dihydrogen citrate salt that has improved thermal stability and solubility as compared to the free form is studied. One-dimensional 1 H, 13 C and 15 N and two-dimensional 1 H- 13 C and 14 N- 1 H heteronuclear correlation NMR experiments under moderate and fast MAS together with GIPAW (CASTEP) calculations enable the assignment of the 1 H, 13 C and 14 N/ 15 N resonances. A two-dimensional 1 H- 1 H double-quantum (DQ) -single-quantum (SQ) MAS spectrum recorded with BaBa recoupling at 60kHz MAS identifies specific proton-proton proximities associated with citrate-citrate and citrate-diethylcarbamazine intermolecular interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

Structure of the extracellular domain of matrix protein 2 of influenza A virus in complex with a protective monoclonal antibody.

PubMed

Cho, Ki Joon; Schepens, Bert; Seok, Jong Hyeon; Kim, Sella; Roose, Kenny; Lee, Ji-Hye; Gallardo, Rodrigo; Van Hamme, Evelien; Schymkowitz, Joost; Rousseau, Frederic; Fiers, Walter; Saelens, Xavier; Kim, Kyung Hyun

2015-04-01

The extracellular domain of influenza A virus matrix protein 2 (M2e) is conserved and is being evaluated as a quasiuniversal influenza A vaccine candidate. We describe the crystal structure at 1.6 Å resolution of M2e in complex with the Fab fragment of an M2e-specific monoclonal antibody that protects against influenza A virus challenge. This antibody binds M2 expressed on the surfaces of cells infected with influenza A virus. Five out of six complementary determining regions interact with M2e, and three highly conserved M2e residues are critical for this interaction. In this complex, M2e adopts a compact U-shaped conformation stabilized in the center by the highly conserved tryptophan residue in M2e. This is the first description of the three-dimensional structure of M2e. M2e of influenza A is under investigation as a universal influenza A vaccine, but its three-dimensional structure is unknown. We describe the structure of M2e stabilized with an M2e-specific monoclonal antibody that recognizes natural M2. We found that the conserved tryptophan is positioned in the center of the U-shaped structure of M2e and stabilizes its conformation. The structure also explains why previously reported in vivo escape viruses, selected with a similar monoclonal antibody, carried proline residue substitutions at position 10 in M2. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Interactions of DNA binding proteins with G-Quadruplex structures at the single molecule level

NASA Astrophysics Data System (ADS)

Ray, Sujay

Guanine-rich nucleic acid (DNA/RNA) sequences can form non-canonical secondary structures, known as G-quadruplex (GQ). Numerous in vivo and in vitro studies have demonstrated formation of these structures in telomeric and non-telomeric regions of the genome. Telomeric GQs protect the chromosome ends whereas non-telomeric GQs either act as road blocks or recognition sites for DNA metabolic machinery. These observations suggest the significance of these structures in regulation of different metabolic processes, such as replication and repair. GQs are typically thermodynamically more stable than the corresponding Watson-Crick base pairing formed by G-rich and C-rich strands, making protein activity a crucial factor for their destabilization. Inside the cell, GQs interact with different proteins and their enzymatic activity is the determining factor for their stability. We studied interactions of several proteins with GQs to understand the underlying principles of protein-GQ interactions using single-molecule FRET and other biophysical techniques. Replication Protein-A (RPA), a single stranded DNA (ssDNA) binding protein, is known to posses GQ unfolding activity. First, we compared the thermal stability of three potentially GQ-forming DNA sequences (PQS) to their stability against RPA-mediated unfolding. One of these sequences is the human telomeric repeat and the other two, located in the promoter region of tyrosine hydroxylase gene, are highly heterogeneous sequences that better represent PQS in the genome. The thermal stability of these structures do not necessarily correlate with their stability against protein-mediated unfolding. We conclude that thermal stability is not necessarily an adequate criterion for predicting the physiological viability of GQ structures. To determine the critical structural factors that influence protein-GQ interactions we studied two groups of GQ structures that have systematically varying loop lengths and number of G-tetrad layers. We observed a linear increase in the steady-state stability of the GQ against RPA-mediated unfolding with increasing number of layers or decreasing loop length. The stability demonstrated by different GQ structures varied by at least three orders of magnitude. Finally, we studied another protein-GQ system where a protein complex works synergistically with a GQ to suppress DNA damage signals by preventing RPA to bind to telomeric DNA. Human telomeres that terminate with a single-stranded 3' G-overhang can be recognized as a DNA damage site by RPA. The protection of telomere-1 (POT1) and POT1-interacting protein (TPP1) heterodimer, binds specifically to telomeric DNA and protects it against RPA binding. Using model telomeric DNA, we studied the competition between POT1/TPP1 and RPA to access telomeric GQs in vitro. Under physiological salt and pH conditions, POT1/TPP1 stably load to a minimal DNA sequence adjacent to a folded GQ and unfolds the anti-parallel GQ as the parallel conformation remains folded. We showed that GQ formation of telomeres enhances the ability of POT1/TPP1 to block RPA's access to telomeres by two orders of magnitude and contributes to suppress DNA damage signals.

Free energy determinants of secondary structure formation: III. beta-turns and their role in protein folding.

PubMed

Yang, A S; Hitz, B; Honig, B

1996-06-21

The stability of beta-turns is calculated as a function of sequence and turn type with a Monte Carlo sampling technique. The conformational energy of four internal hydrogen-bonded turn types, I, I', II and II', is obtained by evaluating their gas phase energy with the CHARMM force field and accounting for solvation effects with the Finite Difference Poisson-Boltzmann (FDPB) method. All four turn types are found to be less stable than the coil state, independent of the sequence in the turn. The free-energy penalties associated with turn formation vary between 1.6 kcal/mol and 7.7 kcal/mol, depending on the sequence and turn type. Differences in turn stability arise mainly from intraresidue interactions within the two central residues of the turn. For each combination of the two central residues, except for -Gly-Gly-, the most stable beta-turn type is always found to occur most commonly in native proteins. The fact that a model based on local interactions accounts for the observed preference of specific sequences suggests that long-range tertiary interactions tend to play a secondary role in determining turn conformation. In contrast, for beta-hairpins, long-range interactions appear to dominate. Specifically, due to the right-handed twist of beta-strands, type I' turns for -Gly-Gly- are found to occur with high frequency, even when local energetics would dictate otherwise. The fact that any combination of two residues is found able to adopt a relatively low-energy turn structure explains why the amino acid sequence in turns is highly variable. The calculated free-energy cost of turn formation, when combined with related numbers obtained for alpha-helices and beta-sheets, suggests a model for the initiation of protein folding based on metastable fragments of secondary structure.

An Electrospray Ionization Mass Spectrometry Study on the "In Vacuo" Hetero-Oligomers Formed by the Antimicrobial Peptides, Surfactin and Gramicidin S

NASA Astrophysics Data System (ADS)

Rautenbach, Marina; Vlok, N. Maré; Eyéghé-Bickong, Hans A.; van der Merwe, Marthinus J.; Stander, Marietjie A.

2017-08-01

It was previously observed that the lipopeptide surfactants in surfactin (Srf) have an antagonistic action towards the highly potent antimicrobial cyclodecapeptide, gramicidin S (GS). This study reports on some of the molecular aspects of the antagonism as investigated through complementary electrospray ionization mass spectrometry techniques. We were able to detect stable 1:1 and 2:1 hetero-oligomers in a mixture of surfactin and gramicidin S. The noncovalent interaction between GS and Srf, with the proposed equilibrium: GS Srf↔GS+Srf correlated to apparent K d values of 6-9 μM in gas-phase and 1 μM in aqueous solution. The apparent K d values decreased with a longer incubation time and indicated a slow oligomerization equilibrium. Furthermore, the low μM K d app values of GS Srf↔GS+Srf fell within the biological concentration range and related to the 2- to 3-fold increase in [GS] needed for bacterial growth inhibition in the presence of Srf. Competition studies indicated that neither Na+ nor Ca2+ had a major effect on the stability of preformed heterodimers and that GS in fact out-competed Ca2+ and Na+ from Srf. Traveling wave ion mobility mass spectrometry revealed near symmetrical peaks of the heterodimers correlating to a compact dimer conformation that depend on specific interactions. Collision-induced dissociation studies indicated that the peptide interaction is most probably between one Orn residue in GS and the Asp residue, but not the Glu residue in Srf. We propose that flanking hydrophobic residues in both peptides stabilize the antagonistic and inactive peptide hetero-oligomers and shield the specific polar interactions in an aqueous environment.

Review of alternative fuels data bases

NASA Technical Reports Server (NTRS)

Harsha, P. T.; Edelman, R. B.

1983-01-01

Based on an analysis of the interaction of fuel physical and chemical properties with combustion characteristics and indicators, a ranking of the importance of various fuel properties with respect to the combustion process was established. This ranking was used to define a suite of specific experiments whose objective is the development of an alternative fuels design data base. Combustion characteristics and indicators examined include droplet and spray formation, droplet vaporization and burning, ignition and flame stabilization, flame temperature, laminar flame speed, combustion completion, soot emissions, NOx and SOx emissions, and the fuels' thermal and oxidative stability and fouling and corrosion characteristics. Key fuel property data is found to include composition, thermochemical data, chemical kinetic rate information, and certain physical properties.

Organic Carbamates in Drug Design and Medicinal Chemistry

PubMed Central

2016-01-01

The carbamate group is a key structural motif in many approved drugs and prodrugs. There is an increasing use of carbamates in medicinal chemistry and many derivatives are specifically designed to make drug–target interactions through their carbamate moiety. In this Perspective, we present properties and stabilities of carbamates, reagents and chemical methodologies for the synthesis of carbamates, and recent applications of carbamates in drug design and medicinal chemistry. PMID:25565044

Organic carbamates in drug design and medicinal chemistry.

PubMed

Ghosh, Arun K; Brindisi, Margherita

2015-04-09

The carbamate group is a key structural motif in many approved drugs and prodrugs. There is an increasing use of carbamates in medicinal chemistry and many derivatives are specifically designed to make drug-target interactions through their carbamate moiety. In this Perspective, we present properties and stabilities of carbamates, reagents and chemical methodologies for the synthesis of carbamates, and recent applications of carbamates in drug design and medicinal chemistry.

Conformational dynamics of L-lysine, L-arginine, L-ornithine binding protein reveals ligand-dependent plasticity.

PubMed

Silva, Daniel-Adriano; Domínguez-Ramírez, Lenin; Rojo-Domínguez, Arturo; Sosa-Peinado, Alejandro

2011-07-01

The molecular basis of multiple ligand binding affinity for amino acids in periplasmic binding proteins (PBPs) and in the homologous domain for class C G-protein coupled receptors is an unsolved question. Here, using unrestrained molecular dynamic simulations, we studied the ligand binding mechanism present in the L-lysine, L-arginine, L-ornithine binding protein. We developed an analysis based on dihedral angles for the description of the conformational changes upon ligand binding. This analysis has an excellent correlation with each of the two main movements described by principal component analysis (PCA) and it's more convenient than RMSD measurements to describe the differences in the conformational ensembles observed. Furthermore, an analysis of hydrogen bonds showed specific interactions for each ligand studied as well as the ligand interaction with the aromatic residues Tyr-14 and Phe-52. Using uncharged histidine tautomers, these interactions are not observed. On the basis of these results, we propose a model in which hydrogen bond interactions place the ligand in the correct orientation to induce a cation-π interaction with Tyr-14 and Phe-52 thereby stabilizing the closed state. Our results also show that this protein adopts slightly different closed conformations to make available specific hydrogen bond interactions for each ligand thus, allowing a single mechanism to attain multiple ligand specificity. These results shed light on the experimental evidence for ligand-dependent conformational plasticity not explained by the previous crystallographic data. Copyright © 2011 Wiley-Liss, Inc.

Deriving Heterospecific Self-Assembling Protein-Protein Interactions Using a Computational Interactome Screen.

PubMed

Crooks, Richard O; Baxter, Daniel; Panek, Anna S; Lubben, Anneke T; Mason, Jody M

2016-01-29

Interactions between naturally occurring proteins are highly specific, with protein-network imbalances associated with numerous diseases. For designed protein-protein interactions (PPIs), required specificity can be notoriously difficult to engineer. To accelerate this process, we have derived peptides that form heterospecific PPIs when combined. This is achieved using software that generates large virtual libraries of peptide sequences and searches within the resulting interactome for preferentially interacting peptides. To demonstrate feasibility, we have (i) generated 1536 peptide sequences based on the parallel dimeric coiled-coil motif and varied residues known to be important for stability and specificity, (ii) screened the 1,180,416 member interactome for predicted Tm values and (iii) used predicted Tm cutoff points to isolate eight peptides that form four heterospecific PPIs when combined. This required that all 32 hypothetical off-target interactions within the eight-peptide interactome be disfavoured and that the four desired interactions pair correctly. Lastly, we have verified the approach by characterising all 36 pairs within the interactome. In analysing the output, we hypothesised that several sequences are capable of adopting antiparallel orientations. We subsequently improved the software by removing sequences where doing so led to fully complementary electrostatic pairings. Our approach can be used to derive increasingly large and therefore complex sets of heterospecific PPIs with a wide range of potential downstream applications from disease modulation to the design of biomaterials and peptides in synthetic biology. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Roles of Specific Membrane Lipid Domains in EGF Receptor Activation and Cell Adhesion Molecule Stabilization in a Developing Olfactory System

PubMed Central

Gibson, Nicholas J.; Tolbert, Leslie P.; Oland, Lynne A.

2009-01-01

Background Reciprocal interactions between glial cells and olfactory receptor neurons (ORNs) cause ORN axons entering the brain to sort, to fasciculate into bundles destined for specific glomeruli, and to form stable protoglomeruli in the developing olfactory system of an experimentally advantageous animal species, the moth Manduca sexta. Epidermal growth factor receptors (EGFRs) and the cell adhesion molecules (IgCAMs) neuroglian and fasciclin II are known to be important players in these processes. Methodology/Principal Findings We report in situ and cell-culture studies that suggest a role for glycosphingolipid-rich membrane subdomains in neuron-glia interactions. Disruption of these subdomains by the use of methyl-β-cyclodextrin results in loss of EGFR activation, depletion of fasciclin II in ORN axons, and loss of neuroglian stabilization in the membrane. At the cellular level, disruption leads to aberrant ORN axon trajectories, small antennal lobes, abnormal arrays of olfactory glomerul, and loss of normal glial cell migration. Conclusions/Significance We propose that glycosphingolipid-rich membrane subdomains (possible membrane rafts or platforms) are essential for IgCAM-mediated EGFR activation and for anchoring of neuroglian to the cytoskeleton, both required for normal extension and sorting of ORN axons. PMID:19787046

The split Renilla luciferase complementation assay is useful for identifying the interaction of Epstein-Barr virus protein kinase BGLF4 and a heat shock protein Hsp90.

PubMed

Wang, J; Guo, W; Long, C; Zhou, H; Wang, H; Sun, X

2016-03-01

Protein-protein interactions can regulate different cellular processes, such as transcription, translation, and oncogenic transformation. The split Renilla luciferase complementation assay (SRLCA) is one of the techniques that detect protein-protein interactions. The SRLCA is based on the complementation of the LN and LC non-functional halves of Renilla luciferase fused to possibly interacting proteins which after interaction form a functional enzyme and emit luminescence. The BGLF4 of Epstein-Barr virus (EBV) is a viral protein kinase that is expressed during the early and late stages of lytic cycles, which can regulate multiple cellular and viral substrates to optimize the DNA replication environment. The heat shock protein Hsp90 is a molecular chaperone that maintains the integrity of structure and function of various interacting proteins, which can form a complex with BGLF4 and stabilize its expression in cells. The interaction between BGLF4 and Hsp90 could be specifically detected through the SRLCA. The region of aa 250-295 of BGLF4 is essential for the BGLF4/Hsp90 interaction and the mutation of Phe-254, Leu-266, and Leu-267 can disrupt this interaction. These results suggest that the SRLCA can specifically detect the BGLF4/Hsp90 interaction and provide a reference to develop inhibitors that disrupt the BGLF4/Hsp90 interaction.

Activity and conformation of lysozyme in molecular solvents, protic ionic liquids (PILs) and salt-water systems.

PubMed

Wijaya, Emmy C; Separovic, Frances; Drummond, Calum J; Greaves, Tamar L

2016-09-21

Improving protein stabilisation is important for the further development of many applications in the pharmaceutical, specialty chemical, consumer product and agricultural sectors. However, protein stabilization is highly dependent on the solvent environment and, hence, it is very complex to tailor protein-solvent combinations for stable protein maintenance. Understanding solvent features that govern protein stabilization will enable selection or design of suitable media with favourable solution environments to retain protein native conformation. In this work the structural conformation and activity of lysozyme in 29 solvent systems were investigated to determine the role of various solvent features on the stability of the enzyme. The solvent systems consisted of 19 low molecular weight polar solvents and 4 protic ionic liquids (PILs), both at different water content levels, and 6 aqueous salt solutions. Small angle X-ray scattering, Fourier transform infrared spectroscopy and UV-vis spectroscopy were used to investigate the tertiary and secondary structure of lysozyme along with the corresponding activity in various solvation systems. At low non-aqueous solvent concentrations (high water content), the presence of solvents and salts generally maintained lysozyme in its native structure and enhanced its activity. Due to the presence of a net surface charge on lysozyme, electrostatic interactions in PIL-water systems and salt solutions enhanced lysozyme activity more than the specific hydrogen-bond interactions present in non-ionic molecular solvents. At higher solvent concentrations (lower water content), solvents with a propensity to exhibit the solvophobic effect, analogous to the hydrophobic effect in water, retained lysozyme native conformation and activity. This solvophobic effect was observed particularly for solvents which contained hydroxyl moieties. Preferential solvophobic effects along with bulky chemical structures were postulated to result in less competition with water at the specific hydration layer around the protein, thus reducing protein-solvent interactions and retaining lysozyme's native conformation. The structure-property links established in this study are considered to be applicable to other proteins.

Stability, Bistability, and Critical Thresholds in Fire-prone Forested Landscapes: How Frequency and Intensity of Disturbance Interact and Influence Forest Cover

NASA Astrophysics Data System (ADS)

Miller, A. D.

2015-12-01

Many aspects of disturbance processes can have large impacts on the composition of plant communities, and associated changes in land cover type in turn have biogeochemical feedbacks to climate. In particular, changes to disturbance regimes can potentially change the number and stability of equilibrial states, and plant community states can differ dramatically in their carbon (C) dynamics, energy balance, and hydrology. Using the Klamath region of northern California as a model system, we present a theoretical analysis of how changes to climate and associated fire dynamics can disrupt high-carbon, long-lived conifer forests and replace them with shrub-chaparral communities that have much lower biomass and are more pyrogenic. Specifically, we develop a tractable model of plant community dynamics, structured by size class, life-history traits, lottery-type competition, and species-specific responses to disturbance. We assess the stability of different states in terms of disturbance frequency and intensity, and quantitatively partition long-term low-density population growth rates into mechanisms that influence critical transitions from stable to bistable behavior. Our findings show how different aspects of disturbance act and interact to control competitive outcomes and stable states, hence ecosystem-atmosphere C exchange. Forests tend to dominate in low frequency and intensity regimes, while shrubs dominate at high fire frequency and intensity. In other regimes, the system is bistable, and the fate of the system depends both on initial conditions and random chance. Importantly, the system can cross a critical threshold where hysteresis prevents easy return to the prior forested state. We conclude that changes in disturbance-recovery dynamics driven by projected climate change can shift this system away from forest dominated in the direction of shrub-dominated landscape. This will result in a large net C release from the landscape, and alter biophysical ecosystem-climate interactions.

Contribution of Hydrophobic Interactions to Protein Stability

PubMed Central

Pace, C. Nick; Fu, Hailong; Fryar, Katrina Lee; Landua, John; Trevino, Saul R.; Shirley, Bret A.; Hendricks, Marsha McNutt; Iimura, Satoshi; Gajiwala, Ketan; Scholtz, J. Martin; Grimsley, Gerald R.

2011-01-01

Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin head piece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compare our results with previous studies and reach the following conclusions. 1. Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.6 ± 0.3 kcal/mole per –CH2– group), than to the stability of a large protein, VlsE (1.6 ± 0.3 kcal/mol per –CH2– group). 2. Hydrophobic interactions make the major contribution to the stability of VHP (40 kcal/mol) and the major contributors are (in kcal/mol): Phe 18 (3.9), Met 13 (3.1), Phe 7 (2.9), Phe 11 (2.7), and Leu 21 (2.7). 3. Based on Δ(ΔG) values for 148 hydrophobic mutants in 13 proteins, burying a –CH2– group on folding contributes, on average, 1.1 ± 0.5 kcal/mol to protein stability. 4. The experimental Δ(ΔG) values for aliphatic side chains (Ala, Val, Ile, and Leu) are in good agreement with their ΔGtr values from water to cyclohexane. 5. For 22 proteins with 36 to 534 residues, hydrophobic interactions contribute 60 ± 4% and hydrogen bonds 40 ± 4% to protein stability. 6. Conformational entropy contributes about 2.4 kcal/mol per residue to protein instability. The globular conformation of proteins is stabilized predominately by hydrophobic interactions. PMID:21377472

Specificity in transition state binding: the Pauling model revisited.

PubMed

Amyes, Tina L; Richard, John P

2013-03-26

Linus Pauling proposed that the large rate accelerations for enzymes are caused by the high specificity of the protein catalyst for binding the reaction transition state. The observation that stable analogues of the transition states for enzymatic reactions often act as tight-binding inhibitors provided early support for this simple and elegant proposal. We review experimental results that support the proposal that Pauling's model provides a satisfactory explanation for the rate accelerations for many heterolytic enzymatic reactions through high-energy reaction intermediates, such as proton transfer and decarboxylation. Specificity in transition state binding is obtained when the total intrinsic binding energy of the substrate is significantly larger than the binding energy observed at the Michaelis complex. The results of recent studies that aimed to characterize the specificity in binding of the enolate oxygen at the transition state for the 1,3-isomerization reaction catalyzed by ketosteroid isomerase are reviewed. Interactions between pig heart succinyl-coenzyme A:3-oxoacid coenzyme A transferase (SCOT) and the nonreacting portions of coenzyme A (CoA) are responsible for a rate increase of 3 × 10(12)-fold, which is close to the estimated total 5 × 10(13)-fold enzymatic rate acceleration. Studies that partition the interactions between SCOT and CoA into their contributing parts are reviewed. Interactions of the protein with the substrate phosphodianion group provide an ~12 kcal/mol stabilization of the transition state for the reactions catalyzed by triosephosphate isomerase, orotidine 5'-monophosphate decarboxylase, and α-glycerol phosphate dehydrogenase. The interactions of these enzymes with the substrate piece phosphite dianion provide a 6-8 kcal/mol stabilization of the transition state for reaction of the appropriate truncated substrate. Enzyme activation by phosphite dianion reflects the higher dianion affinity for binding to the enzyme-transition state complex compared with that of the free enzyme. Evidence is presented that supports a model in which the binding energy of the phosphite dianion piece, or the phosphodianion group of the whole substrate, is utilized to drive an enzyme conformational change from an inactive open form E(O) to an active closed form E(C), by closure of a phosphodianion gripper loop. Members of the enolase and haloalkanoic acid dehalogenase superfamilies use variable capping domains to interact with nonreacting portions of the substrate and sequester the substrate from interaction with bulk solvent. Interactions of this capping domain with the phenyl group of mandelate have been shown to activate mandelate racemase for catalysis of deprotonation of α-carbonyl carbon. We propose that an important function of these capping domains is to utilize the binding interactions with nonreacting portions of the substrate to activate the enzyme for catalysis.

Specificity in Transition State Binding: The Pauling Model Revisited

PubMed Central

Amyes, Tina L.; Richard, John P.

2013-01-01

Linus Pauling proposed that the large rate accelerations for enzymes are due to the high specificity of the protein catalyst for binding the reaction transition state. The observation that stable analogs of the transition states for enzymatic reactions often act as tight-binding binding inhibitors provided early support for this simple and elegant proposal. We review experimental results which support the proposal that Pauling’s model provides a satisfactory explanation for the rate accelerations for many heterolytic enzymatic reactions through high energy reaction intermediates, such as proton transfer and decarboxylation. Specificity in transition state binding is obtained when the total intrinsic binding energy of the substrate is significantly larger than the binding energy observed at the Michaelis complex. The results of recent studies to characterize the specificity in binding of the enolate oxygen at the transition state for the 1,3-isomerization reaction catalyzed by ketosteroid isomerase are reviewed. Interactions between pig heart succinyl-CoA:3-oxoacid coenzyme A transferase (SCOT) and the nonreacting portions of CoA are responsible for a rate increase of 3 × 1012-fold, which is close to the estimated total 5 × 1013-fold enzymatic rate acceleration. Studies that partition the interactions between SCOT and CoA into their contributing parts are reviewed. Interactions of the protein with the substrate phosphodianion group provide a ca. 12 kcal/mol stabilization of the transition state for the reactions catalyzed by triosephosphate isomerase, orotidine 5′-monophosphate decarboxylase and α-glycerol phosphate dehydrogenase. The interactions of these enzymes with the substrate piece phosphite dianion provide a 6 – 8 kcal/mol stabilization of the transition state for reaction of the appropriate truncated substrate. Enzyme activation by phosphite dianion reflects the higher dianion affinity for binding to the enzyme-transition state complex compared with the free enzyme. Evidence is presented that supports a model in which the binding energy of the phosphite dianion piece, or the phosphodianion group of the whole substrate, is utilized to drive an enzyme conformational change from an inactive open form EO to an active closed form EC, by closure of a phosphodianion gripper loop. Members of the enolase and haloalkanoic acid dehalogenase superfamilies use variable capping domains to interact with nonreacting portions of the substrate and sequester the substrate from interaction with bulk solvent. Interactions of this capping domain with the phenyl group of mandelate have been shown to activate mandelate racemase for catalysis of deprotonation of α-carbonyl carbon. We propose that an important function of these capping domains is to utilize the binding interactions with nonreacting portions of the substrate to activate the enzyme for catalysis. PMID:23327224

Handling Qualities Specifications for U.S. Military Helicopters

NASA Technical Reports Server (NTRS)

Key, David L.

1982-01-01

Inadequacies in the military specification for helicopter handling qualities, MIL-H-8501A, have long been recognized, and the latest procurements by the U.S. Army used special Prime Item Development Specifications (PIDS). This paper assesses the efficacy of these PIDS and suggests that changes should be made. In particular, the structure developed in MIL-F-8785B (ASG) (the specification for flying qualities of piloted airplanes) should be incorporated. Improved requirements must be based on a systematic data base and concentrated on topics most important in preliminary design: static and dynamic stability, control power and sensitivity, and interaction with controllers and displays. Emphasis should be on current military helicopter missions and helicopter idiosyncrasies such as cross-coupling, nonlinearities, and higher-order dynamics.

The acoustic sensor for rapid analysis of bacterial cells in the conductive suspensions.

PubMed

Borodina, I A; Zaitsev, B D; Guliy, O; Teplykh, A A; Shikhabudinov, A M

2017-11-01

The possibility of using the acoustic sensor on the basis of a two-channel delay line for rapid analysis of bacterial cells in the conductive suspensions was investigated. The dependencies of change in phase and insertion loss of output signal of the sensor on conductivity of buffer solution with various concentrations of cells due to a specific interaction "bacterial cells - mini-antibodies" for electrically open and electrically shorted channels of delay line were measured. It has been found that these changes have the most values for the electrically open channel. It has been also shown that the sensor rapidly responds to the specific interaction and the time stabilization of the phase and insertion loss of output signal is less than 10min. Copyright © 2017 Elsevier B.V. All rights reserved.

Highly Dynamic Anion-Quadrupole Networks in Proteins.

PubMed

Kapoor, Karan; Duff, Michael R; Upadhyay, Amit; Bucci, Joel C; Saxton, Arnold M; Hinde, Robert J; Howell, Elizabeth E; Baudry, Jerome

2016-11-01

The dynamics of anion-quadrupole (or anion-π) interactions formed between negatively charged (Asp/Glu) and aromatic (Phe) side chains are for the first time computationally characterized in RmlC (Protein Data Bank entry 1EP0 ), a homodimeric epimerase. Empirical force field-based molecular dynamics simulations predict anion-quadrupole pairs and triplets (anion-anion-π and anion-π-π) are formed by the protein during the simulated trajectory, which suggests that the anion-quadrupole interactions may provide a significant contribution to the overall stability of the protein, with an average of -1.6 kcal/mol per pair. Some anion-π interactions are predicted to form during the trajectory, extending the number of anion-quadrupole interactions beyond those predicted from crystal structure analysis. At the same time, some anion-π pairs observed in the crystal structure exhibit marginal stability. Overall, most anion-π interactions alternate between an "on" state, with significantly stabilizing energies, and an "off" state, with marginal or null stabilizing energies. The way proteins possibly compensate for transient loss of anion-quadrupole interactions is characterized in the RmlC aspartate 84-phenylalanine 112 anion-quadrupole pair observed in the crystal structure. A double-mutant cycle analysis of the thermal stability suggests a possible loss of anion-π interactions compensated by variations of hydration of the residues and formation of compensating electrostatic interactions. These results suggest that near-planar anion-quadrupole pairs can exist, sometimes transiently, which may play a role in maintaining the structural stability and function of the protein, in an otherwise very dynamic interplay of a nonbonded interaction network as well as solvent effects.

Specific RNP capture with antisense LNA/DNA mixmers

PubMed Central

Rogell, Birgit; Fischer, Bernd; Rettel, Mandy; Krijgsveld, Jeroen; Castello, Alfredo; Hentze, Matthias W.

2017-01-01

RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe “specific ribonucleoprotein (RNP) capture,” a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein–RNA interactions taking place at “zero distance.” Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins. PMID:28476952

Specific RNP capture with antisense LNA/DNA mixmers.

PubMed

Rogell, Birgit; Fischer, Bernd; Rettel, Mandy; Krijgsveld, Jeroen; Castello, Alfredo; Hentze, Matthias W

2017-08-01

RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance." Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins. © 2017 Rogell et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

An RNA-binding compound that stabilizes the HIV-1 gRNA packaging signal structure and specifically blocks HIV-1 RNA encapsidation.

PubMed

Ingemarsdotter, Carin K; Zeng, Jingwei; Long, Ziqi; Lever, Andrew M L; Kenyon, Julia C

2018-03-14

NSC260594, a quinolinium derivative from the NCI diversity set II compound library, was previously identified in a target-based assay as an inhibitor of the interaction between the HIV-1 (ψ) stem-loop 3 (SL3) RNA and Gag. This compound was shown to exhibit potent antiviral activity. Here, the effects of this compound on individual stages of the viral lifecycle were examined by qRT-PCR, ELISA and Western blot, to see if its actions were specific to the viral packaging stage. The structural effects of NSC260594 binding to the HIV-1 gRNA were also examined by SHAPE and dimerization assays. Treatment of cells with NSC260594 did not reduce the number of integration events of incoming virus, and treatment of virus producing cells did not affect the level of intracellular Gag protein or viral particle release as determined by immunoblot. However, NSC260594 reduced the incorporation of gRNA into virions by up to 82%, without affecting levels of gRNA inside the cell. This reduction in packaging correlated closely with the reduction in infectivity of the released viral particles. To establish the structural effects of NSC260594 on the HIV-1 gRNA, we performed SHAPE analyses to pinpoint RNA structural changes. NSC260594 had a stabilizing effect on the wild type RNA that was not confined to SL3, but that was propagated across the structure. A packaging mutant lacking SL3 did not show this effect. NSC260594 acts as a specific inhibitor of HIV-1 RNA packaging. No other viral functions are affected. Its action involves preventing the interaction of Gag with SL3 by stabilizing this small RNA stem-loop which then leads to stabilization of the global packaging signal region (psi or ψ). This confirms data, previously only shown in analyses of isolated SL3 oligonucleotides, that SL3 is structurally labile in the presence of Gag and that this is critical for the complete psi region to be able to adopt different conformations. Since replication is otherwise unaffected by NSC260594 the flexibility of SL3 appears to be a unique requirement for genome encapsidation and identifies this process as a highly specific drug target. This study is proof of principle that development of a new class of antiretroviral drugs that specifically target viral packaging by binding to the viral genomic RNA is achievable.

Distributed and Centralized Conflict Management Under Traffic Flow Management Constraints

NASA Technical Reports Server (NTRS)

Feron, Eric; Bilimoria, Karl (Technical Monitor)

2001-01-01

The past year's activity has concentrated on the following two activities: (1) Refining and completing our study on the stability of interacting flows of aircraft when they have to resolve conflicts in a decentralized and sequential manner. More specifically, it was felt that some of the modeling assumptions made during previous research (such offset maneuvering models) could be improved to include more realistic models such as heading changes when analyzing interacting flow stability problems. We extended our analysis to achieve this goal. The results of this study have been submitted for presentation at the 2002 American Control Conference; (2) Examining the issues associated with delay propagation across multiple enroute sectors. This study was initiated at NASA in cooperation with Dr. Karl Bilimoria. Considering a set of adjacent sectors, this ongoing study concentrates on the effect of various traffic flow management strategies on the propagation of delays and congestion across sectors. The problem description and findings so far are reported in the attached working paper "Enroute sector buffering capacity."

Bacterial Lipopolysaccharide Destabilizes Influenza Viruses.

PubMed

Bandoro, Christopher; Runstadler, Jonathan A

2017-01-01

Depending on the specific viral pathogen, commensal bacteria can promote or reduce the severity of viral infection and disease progression in their hosts. Influenza A virus (IAV) has a broad host range, comprises many subtypes, and utilizes different routes of transmission, including the fecal-oral route in wild birds. It has been previously demonstrated that commensal bacteria can interact with the host's immune system to protect against IAV pathogenesis. However, it is unclear whether bacteria and their products may be interacting directly with IAV to impact virion stability. Herein we show that gastrointestinal (GI) tract bacterial isolates in an in vitro system significantly reduce the stability of IAV. Moreover, bacterial lipopolysaccharide (LPS), found on the exterior surfaces of bacteria, was sufficient to significantly decrease the stability of both human and avian viral strains in a temperature-dependent manner, including at the relevant temperatures of their respective hosts and the external aquatic habitat. The subtype and host origin of the viruses were shown to affect the extent to which IAV was susceptible to LPS. Furthermore, using a receptor binding assay and transmission electron microscopy, we observed that LPS binds to and alters the morphology of influenza virions, suggesting that direct interaction with the viral surface contributes to the observed antiviral effect of LPS on influenza. IMPORTANCE Influenza A virus (IAV), transmitted primarily via the fecal-oral route in wild birds, encounters high concentrations of bacteria and their products. Understanding the extent to which bacteria affect the infectivity of IAV will lead to a broader understanding of viral ecology in reservoir hosts and may lead to insights for the development of therapeutics in respiratory infection. Herein we show that bacteria and lipopolysaccharide (LPS) interact with and destabilize influenza virions. Moreover, we show that LPS reduces the long-term persistence and freeze-thaw stability of IAV, which is important information for modeling the movement and emergence of novel strains from animal hosts. Our results, demonstrating that the subtype and host origin of a virus also influence its susceptibility to LPS, raise key questions about the fitness of viruses in reservoir hosts, their potential to transmit to humans, and the importance of bacterial-viral interactions in viral ecology.

Bacterial Lipopolysaccharide Destabilizes Influenza Viruses

PubMed Central

2017-01-01

ABSTRACT Depending on the specific viral pathogen, commensal bacteria can promote or reduce the severity of viral infection and disease progression in their hosts. Influenza A virus (IAV) has a broad host range, comprises many subtypes, and utilizes different routes of transmission, including the fecal-oral route in wild birds. It has been previously demonstrated that commensal bacteria can interact with the host’s immune system to protect against IAV pathogenesis. However, it is unclear whether bacteria and their products may be interacting directly with IAV to impact virion stability. Herein we show that gastrointestinal (GI) tract bacterial isolates in an in vitro system significantly reduce the stability of IAV. Moreover, bacterial lipopolysaccharide (LPS), found on the exterior surfaces of bacteria, was sufficient to significantly decrease the stability of both human and avian viral strains in a temperature-dependent manner, including at the relevant temperatures of their respective hosts and the external aquatic habitat. The subtype and host origin of the viruses were shown to affect the extent to which IAV was susceptible to LPS. Furthermore, using a receptor binding assay and transmission electron microscopy, we observed that LPS binds to and alters the morphology of influenza virions, suggesting that direct interaction with the viral surface contributes to the observed antiviral effect of LPS on influenza. IMPORTANCE Influenza A virus (IAV), transmitted primarily via the fecal-oral route in wild birds, encounters high concentrations of bacteria and their products. Understanding the extent to which bacteria affect the infectivity of IAV will lead to a broader understanding of viral ecology in reservoir hosts and may lead to insights for the development of therapeutics in respiratory infection. Herein we show that bacteria and lipopolysaccharide (LPS) interact with and destabilize influenza virions. Moreover, we show that LPS reduces the long-term persistence and freeze-thaw stability of IAV, which is important information for modeling the movement and emergence of novel strains from animal hosts. Our results, demonstrating that the subtype and host origin of a virus also influence its susceptibility to LPS, raise key questions about the fitness of viruses in reservoir hosts, their potential to transmit to humans, and the importance of bacterial-viral interactions in viral ecology. PMID:29034326

Stabilization of magnetic skyrmions by RKKY interactions

NASA Astrophysics Data System (ADS)

Bezvershenko, Alla V.; Kolezhuk, Alexei K.; Ivanov, Boris A.

2018-02-01

We study the stabilization of an isolated magnetic skyrmion in a magnetic monolayer on a nonmagnetic conducting substrate via the Ruderman-Kittel-Kasuya-Yosida (RKKY) exchange interaction. Two different types of the substrate are considered, usual normal metal and single-layer graphene. While the full stability analysis for skyrmions in the presence of the RKKY coupling requires a separate effort that is outside the scope of this work, we are able to study the radial stability (stability of a skyrmion against collapse) using variational energy estimates obtained within first-order perturbation theory, with the unperturbed Hamiltonian describing the isotropic Heisenberg magnet, and the two perturbations being the RKKY exchange and the easy-axis anisotropy. We show that a proper treatment of the long-range nature of the RKKY interaction leads to a qualitatively different stabilization scenario compared to previous studies, where solitons were stabilized by the frustrated exchange coupling (leading to terms with the fourth power of the magnetization gradients) or by the Dzyaloshinskii-Moriya interaction (described by terms linear in the magnetization gradients). In the case of a metallic substrate, the skyrmion stabilization is possible under restrictive conditions on the Fermi surface parameters, while in the case of a graphene substrate the stabilization is naturally achieved in several geometries with a lattice-matching of graphene and magnetic layer.

Amorphous stabilization and dissolution enhancement of amorphous ternary solid dispersions: combination of polymers showing drug-polymer interaction for synergistic effects.

PubMed

Prasad, Dev; Chauhan, Harsh; Atef, Eman

2014-11-01

The purpose of this study was to understand the combined effect of two polymers showing drug-polymer interactions on amorphous stabilization and dissolution enhancement of indomethacin (IND) in amorphous ternary solid dispersions. The mechanism responsible for the enhanced stability and dissolution of IND in amorphous ternary systems was studied by exploring the miscibility and intermolecular interactions between IND and polymers through thermal and spectroscopic analysis. Eudragit E100 and PVP K90 at low concentrations (2.5%-40%, w/w) were used to prepare amorphous binary and ternary solid dispersions by solvent evaporation. Stability results showed that amorphous ternary solid dispersions have better stability compared with amorphous binary solid dispersions. The dissolution of IND from the ternary dispersion was substantially higher than the binary dispersions as well as amorphous drug. Melting point depression of physical mixtures reveals that the drug was miscible in both the polymers; however, greater miscibility was observed in ternary physical mixtures. The IR analysis confirmed intermolecular interactions between IND and individual polymers. These interactions were found to be intact in ternary systems. These results suggest that the combination of two polymers showing drug-polymer interaction offers synergistic enhancement in amorphous stability and dissolution in ternary solid dispersions. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Role of water mediated interactions in protein-protein recognition landscapes.

PubMed

Papoian, Garegin A; Ulander, Johan; Wolynes, Peter G

2003-07-30

The energy landscape picture of protein folding and binding is employed to optimize a number of pair potentials for direct and water-mediated interactions in protein complex interfaces. We find that water-mediated interactions greatly complement direct interactions in discriminating against various types of trap interactions that model those present in the cell. We highlight the context dependent nature of knowledge-based binding potentials, as contrasted with the situation for autonomous folding. By performing a Principal Component Analysis (PCA) of the corresponding interaction matrixes, we rationalize the strength of the recognition signal for each combination of the contact type and reference trap states using the differential in the idealized "canonical" amino acid compositions of native and trap layers. The comparison of direct and water-mediated contact potential matrixes emphasizes the importance of partial solvation in stabilizing charged groups in the protein interfaces. Specific water-mediated interresidue interactions are expected to influence significantly the kinetics as well as thermodynamics of protein association.

Modulating the DNA affinity of Elk-1 with computationally selected mutations.

PubMed

Park, Sheldon; Boder, Eric T; Saven, Jeffery G

2005-04-22

In order to regulate gene expression, transcription factors must first bind their target DNA sequences. The affinity of this binding is determined by both the network of interactions at the interface and the entropy change associated with the complex formation. To study the role of structural fluctuation in fine-tuning DNA affinity, we performed molecular dynamics simulations of two highly homologous proteins, Elk-1 and SAP-1, that exhibit different sequence specificity. Simulation studies show that several residues in Elk have significantly higher main-chain root-mean-square deviations than their counterparts in SAP. In particular, a single residue, D69, may contribute to Elk's lower DNA affinity for P(c-fos) by structurally destabilizing the carboxy terminus of the recognition helix. While D69 does not contact DNA directly, the increased mobility in the region may contribute to its weaker binding. We measured the ability of single point mutants of Elk to bind P(c-fos) in a reporter assay, in which D69 of wild-type Elk has been mutated to other residues with higher helix propensity in order to stabilize the local conformation. The gains in transcriptional activity and the free energy of binding suggested from these measurements correlate well with stability gains computed from helix propensity and charge-macrodipole interactions. The study suggests that residues that are distal to the binding interface may indirectly modulate the binding affinity by stabilizing the protein scaffold required for efficient DNA interaction.

Synchronisation and stability in river metapopulation networks.

PubMed

Yeakel, J D; Moore, J W; Guimarães, P R; de Aguiar, M A M

2014-03-01

Spatial structure in landscapes impacts population stability. Two linked components of stability have large consequences for persistence: first, statistical stability as the lack of temporal fluctuations; second, synchronisation as an aspect of dynamic stability, which erodes metapopulation rescue effects. Here, we determine the influence of river network structure on the stability of riverine metapopulations. We introduce an approach that converts river networks to metapopulation networks, and analytically show how fluctuation magnitude is influenced by interaction structure. We show that river metapopulation complexity (in terms of branching prevalence) has nonlinear dampening effects on population fluctuations, and can also buffer against synchronisation. We conclude by showing that river transects generally increase synchronisation, while the spatial scale of interaction has nonlinear effects on synchronised dynamics. Our results indicate that this dual stability - conferred by fluctuation and synchronisation dampening - emerges from interaction structure in rivers, and this may strongly influence the persistence of river metapopulations. © 2013 John Wiley & Sons Ltd/CNRS.

Lipid Interaction Sites on Channels, Transporters and Receptors: Recent Insights from Molecular Dynamics Simulations

PubMed Central

Hedger, George; Sansom, Mark S. P.

2017-01-01

Lipid molecules are able to selectively interact with specific sites on integral membrane proteins, and modulate their structure and function. Identification and characterisation of these sites is of importance for our understanding of the molecular basis of membrane protein function and stability, and may facilitate the design of lipid-like drug molecules. Molecular dynamics simulations provide a powerful tool for the identification of these sites, complementing advances in membrane protein structural biology and biophysics. We describe recent notable biomolecular simulation studies which have identified lipid interaction sites on a range of different membrane proteins. The sites identified in these simulation studies agree well with those identified by complementary experimental techniques. This demonstrates the power of the molecular dynamics approach in the prediction and characterization of lipid interaction sites on integral membrane proteins. PMID:26946244

Targeting Allosteric Control Mechanisms in Heat Shock Protein 70 (Hsp70).

PubMed

Li, Xiaokai; Shao, Hao; Taylor, Isabelle R; Gestwicki, Jason E

2016-01-01

Heat shock protein 70 (Hsp70) is a molecular chaperone that plays critical roles in protein homeostasis. Hsp70's chaperone activity is coordinated by intra-molecular interactions between its two domains, as well as inter-molecular interactions between Hsp70 and its co-chaperones. Each of these contacts represents a potential opportunity for the development of chemical inhibitors. To illustrate this concept, we review three classes of recently identified molecules that bind distinct pockets on Hsp70. Although all three compounds share the ability to interrupt core biochemical functions of Hsp70, they stabilize different conformers. Accordingly, each compound appears to interrupt a specific subset of inter- and intra-molecular interactions. Thus, an accurate definition of an Hsp70 inhibitor may require a particularly detailed understanding of the molecule's binding site and its effects on protein-protein interactions.

Capturing RNA Folding Free Energy with Coarse-Grained Molecular Dynamics Simulations

PubMed Central

Bell, David R.; Cheng, Sara Y.; Salazar, Heber; Ren, Pengyu

2017-01-01

We introduce a coarse-grained RNA model for molecular dynamics simulations, RACER (RnA CoarsE-gRained). RACER achieves accurate native structure prediction for a number of RNAs (average RMSD of 2.93 Å) and the sequence-specific variation of free energy is in excellent agreement with experimentally measured stabilities (R2 = 0.93). Using RACER, we identified hydrogen-bonding (or base pairing), base stacking, and electrostatic interactions as essential driving forces for RNA folding. Also, we found that separating pairing vs. stacking interactions allowed RACER to distinguish folded vs. unfolded states. In RACER, base pairing and stacking interactions each provide an approximate stability of 3–4 kcal/mol for an A-form helix. RACER was developed based on PDB structural statistics and experimental thermodynamic data. In contrast with previous work, RACER implements a novel effective vdW potential energy function, which led us to re-parameterize hydrogen bond and electrostatic potential energy functions. Further, RACER is validated and optimized using a simulated annealing protocol to generate potential energy vs. RMSD landscapes. Finally, RACER is tested using extensive equilibrium pulling simulations (0.86 ms total) on eleven RNA sequences (hairpins and duplexes). PMID:28393861

Regulation of chromatin organization and inducible gene expression by a Drosophila insulator

PubMed Central

Wood, Ashley M.; Van Bortle, Kevin; Ramos, Edward; Takenaka, Naomi; Rohrbaugh, Margaret; Jones, Brian C.; Jones, Keith C.; Corces, Victor G.

2011-01-01

SUMMARY Insulators are multi-protein-DNA complexes thought to affect gene expression by mediating inter- and intra-chromosomal interactions. Drosophila insulators contain specific DNA binding proteins plus common components, such as CP190, that facilitate these interactions. Here we examine changes in the distribution of Drosophila insulator proteins during the heat-shock and ecdysone responses. We find that CP190 recruitment to insulator sites is the main regulatable step in controlling insulator function during heat shock. In contrast, both CP190 and DNA binding protein recruitment are regulated during the ecdysone response. CP190 is necessary to stabilize specific chromatin loops and for proper activation of transcription of genes regulated by this hormone. These findings suggest that cells may regulate recruitment of insulator proteins to the DNA in order to activate insulator activity at specific sites and create distinct patterns of nuclear organization that are necessary to achieve proper gene expression in response to different stimuli. PMID:21981916

Regulation of chromatin organization and inducible gene expression by a Drosophila insulator.

PubMed

Wood, Ashley M; Van Bortle, Kevin; Ramos, Edward; Takenaka, Naomi; Rohrbaugh, Margaret; Jones, Brian C; Jones, Keith C; Corces, Victor G

2011-10-07

Insulators are multiprotein-DNA complexes thought to affect gene expression by mediating inter- and intrachromosomal interactions. Drosophila insulators contain specific DNA-binding proteins plus common components, such as CP190, that facilitate these interactions. Here, we examine changes in the distribution of Drosophila insulator proteins during the heat-shock and ecdysone responses. We find that CP190 recruitment to insulator sites is the main regulatable step in controlling insulator function during heat shock. In contrast, both CP190 and DNA-binding protein recruitment are regulated during the ecdysone response. CP190 is necessary to stabilize specific chromatin loops and for proper activation of transcription of genes regulated by this hormone. These findings suggest that cells may regulate recruitment of insulator proteins to DNA to activate insulator activity at specific sites and create distinct patterns of nuclear organization that are necessary to achieve proper gene expression in response to different stimuli. Copyright © 2011 Elsevier Inc. All rights reserved.

Perturbations to trophic interactions and the stability of complex food webs

PubMed Central

O'Gorman, Eoin J.; Emmerson, Mark C.

2009-01-01

The pattern of predator–prey interactions is thought to be a key determinant of ecosystem processes and stability. Complex ecological networks are characterized by distributions of interaction strengths that are highly skewed, with many weak and few strong interactors present. Theory suggests that this pattern promotes stability as weak interactors dampen the destabilizing potential of strong interactors. Here, we present an experimental test of this hypothesis and provide empirical evidence that the loss of weak interactors can destabilize communities in nature. We ranked 10 marine consumer species by the strength of their trophic interactions. We removed the strongest and weakest of these interactors from experimental food webs containing >100 species. Extinction of strong interactors produced a dramatic trophic cascade and reduced the temporal stability of key ecosystem process rates, community diversity and resistance to changes in community composition. Loss of weak interactors also proved damaging for our experimental ecosystems, leading to reductions in the temporal and spatial stability of ecosystem process rates, community diversity, and resistance. These results highlight the importance of conserving species to maintain the stabilizing pattern of trophic interactions in nature, even if they are perceived to have weak effects in the system. PMID:19666606

Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes

PubMed Central

Dodds, Peter N.; Lawrence, Gregory J.; Catanzariti, Ann-Maree; Teh, Trazel; Wang, Ching-I. A.; Ayliffe, Michael A.; Kobe, Bostjan; Ellis, Jeffrey G.

2006-01-01

Plant resistance proteins (R proteins) recognize corresponding pathogen avirulence (Avr) proteins either indirectly through detection of changes in their host protein targets or through direct R–Avr protein interaction. Although indirect recognition imposes selection against Avr effector function, pathogen effector molecules recognized through direct interaction may overcome resistance through sequence diversification rather than loss of function. Here we show that the flax rust fungus AvrL567 genes, whose products are recognized by the L5, L6, and L7 R proteins of flax, are highly diverse, with 12 sequence variants identified from six rust strains. Seven AvrL567 variants derived from Avr alleles induce necrotic responses when expressed in flax plants containing corresponding resistance genes (R genes), whereas five variants from avr alleles do not. Differences in recognition specificity between AvrL567 variants and evidence for diversifying selection acting on these genes suggest they have been involved in a gene-specific arms race with the corresponding flax R genes. Yeast two-hybrid assays indicate that recognition is based on direct R–Avr protein interaction and recapitulate the interaction specificity observed in planta. Biochemical analysis of Escherichia coli-produced AvrL567 proteins shows that variants that escape recognition nevertheless maintain a conserved structure and stability, suggesting that the amino acid sequence differences directly affect the R–Avr protein interaction. We suggest that direct recognition associated with high genetic diversity at corresponding R and Avr gene loci represents an alternative outcome of plant–pathogen coevolution to indirect recognition associated with simple balanced polymorphisms for functional and nonfunctional R and Avr genes. PMID:16731621

Deep convolutional neural networks for pan-specific peptide-MHC class I binding prediction.

PubMed

Han, Youngmahn; Kim, Dongsup

2017-12-28

Computational scanning of peptide candidates that bind to a specific major histocompatibility complex (MHC) can speed up the peptide-based vaccine development process and therefore various methods are being actively developed. Recently, machine-learning-based methods have generated successful results by training large amounts of experimental data. However, many machine learning-based methods are generally less sensitive in recognizing locally-clustered interactions, which can synergistically stabilize peptide binding. Deep convolutional neural network (DCNN) is a deep learning method inspired by visual recognition process of animal brain and it is known to be able to capture meaningful local patterns from 2D images. Once the peptide-MHC interactions can be encoded into image-like array(ILA) data, DCNN can be employed to build a predictive model for peptide-MHC binding prediction. In this study, we demonstrated that DCNN is able to not only reliably predict peptide-MHC binding, but also sensitively detect locally-clustered interactions. Nonapeptide-HLA-A and -B binding data were encoded into ILA data. A DCNN, as a pan-specific prediction model, was trained on the ILA data. The DCNN showed higher performance than other prediction tools for the latest benchmark datasets, which consist of 43 datasets for 15 HLA-A alleles and 25 datasets for 10 HLA-B alleles. In particular, the DCNN outperformed other tools for alleles belonging to the HLA-A3 supertype. The F1 scores of the DCNN were 0.86, 0.94, and 0.67 for HLA-A*31:01, HLA-A*03:01, and HLA-A*68:01 alleles, respectively, which were significantly higher than those of other tools. We found that the DCNN was able to recognize locally-clustered interactions that could synergistically stabilize peptide binding. We developed ConvMHC, a web server to provide user-friendly web interfaces for peptide-MHC class I binding predictions using the DCNN. ConvMHC web server can be accessible via http://jumong.kaist.ac.kr:8080/convmhc . We developed a novel method for peptide-HLA-I binding predictions using DCNN trained on ILA data that encode peptide binding data and demonstrated the reliable performance of the DCNN in nonapeptide binding predictions through the independent evaluation on the latest IEDB benchmark datasets. Our approaches can be applied to characterize locally-clustered patterns in molecular interactions, such as protein/DNA, protein/RNA, and drug/protein interactions.

The role of stabilization centers in protein thermal stability

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

Magyar, Csaba; Gromiha, M. Michael; Sávoly, Zoltán

2016-02-26

The definition of stabilization centers was introduced almost two decades ago. They are centers of noncovalent long range interaction clusters, believed to have a role in maintaining the three-dimensional structure of proteins by preventing their decay due to their cooperative long range interactions. Here, this hypothesis is investigated from the viewpoint of thermal stability for the first time, using a large protein thermodynamics database. The positions of amino acids belonging to stabilization centers are correlated with available experimental thermodynamic data on protein thermal stability. Our analysis suggests that stabilization centers, especially solvent exposed ones, do contribute to the thermal stabilizationmore » of proteins. - Highlights: • Stabilization centers contribute to thermal stabilization of protein structures. • Stabilization center content correlates with melting temperature of proteins. • Exposed stabilization center content correlates with stability even in hyperthermophiles. • Stability changing mutations are frequently found at stabilization centers.« less

Thermodynamic Origins of Monovalent Facilitated RNA Folding

PubMed Central

Holmstrom, Erik D.; Fiore, Julie L.; Nesbitt, David J.

2012-01-01

Cations have long been associated with formation of native RNA structure and are commonly thought to stabilize the formation of tertiary contacts by favorably interacting with the electrostatic potential of the RNA, giving rise to an “ion atmosphere”. A significant amount of information regarding the thermodynamics of structural transitions in the presence of an ion atmosphere has accumulated and suggests stabilization is dominated by entropic terms. This work provides an analysis of how RNA–cation interactions affect the entropy and enthalpy associated with an RNA tertiary transition. Specifically, temperature-dependent single-molecule fluorescence resonance energy transfer studies have been exploited to determine the free energy (ΔG°), enthalpy (ΔH°), and entropy (ΔS°) of folding for an isolated tetraloop–receptor tertiary interaction as a function of Na+ concentration. Somewhat unexpectedly, increasing the Na+ concentration changes the folding enthalpy from a strongly exothermic process [e.g., ΔH° = −26(2) kcal/mol at 180 mM] to a weakly exothermic process [e.g., ΔH° = −4(1) kcal/mol at 630 mM]. As a direct corollary, it is the strong increase in folding entropy [Δ(ΔS°) > 0] that compensates for this loss of exothermicity for the achievement of more favorable folding [Δ(ΔG°) < 0] at higher Na+ concentrations. In conjunction with corresponding measurements of the thermodynamics of the transition state barrier, these data provide a detailed description of the folding pathway associated with the GAAA tetraloop–receptor interaction as a function of Na+ concentration. The results support a potentially universal mechanism for monovalent facilitated RNA folding, whereby an increasing monovalent concentration stabilizes tertiary structure by reducing the entropic penalty for folding. PMID:22448852

Predicting the tolerated sequences for proteins and protein interfaces using RosettaBackrub flexible backbone design.

PubMed

Smith, Colin A; Kortemme, Tanja

2011-01-01

Predicting the set of sequences that are tolerated by a protein or protein interface, while maintaining a desired function, is useful for characterizing protein interaction specificity and for computationally designing sequence libraries to engineer proteins with new functions. Here we provide a general method, a detailed set of protocols, and several benchmarks and analyses for estimating tolerated sequences using flexible backbone protein design implemented in the Rosetta molecular modeling software suite. The input to the method is at least one experimentally determined three-dimensional protein structure or high-quality model. The starting structure(s) are expanded or refined into a conformational ensemble using Monte Carlo simulations consisting of backrub backbone and side chain moves in Rosetta. The method then uses a combination of simulated annealing and genetic algorithm optimization methods to enrich for low-energy sequences for the individual members of the ensemble. To emphasize certain functional requirements (e.g. forming a binding interface), interactions between and within parts of the structure (e.g. domains) can be reweighted in the scoring function. Results from each backbone structure are merged together to create a single estimate for the tolerated sequence space. We provide an extensive description of the protocol and its parameters, all source code, example analysis scripts and three tests applying this method to finding sequences predicted to stabilize proteins or protein interfaces. The generality of this method makes many other applications possible, for example stabilizing interactions with small molecules, DNA, or RNA. Through the use of within-domain reweighting and/or multistate design, it may also be possible to use this method to find sequences that stabilize particular protein conformations or binding interactions over others.

A peek into tropomyosin binding and unfolding on the actin filament.

PubMed

Singh, Abhishek; Hitchcock-Degregori, Sarah E

2009-07-24

Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin. Tropomyosin's periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle alpha-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven) increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering), and chain dissociation (analyzed using circular dichroism). This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest that local disorder is a property typical of coiled coil binding sites and proteins that have multiple binding partners, of which tropomyosin is one type.

Elucidating sequence and solvent specific design targets to protect and stabilize enzymes for biocatalysis in ionic liquids.

PubMed

Sprenger, K G; Plaks, J G; Kaar, J L; Pfaendtner, J

2017-07-05

For many different frameworks, the structure, function, and dynamics of an enzyme is largely determined by the nature of its interactions with the surrounding host environment, thus a molecular level understanding of enzyme/host interactions is essential to the design of new processes and applications. Ionic liquid (IL) solvents are a popular class of solvents in which to study enzyme behavior, yet it is still not possible to predict how a given enzyme will behave in a given IL solvent. Furthermore, a dearth of experimental data with which to evaluate simulation force fields has prevented the full integration of experimental and computational techniques to gain a complete picture of enzyme/IL interactions. Utilizing recently published crystallographic data of an enzyme in complex with an IL, this study aims to validate the use of current molecular force fields for studying enzyme/IL interactions, and to provide new mechanistic insight into enzyme stabilization in IL solvents. Classical molecular dynamics (MD) simulations have been performed on both the folded and unfolded state of Bacillus subtilis lipase A and a quadruple-mutant version of lipase A, in solutions of aqueous 1-butyl-3-methylimidazolium chloride. Results show classical MD simulations can predict the preferred surface binding locations of IL cations as well as reductions in IL anion binding to mutated surface residues with high accuracy. The results also point to a mechanistic difference between IL binding to the folded and unfolded state of an enzyme, which we call the "counter-ion effect". These findings could have important implications for future rational design efforts to stabilize enzymes in non-conventional media.

Biomechanical concepts applicable to minimally invasive fracture repair in small animals.

PubMed

Chao, Peini; Lewis, Daniel D; Kowaleski, Michael P; Pozzi, Antonio

2012-09-01

Understanding the basic biomechanical principles of surgical stabilization of fractures is essential for developing an appropriate preoperative plan as well as making prudent intraoperative decisions. This article aims to provide basic biomechanical knowledge essential to the understanding of the complex interaction between the mechanics and biology of fracture healing. The type of healing and the outcome can be influenced by several mechanical factors, which depend on the interaction between bone and implant. The surgeon should understand the mechanical principles of fracture fixation and be able to choose the best type of fixation for each specific fracture. Copyright © 2012 Elsevier Inc. All rights reserved.

Context-dependent effects of asparagine glycosylation on Pin WW folding kinetics and thermodynamics.

PubMed

Price, Joshua L; Shental-Bechor, Dalit; Dhar, Apratim; Turner, Maurice J; Powers, Evan T; Gruebele, Martin; Levy, Yaakov; Kelly, Jeffery W

2010-11-03

Asparagine glycosylation is one of the most common and important post-translational modifications of proteins in eukaryotic cells. N-glycosylation occurs when a triantennary glycan precursor is transferred en bloc to a nascent polypeptide (harboring the N-X-T/S sequon) as the peptide is cotranslationally translocated into the endoplasmic reticulum (ER). In addition to facilitating binding interactions with components of the ER proteostasis network, N-glycans can also have intrinsic effects on protein folding by directly altering the folding energy landscape. Previous work from our laboratories (Hanson et al. Proc. Natl. Acad. Sci. U.S.A. 2009, 109, 3131-3136; Shental-Bechor, D.; Levy, Y. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 8256-8261) suggested that the three sugar residues closest to the protein are sufficient for accelerating protein folding and stabilizing the resulting structure in vitro; even a monosaccharide can have a dramatic effect. The highly conserved nature of these three proximal sugars in N-glycans led us to speculate that introducing an N-glycosylation site into a protein that is not normally glycosylated would stabilize the protein and increase its folding rate in a manner that does not depend on the presence of specific stabilizing protein-saccharide interactions. Here, we test this hypothesis experimentally and computationally by incorporating an N-linked GlcNAc residue at various positions within the Pin WW domain, a small β-sheet-rich protein. The results show that an increased folding rate and enhanced thermodynamic stability are not general, context-independent consequences of N-glycosylation. Comparison between computational predictions and experimental observations suggests that generic glycan-based excluded volume effects are responsible for the destabilizing effect of glycosylation at highly structured positions. However, this reasoning does not adequately explain the observed destabilizing effect of glycosylation within flexible loops. Our data are consistent with the hypothesis that specific, evolved protein-glycan contacts must also play an important role in mediating the beneficial energetic effects on protein folding that glycosylation can confer.

Ubiquitin-specific protease 8 links the PTEN-Akt-AIP4 pathway to the control of FLIPS stability and TRAIL sensitivity in glioblastoma multiforme.

PubMed

Panner, Amith; Crane, Courtney A; Weng, Changjiang; Feletti, Alberto; Fang, Shanna; Parsa, Andrew T; Pieper, Russell O

2010-06-15

The antiapoptotic protein FLIP(S) is a key suppressor of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human glioblastoma multiforme (GBM) cells. We previously reported that a novel phosphatase and tensin homologue (PTEN)-Akt-atrophin-interacting protein 4 (AIP4) pathway regulates FLIP(S) ubiquitination and stability, although the means by which PTEN and Akt were linked to AIP4 activity were unclear. Here, we report that a second regulator of ubiquitin metabolism, the ubiquitin-specific protease 8 (USP8), is a downstream target of Akt, and that USP8 links Akt to AIP4 and the regulation of FLIP(S) stability and TRAIL resistance. In human GBM xenografts, levels of USP8 correlated inversely with pAkt levels, and genetic or pharmacologic manipulation of Akt regulated USP8 levels in an inverse manner. Overexpression of wild-type USP8, but not catalytically inactive USP8, increased FLIP(S) ubiquitination, decreased FLIP(S) half-life, decreased FLIP(S) steady-state levels, and decreased TRAIL resistance, whereas short interfering RNA (siRNA)-mediated suppression of USP8 levels had the opposite effect. Because high levels of the USP8 deubiquitinase correlated with high levels of FLIP(S) ubiquitination, USP8 seemed to control FLIP(S) ubiquitination through an intermediate target. Consistent with this idea, overexpression of wild-type USP8 decreased the ubiquitination of the FLIP(S) E3 ubiquitin ligase AIP4, an event previously shown to increase AIP4-FLIP(S) interaction, whereas siRNA-mediated suppression of USP8 increased AIP4 ubiquitination. Furthermore, the suppression of FLIP(S) levels by USP8 overexpression was reversed by the introduction of siRNA targeting AIP4. These results show that USP8, a downstream target of Akt, regulates the ability of AIP4 to control FLIP(S) stability and TRAIL sensitivity.

The Contribution of Interchain Salt Bridges to Triple-Helical Stability in Collagen

PubMed Central

Gurry, Thomas; Nerenberg, Paul S.; Stultz, Collin M.

2010-01-01

Abstract Studies on collagen and collagen-like peptides suggest that triple-helical stability can vary along the amino acid chain. In this regard, it has been shown that lysine residues in the Y position and acidic residues in the X′ position of (GPO)3GXYGX′Y′(GPO)3 peptides lead to triple-helical structures with melting temperatures similar to (GPO)8 (where O is hydroxyproline), which is generally regarded as the most stable collagen-like sequence of this length. This enhanced stability has been attributed to the formation of salt bridges between adjacent collagen chains. In this study, we explore the relationship between interchain salt bridge formation and triple-helical stability using detailed molecular simulations. Although our results confirm that salt bridges promote triple-helical stability, we find that not all salt bridges are created equal. In particular, lysine-glutamate salt bridges are most stabilizing when formed between residues in the middle strand (B) and the trailing strand (C), whereas lysine-aspartate salt bridges are most stabilizing when formed between residues in the leading (A) and middle (B) strand—the latter observation being consistent with recent NMR data on a heterotrimeric model peptide. Overall, we believe these data clarify the role of salt bridges in modulating triple-helical stability and can be used to guide the design of collagen-like peptides that have specific interchain interactions. PMID:20513408

Sensitivity of system stability to model structure

USGS Publications Warehouse

Hosack, G.R.; Li, H.W.; Rossignol, P.A.

2009-01-01

A community is stable, and resilient, if the levels of all community variables can return to the original steady state following a perturbation. The stability properties of a community depend on its structure, which is the network of direct effects (interactions) among the variables within the community. These direct effects form feedback cycles (loops) that determine community stability. Although feedback cycles have an intuitive interpretation, identifying how they form the feedback properties of a particular community can be intractable. Furthermore, determining the role that any specific direct effect plays in the stability of a system is even more daunting. Such information, however, would identify important direct effects for targeted experimental and management manipulation even in complex communities for which quantitative information is lacking. We therefore provide a method that determines the sensitivity of community stability to model structure, and identifies the relative role of particular direct effects, indirect effects, and feedback cycles in determining stability. Structural sensitivities summarize the degree to which each direct effect contributes to stabilizing feedback or destabilizing feedback or both. Structural sensitivities prove useful in identifying ecologically important feedback cycles within the community structure and for detecting direct effects that have strong, or weak, influences on community stability. The approach may guide the development of management intervention and research design. We demonstrate its value with two theoretical models and two empirical examples of different levels of complexity. ?? 2009 Elsevier B.V. All rights reserved.

Trends in Thermostability Provide Information on the Nature of Substrate, Inhibitor, and Lipid Interactions with Mitochondrial Carriers*

PubMed Central

Crichton, Paul G.; Lee, Yang; Ruprecht, Jonathan J.; Cerson, Elizabeth; Thangaratnarajah, Chancievan; King, Martin S.; Kunji, Edmund R. S.

2015-01-01

Mitochondrial carriers, including uncoupling proteins, are unstable in detergents, which hampers structural and mechanistic studies. To investigate carrier stability, we have purified ligand-free carriers and assessed their stability with a fluorescence-based thermostability assay that monitors protein unfolding with a thiol-reactive dye. We find that mitochondrial carriers from both mesophilic and thermophilic organisms exhibit poor stability in mild detergents, indicating that instability is inherent to the protein family. Trends in the thermostability of yeast ADP/ATP carrier AAC2 and ovine uncoupling protein UCP1 allow optimal conditions for stability in detergents to be established but also provide mechanistic insights into the interactions of lipids, substrates, and inhibitors with these proteins. Both proteins exhibit similar stability profiles across various detergents, where stability increases with the size of the associated detergent micelle. Detailed analysis shows that lipids stabilize carriers indirectly by increasing the associated detergent micelle size, but cardiolipin stabilizes by direct interactions as well. Cardiolipin reverses destabilizing effects of ADP and bongkrekic acid on AAC2 and enhances large stabilizing effects of carboxyatractyloside, revealing that this lipid interacts in the m-state and possibly other states of the transport cycle, despite being in a dynamic interface. Fatty acid activators destabilize UCP1 in a similar way, which can also be prevented by cardiolipin, indicating that they interact like transport substrates. Our controls show that carriers can be soluble but unfolded in some commonly used detergents, such as the zwitterionic Fos-choline-12, which emphasizes the need for simple validation assays like the one used here. PMID:25653283

Optimized hydrophobic interactions and hydrogen bonding at the target-ligand interface leads the pathways of drug-designing.

PubMed

Patil, Rohan; Das, Suranjana; Stanley, Ashley; Yadav, Lumbani; Sudhakar, Akulapalli; Varma, Ashok K

2010-08-16

Weak intermolecular interactions such as hydrogen bonding and hydrophobic interactions are key players in stabilizing energetically-favored ligands, in an open conformational environment of protein structures. However, it is still poorly understood how the binding parameters associated with these interactions facilitate a drug-lead to recognize a specific target and improve drugs efficacy. To understand this, comprehensive analysis of hydrophobic interactions, hydrogen bonding and binding affinity have been analyzed at the interface of c-Src and c-Abl kinases and 4-amino substituted 1H-pyrazolo [3, 4-d] pyrimidine compounds. In-silico docking studies were performed, using Discovery Studio software modules LigandFit, CDOCKER and ZDOCK, to investigate the role of ligand binding affinity at the hydrophobic pocket of c-Src and c-Abl kinase. Hydrophobic and hydrogen bonding interactions of docked molecules were compared using LigPlot program. Furthermore, 3D-QSAR and MFA calculations were scrutinized to quantify the role of weak interactions in binding affinity and drug efficacy. The in-silico method has enabled us to reveal that a multi-targeted small molecule binds with low affinity to its respective targets. But its binding affinity can be altered by integrating the conformationally favored functional groups at the active site of the ligand-target interface. Docking studies of 4-amino-substituted molecules at the bioactive cascade of the c-Src and c-Abl have concluded that 3D structural folding at the protein-ligand groove is also a hallmark for molecular recognition of multi-targeted compounds and for predicting their biological activity. The results presented here demonstrate that hydrogen bonding and optimized hydrophobic interactions both stabilize the ligands at the target site, and help alter binding affinity and drug efficacy.

Optimized Hydrophobic Interactions and Hydrogen Bonding at the Target-Ligand Interface Leads the Pathways of Drug-Designing

PubMed Central

Stanley, Ashley; Yadav, Lumbani; Sudhakar, Akulapalli; Varma, Ashok K.

2010-01-01

Background Weak intermolecular interactions such as hydrogen bonding and hydrophobic interactions are key players in stabilizing energetically-favored ligands, in an open conformational environment of protein structures. However, it is still poorly understood how the binding parameters associated with these interactions facilitate a drug-lead to recognize a specific target and improve drugs efficacy. To understand this, comprehensive analysis of hydrophobic interactions, hydrogen bonding and binding affinity have been analyzed at the interface of c-Src and c-Abl kinases and 4-amino substituted 1H-pyrazolo [3, 4-d] pyrimidine compounds. Methodology In-silico docking studies were performed, using Discovery Studio software modules LigandFit, CDOCKER and ZDOCK, to investigate the role of ligand binding affinity at the hydrophobic pocket of c-Src and c-Abl kinase. Hydrophobic and hydrogen bonding interactions of docked molecules were compared using LigPlot program. Furthermore, 3D-QSAR and MFA calculations were scrutinized to quantify the role of weak interactions in binding affinity and drug efficacy. Conclusions The in-silico method has enabled us to reveal that a multi-targeted small molecule binds with low affinity to its respective targets. But its binding affinity can be altered by integrating the conformationally favored functional groups at the active site of the ligand-target interface. Docking studies of 4-amino-substituted molecules at the bioactive cascade of the c-Src and c-Abl have concluded that 3D structural folding at the protein-ligand groove is also a hallmark for molecular recognition of multi-targeted compounds and for predicting their biological activity. The results presented here demonstrate that hydrogen bonding and optimized hydrophobic interactions both stabilize the ligands at the target site, and help alter binding affinity and drug efficacy. PMID:20808434

Reliability of a Novel Social Activity Questionnaire: Perceived Social Support and Verbal Interaction in the Wisconsin Registry for Alzheimer's Prevention.

PubMed

Zuelsdorff, Megan L; Koscik, Rebecca L; Okonkwo, Ozioma C; Peppard, Paul E; Hermann, Bruce P; Sager, Mark A; Johnson, Sterling C; Engelman, Corinne D

2018-02-01

Social activity is associated with healthy aging and preserved cognition. Such activity includes a confluence of social support and verbal interaction, each influencing cognition through rarely parsed, mechanistically distinct pathways. We created a novel verbal interaction measure for the Wisconsin Registry for Alzheimer's Prevention (WRAP) and assessed reliability of resultant data, a first step toward mechanism-driven examination of social activity as a modifiable predictor of cognitive health. Two WRAP subsamples completed a test-retest study to determine 8-week stability ( n = 107) and 2-year stability ( n = 136) of verbal interaction, and 2-year stability of perceived social support. Reliability was determined using quadratic-weighted kappa, percent agreement, or correlation coefficients. Reliability was fair to almost perfect. The association between social support and interaction quantity decreased with age. Social activity data demonstrate moderate to excellent temporal stability. Moreover, in older individuals, social support and verbal interaction represent two distinct dimensions of social activity.

Weak interactions, omnivory and emergent food-web properties.

PubMed

Emmerson, Mark; Yearsley, Jon M

2004-02-22

Empirical studies have shown that, in real ecosystems, species-interaction strengths are generally skewed in their distribution towards weak interactions. Some theoretical work also suggests that weak interactions, especially in omnivorous links, are important for the local stability of a community at equilibrium. However, the majority of theoretical studies use uniform distributions of interaction strengths to generate artificial communities for study. We investigate the effects of the underlying interaction-strength distribution upon the return time, permanence and feasibility of simple Lotka-Volterra equilibrium communities. We show that a skew towards weak interactions promotes local and global stability only when omnivory is present. It is found that skewed interaction strengths are an emergent property of stable omnivorous communities, and that this skew towards weak interactions creates a dynamic constraint maintaining omnivory. Omnivory is more likely to occur when omnivorous interactions are skewed towards weak interactions. However, a skew towards weak interactions increases the return time to equilibrium, delays the recovery of ecosystems and hence decreases the stability of a community. When no skew is imposed, the set of stable omnivorous communities shows an emergent distribution of skewed interaction strengths. Our results apply to both local and global concepts of stability and are robust to the definition of a feasible community. These results are discussed in the light of empirical data and other theoretical studies, in conjunction with their broader implications for community assembly.

Nanopore Force Spectroscopy of Aptamer–Ligand Complexes

PubMed Central

Arnaut, Vera; Langecker, Martin; Simmel, Friedrich C.

2013-01-01

The stability of aptamer–ligand complexes is probed in nanopore-based dynamic force spectroscopy experiments. Specifically, the ATP-binding aptamer is investigated using a backward translocation technique, in which the molecules are initially pulled through an α-hemolysin nanopore from the cis to the trans side of a lipid bilayer membrane, allowed to refold and interact with their target, and then translocated back in the trans–cis direction. From these experiments, the distribution of bound and unbound complexes is determined, which in turn allows determination of the dissociation constant Kd ≈ 0.1 mM of the aptamer and of voltage-dependent unfolding rates. The experiments also reveal differences in binding of the aptamer to AMP, ADP, or ATP ligands. Investigation of an aptamer variant with a stabilized ATP-binding site indicates fast conformational switching of the original aptamer before ATP binding. Nanopore force spectroscopy is also used to study binding of the thrombin-binding aptamer to its target. To detect aptamer–target interactions in this case, the stability of the ligand-free aptamer—containing G-quadruplexes—is tuned via the potassium content of the buffer. Although the presence of thrombin was detected, limitations of the method for aptamers with strong secondary structures and complexes with nanomolar Kd were identified. PMID:24010663

Stabilization of a supersaturated solution of mefenamic acid from a solid dispersion with EUDRAGIT(®) EPO.

PubMed

Kojima, Taro; Higashi, Kenjirou; Suzuki, Toyofumi; Tomono, Kazuo; Moribe, Kunikazu; Yamamoto, Keiji

2012-10-01

The stabilization mechanism of a supersaturated solution of mefenamic acid (MFA) from a solid dispersion with EUDRAGIT(®) EPO (EPO) was investigated. The solid dispersions were prepared by cryogenic grinding method. Powder X-ray diffractometry, in vitro dissolution test, in vivo oral absorption study, infrared spectroscopy, and solid- and solution-state NMR spectroscopies were used to characterize the solid dispersions. Dissolution tests in acetate buffer (pH 5.5) revealed that solid dispersion showed > 200-fold higher concentration of MFA. Supersaturated solution was stable over 1 month and exhibited improved oral bioavailability of MFA in rats, with a 7.8-fold higher area under the plasma concentration-versus-time curve. Solid-state (1)H spin-lattice relaxation time (T(1)) measurement showed that MFA was almost monomolecularly dispersed in the EPO polymer matrix. Intermolecular interaction between MFA and EPO was indicated by solid-state infrared and (13)C-T(1) measurements. Solution-state (1)H-NMR measurement demonstrated that MFA existed in monomolecular state in supersaturated solution. (1)H-T(1) and difference nuclear Overhauser effect measurements indicated that cross relaxation occurred between MFA and EPO due to the small distance between them. The formation and high stability of the supersaturated solution were attributable to the specifically formed intermolecular interactions between MFA and EPO.

Recognition of DNA/RNA bulges by antimicrobial and antitumor metallohelices.

PubMed

Malina, Jaroslav; Scott, Peter; Brabec, Viktor

2015-09-07

Bulged structures have been identified in nucleic acids and have been shown to be linked to biomolecular processes involved in numerous diseases. Thus, chemical agents with affinity for bulged nucleic acids are of general biological significance. Herein, the mechanism of specific recognition and stabilization of bulged DNA and RNA by helical bimetallic species was established through detailed molecular biophysics and biochemistry assays. These agents, known as 'flexicates', are potential mimetics of α-helical peptides in cancer treatment, exhibiting antimicrobial and antitumor effects. The flexicates have positive impacts on the thermal stability of DNA duplexes containing bulges, which means that the flexicates interact with the duplexes containing bulges, and that these interactions stabilize the secondary structures of these duplexes. Notably, the stabilising effect of the flexicates increases with the size of the bulge, the maximal stabilization is observed for the duplexes containing a bulge composed of at least three bases. The flexicates bind most preferentially to the bulges composed of pyrimidines flanked on both sides also by pyrimidines. It is suggested that it is so because these bulges exhibit greatest conformational variability in comparison with other combinations of bases in the bulge loop and bases flanking the bulge. Finally, the results indicate that there is only one dominant binding site for the flexicates on the DNA and RNA bulges and that the flexicates bind directly to the bulge or in its close proximity. It is also shown that the flexicates effectively bind to RNA duplexes containing the bulged region of HIV-1 TAR RNA.

Molecular modeling and structural analysis of nAChR variants uncovers the mechanism of resistance to snake toxins.

PubMed

Gunasekaran, D; Sridhar, J; Suryanarayanan, V; Manimaran, N C; Singh, Sanjeev Kumar

2017-06-01

Nicotinic acetylcholine receptors (nAChRs) are neuromuscular proteins responsible for muscle contraction upon binding with chemical stimulant acetylcholine (ACh). The α-neurotoxins of snake mimic the structure of ACh and attacks nAChRs, which block the flow of ACh and leads to numbness and paralysis. The toxin-binding site of alpha subunit in the nAChRs is highly conserved throughout chordate lineages with few exceptions in resistance organisms. In this study, we have analyzed the sequence and structures of toxin-binding/resistant nAChRs and their interaction stability with toxins through molecular docking and molecular dynamics simulation (MDS). We have reported the potential glycosylation residues within the toxin-binding cleft adding sugar moieties through N-linked glycosylation in resistant organisms. Residue variations at key positions alter the secondary structure of binding cleft, which might interfere with toxin binding and it could be one of the possible explanations for the resistance to snake venoms. Analysis of nAChR-α-neurotoxin complexes has confirmed the key interacting residues. In addition, drastic variation in the binding stability of Mongoose nAChR-α-Bungarotoxin (α-BTX) and human nAChR-α-BTX complexes were found at specific phase of MDS. Our findings suggest that specific mutations in the binding site of toxin are potentially preventing the formation of stable complex of receptor-toxin, which might lead to mechanism of resistance. This in silico study on the binding cleft of nAChR and the findings of interacting residues will assist in designing potential inhibitors as therapeutic targets.

In vitro and in vivo cytotoxic activity of human lactoferricin derived antitumor peptide R-DIM-P-LF11-334 on human malignant melanoma

PubMed Central

Riedl, Sabrina; Rinner, Beate; Schaider, Helmut; Liegl-Atzwanger, Bernadette; Meditz, Katharina; Preishuber-Pflügl, Julia; Grissenberger, Sarah; Lohner, Karl; Zweytick, Dagmar

2017-01-01

Di-peptides derived from the human host defense peptide lactoferricin were previously described to specifically interact with the negatively charged lipid phosphatidylserine exposed by cancer cells. In this study one further derivative, namely R-DIM-P-LF11-334 is shown to exhibit even increased cancer toxicity in vitro and in vivo while non-neoplastic cells are not harmed. In liposomal model systems composed of phosphatidylserine mimicking cancerous and phosphatidylcholine mimicking non-cancerous membranes the specific interaction with the cancer marker PS was confirmed by specific induction of membrane perturbation and permeabilization in presence of the peptide. In vitro studies with cell lines of human malignant melanoma, such as A375, or primary cells of human melanoma metastases to the brain, as MUG Mel1, and non-neoplastic human dermal fibroblasts NHDF revealed high cytotoxic effect of R-DIM-P-LF11-334 on melanoma cells of A375 and MUG Mel1, whereas only minor effect on the dermal fibroblasts NHDF was observed, yielding an about 20-fold killing-specificity for A375 and MUG-Mel1. The LC50 values for melanoma A375 and MUG Mel1 were about 10 μM. Analysis of secondary structure of the peptide revealed an increase in the proportion of β-sheets exclusively in presence of the cancer mimic. Stability studies further indicated a potential adequate stability in blood or under stringent conditions. Importantly the cytotoxic effect on cancer cells was also proven in vivo in mouse xenografts of human melanoma, where peptide treatment induced strong tumor regression and in average a tumor area reduction of 85% compared to tumors of control mice without peptide treatment. PMID:29069749

In vitro and in vivo cytotoxic activity of human lactoferricin derived antitumor peptide R-DIM-P-LF11-334 on human malignant melanoma.

PubMed

Riedl, Sabrina; Rinner, Beate; Schaider, Helmut; Liegl-Atzwanger, Bernadette; Meditz, Katharina; Preishuber-Pflügl, Julia; Grissenberger, Sarah; Lohner, Karl; Zweytick, Dagmar

2017-09-22

Di-peptides derived from the human host defense peptide lactoferricin were previously described to specifically interact with the negatively charged lipid phosphatidylserine exposed by cancer cells. In this study one further derivative, namely R-DIM-P-LF11-334 is shown to exhibit even increased cancer toxicity in vitro and in vivo while non-neoplastic cells are not harmed. In liposomal model systems composed of phosphatidylserine mimicking cancerous and phosphatidylcholine mimicking non-cancerous membranes the specific interaction with the cancer marker PS was confirmed by specific induction of membrane perturbation and permeabilization in presence of the peptide. In vitro studies with cell lines of human malignant melanoma, such as A375, or primary cells of human melanoma metastases to the brain, as MUG Mel1, and non-neoplastic human dermal fibroblasts NHDF revealed high cytotoxic effect of R-DIM-P-LF11-334 on melanoma cells of A375 and MUG Mel1, whereas only minor effect on the dermal fibroblasts NHDF was observed, yielding an about 20-fold killing-specificity for A375 and MUG-Mel1. The LC 50 values for melanoma A375 and MUG Mel1 were about 10 μM. Analysis of secondary structure of the peptide revealed an increase in the proportion of β-sheets exclusively in presence of the cancer mimic. Stability studies further indicated a potential adequate stability in blood or under stringent conditions. Importantly the cytotoxic effect on cancer cells was also proven in vivo in mouse xenografts of human melanoma, where peptide treatment induced strong tumor regression and in average a tumor area reduction of 85% compared to tumors of control mice without peptide treatment.

Impact of excipient interactions on solid dosage form stability.

PubMed

Narang, Ajit S; Desai, Divyakant; Badawy, Sherif

2012-10-01

Drug-excipient interactions in solid dosage forms can affect drug product stability in physical aspects such as organoleptic changes and dissolution slowdown, or chemically by causing drug degradation. Recent research has allowed the distinction in chemical instability resulting from direct drug-excipient interactions and from drug interactions with excipient impurities. A review of chemical instability in solid dosage forms highlights common mechanistic themes applicable to multiple degradation pathways. These common themes include the role of water and microenvironmental pH. In addition, special aspects of solid-state reactions with excipients and/or excipient impurities add to the complexity in understanding and modeling reaction pathways. This paper discusses mechanistic basis of known drug-excipient interactions with case studies and provides an overview of common underlying themes. Recent developments in the understanding of degradation pathways further impact methodologies used in the pharmaceutical industry for prospective stability assessment. This paper discusses these emerging aspects in terms of limitations of drug-excipient compatibility studies, emerging paradigms in accelerated stability testing, and application of mathematical modeling for prediction of drug product stability.

An experimental test of a fundamental food web motif.

PubMed

Rip, Jason M K; McCann, Kevin S; Lynn, Denis H; Fawcett, Sonia

2010-06-07

Large-scale changes to the world's ecosystem are resulting in the deterioration of biostructure-the complex web of species interactions that make up ecological communities. A difficult, yet crucial task is to identify food web structures, or food web motifs, that are the building blocks of this baroque network of interactions. Once identified, these food web motifs can then be examined through experiments and theory to provide mechanistic explanations for how structure governs ecosystem stability. Here, we synthesize recent ecological research to show that generalist consumers coupling resources with different interaction strengths, is one such motif. This motif amazingly occurs across an enormous range of spatial scales, and so acts to distribute coupled weak and strong interactions throughout food webs. We then perform an experiment that illustrates the importance of this motif to ecological stability. We find that weak interactions coupled to strong interactions by generalist consumers dampen strong interaction strengths and increase community stability. This study takes a critical step by isolating a common food web motif and through clear, experimental manipulation, identifies the fundamental stabilizing consequences of this structure for ecological communities.

SPLINTS: small-molecule protein ligand interface stabilizers.

PubMed

Fischer, Eric S; Park, Eunyoung; Eck, Michael J; Thomä, Nicolas H

2016-04-01

Regulatory protein-protein interactions are ubiquitous in biology, and small molecule protein-protein interaction inhibitors are an important focus in drug discovery. Remarkably little attention has been given to the opposite strategy-stabilization of protein-protein interactions, despite the fact that several well-known therapeutics act through this mechanism. From a structural perspective, we consider representative examples of small molecules that induce or stabilize the association of protein domains to inhibit, or alter, signaling for nuclear hormone, GTPase, kinase, phosphatase, and ubiquitin ligase pathways. These SPLINTS (small-molecule protein ligand interface stabilizers) drive interactions that are in some cases physiologically relevant, and in others entirely adventitious. The diverse structural mechanisms employed suggest approaches for a broader and systematic search for such compounds in drug discovery. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structural Basis of Substrate Recognition by Hematopoietic Tyrosine Phosphatase (HePTP)

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

Critton, D.; Tortajada, A; Stetson, G

2008-01-01

Hematopoietic tyrosine phosphatase (HePTP) is one of three members of the kinase interaction motif (KIM) phosphatase family which also includes STEP and PCPTP1. The KIM-PTPs are characterized by a 15 residue sequence, the KIM, which confers specific high-affinity binding to their only known substrates, the MAP kinases Erk and p38, an interaction which is critical for their ability to regulate processes such as T cell differentiation (HePTP) and neuronal signaling (STEP). The KIM-PTPs are also characterized by a unique set of residues in their PTP substrate binding loops, where 4 of the 13 residues are differentially conserved among the KIM-PTPsmore » as compared to more than 30 other class I PTPs. One of these residues, T106 in HePTP, is either an aspartate or asparagine in nearly every other PTP. Using multiple techniques, we investigate the role of these KIM-PTP specific residues in order to elucidate the molecular basis of substrate recognition by HePTP. First, we used NMR spectroscopy to show that Erk2-derived peptides interact specifically with HePTP at the active site. Next, to reveal the molecular details of this interaction, we solved the high-resolution three-dimensional structures of two distinct HePTP-Erk2 peptide complexes. Strikingly, we were only able to obtain crystals of these transient complexes using a KIM-PTP specific substrate-trapping mutant, in which the KIM-PTP specific residue T106 was mutated to an aspartic acid (T106D). The introduced aspartate side chain facilitates the coordination of the bound peptides, thereby stabilizing the active dephosphorylation complex. These structures establish the essential role of HePTP T106 in restricting HePTP specificity to only those substrates which are able to interact with KIM-PTPs via the KIM (e.g., Erk2, p38). Finally, we describe how this interaction of the KIM is sufficient for overcoming the otherwise weak interaction at the active site of KIM-PTPs.« less

Engineering Proteins for Thermostability with iRDP Web Server

PubMed Central

Ghanate, Avinash; Ramasamy, Sureshkumar; Suresh, C. G.

2015-01-01

Engineering protein molecules with desired structure and biological functions has been an elusive goal. Development of industrially viable proteins with improved properties such as stability, catalytic activity and altered specificity by modifying the structure of an existing protein has widely been targeted through rational protein engineering. Although a range of factors contributing to thermal stability have been identified and widely researched, the in silico implementation of these as strategies directed towards enhancement of protein stability has not yet been explored extensively. A wide range of structural analysis tools is currently available for in silico protein engineering. However these tools concentrate on only a limited number of factors or individual protein structures, resulting in cumbersome and time-consuming analysis. The iRDP web server presented here provides a unified platform comprising of iCAPS, iStability and iMutants modules. Each module addresses different facets of effective rational engineering of proteins aiming towards enhanced stability. While iCAPS aids in selection of target protein based on factors contributing to structural stability, iStability uniquely offers in silico implementation of known thermostabilization strategies in proteins for identification and stability prediction of potential stabilizing mutation sites. iMutants aims to assess mutants based on changes in local interaction network and degree of residue conservation at the mutation sites. Each module was validated using an extensively diverse dataset. The server is freely accessible at http://irdp.ncl.res.in and has no login requirements. PMID:26436543

Engineering Proteins for Thermostability with iRDP Web Server.

PubMed

Panigrahi, Priyabrata; Sule, Manas; Ghanate, Avinash; Ramasamy, Sureshkumar; Suresh, C G

2015-01-01

Engineering protein molecules with desired structure and biological functions has been an elusive goal. Development of industrially viable proteins with improved properties such as stability, catalytic activity and altered specificity by modifying the structure of an existing protein has widely been targeted through rational protein engineering. Although a range of factors contributing to thermal stability have been identified and widely researched, the in silico implementation of these as strategies directed towards enhancement of protein stability has not yet been explored extensively. A wide range of structural analysis tools is currently available for in silico protein engineering. However these tools concentrate on only a limited number of factors or individual protein structures, resulting in cumbersome and time-consuming analysis. The iRDP web server presented here provides a unified platform comprising of iCAPS, iStability and iMutants modules. Each module addresses different facets of effective rational engineering of proteins aiming towards enhanced stability. While iCAPS aids in selection of target protein based on factors contributing to structural stability, iStability uniquely offers in silico implementation of known thermostabilization strategies in proteins for identification and stability prediction of potential stabilizing mutation sites. iMutants aims to assess mutants based on changes in local interaction network and degree of residue conservation at the mutation sites. Each module was validated using an extensively diverse dataset. The server is freely accessible at http://irdp.ncl.res.in and has no login requirements.

Thermodynamics of protein destabilization in live cells.

PubMed

Danielsson, Jens; Mu, Xin; Lang, Lisa; Wang, Huabing; Binolfi, Andres; Theillet, François-Xavier; Bekei, Beata; Logan, Derek T; Selenko, Philipp; Wennerström, Håkan; Oliveberg, Mikael

2015-10-06

Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.

Effect of mutation at the interface of Trp-repressor dimeric protein: a steered molecular dynamics simulation.

PubMed

Miño, German; Baez, Mauricio; Gutierrez, Gonzalo

2013-09-01

The strength of key interfacial contacts that stabilize protein-protein interactions have been studied by computer simulation. Experimentally, changes in the interface are evaluated by generating specific mutations at one or more points of the protein structure. Here, such an evaluation is performed by means of steered molecular dynamics and use of a dimeric model of tryptophan repressor and in-silico mutants as a test case. Analysis of four particular cases shows that, in principle, it is possible to distinguish between wild-type and mutant forms by examination of the total energy and force-extension profiles. In particular, detailed atomic level structural analysis indicates that specific mutations at the interface of the dimeric model (positions 19 and 39) alter interactions that appear in the wild-type form of tryptophan repressor, reducing the energy and force required to separate both subunits.

HCM and DCM cardiomyopathy-linked α-tropomyosin mutations influence off-state stability and crossbridge interaction on thin filaments.

PubMed

Farman, Gerrie P; Rynkiewicz, Michael J; Orzechowski, Marek; Lehman, William; Moore, Jeffrey R

2018-06-01

Calcium regulation of cardiac muscle contraction is controlled by the thin-filament proteins troponin and tropomyosin bound to actin. In the absence of calcium, troponin-tropomyosin inhibits myosin-interactions on actin and induces muscle relaxation, whereas the addition of calcium relieves the inhibitory constraint to initiate contraction. Many mutations in thin filament proteins linked to cardiomyopathy appear to disrupt this regulatory switching. Here, we tested perturbations caused by mutant tropomyosins (E40K, DCM; and E62Q, HCM) on intra-filament interactions affecting acto-myosin interactions including those induced further by myosin association. Comparison of wild-type and mutant human α-tropomyosin (Tpm1.1) behavior was carried out using in vitro motility assays and molecular dynamics simulations. Our results show that E62Q tropomyosin destabilizes thin filament off-state function by increasing calcium-sensitivity, but without apparent affect on global tropomyosin structure by modifying coiled-coil rigidity. In contrast, the E40K mutant tropomyosin appears to stabilize the off-state, demonstrates increased tropomyosin flexibility, while also decreasing calcium-sensitivity. In addition, the E40K mutation reduces thin filament velocity at low myosin concentration while the E62Q mutant tropomyosin increases velocity. Corresponding molecular dynamics simulations indicate specific residue interactions that are likely to redefine underlying molecular regulatory mechanisms, which we propose explain the altered contractility evoked by the disease-causing mutations. Copyright © 2018 Elsevier Inc. All rights reserved.

AMMI adjustment for statistical analysis of an international wheat yield trial.

PubMed

Crossa, J; Fox, P N; Pfeiffer, W H; Rajaram, S; Gauch, H G

1991-01-01

Multilocation trials are important for the CIMMYT Bread Wheat Program in producing high-yielding, adapted lines for a wide range of environments. This study investigated procedures for improving predictive success of a yield trial, grouping environments and genotypes into homogeneous subsets, and determining the yield stability of 18 CIMMYT bread wheats evaluated at 25 locations. Additive Main effects and Multiplicative Interaction (AMMI) analysis gave more precise estimates of genotypic yields within locations than means across replicates. This precision facilitated formation by cluster analysis of more cohesive groups of genotypes and locations for biological interpretation of interactions than occurred with unadjusted means. Locations were clustered into two subsets for which genotypes with positive interactions manifested in high, stable yields were identified. The analyses highlighted superior selections with both broad and specific adaptation.

Interactions between Casein Kinase Iε (CKIε) and Two Substrates from Disparate Signaling Pathways Reveal Mechanisms for Substrate-Kinase Specificity

PubMed Central

Dahlberg, Caroline Lund; Nguyen, Elizabeth Z.; Goodlett, David; Kimelman, David

2009-01-01

Background Members of the Casein Kinase I (CKI) family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIε and two substrates from different signaling pathways. Methodology/Principal Findings CKIε, but not CKIα, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIα's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIε does not determine Dishevelled's and Period's preference for CKIε nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIε with its substrates. We demonstrate that autophosphorylation of CKIε's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding. Conclusions/Significance The biochemical interactions between CKIε and Disheveled, Period, and its own C-terminus lead to models that explain CKIε's specificity and regulation. PMID:19274088

Insights into the binding specificity of wild type and mutated wheat germ agglutinin towards Neu5Acα(2-3)Gal: a study by in silico mutations and molecular dynamics simulations.

PubMed

Parasuraman, Ponnusamy; Murugan, Veeramani; Selvin, Jeyasigamani F A; Gromiha, M Michael; Fukui, Kazuhiko; Veluraja, Kasinadar

2014-08-01

Wheat germ agglutinin (WGA) is a plant lectin, which specifically recognizes the sugars NeuNAc and GlcNAc. Mutated WGA with enhanced binding specificity can be used as biomarkers for cancer. In silico mutations are performed at the active site of WGA to enhance the binding specificity towards sialylglycans, and molecular dynamics simulations of 20 ns are carried out for wild type and mutated WGAs (WGA1, WGA2, and WGA3) in complex with sialylgalactose to examine the change in binding specificity. MD simulations reveal the change in binding specificity of wild type and mutated WGAs towards sialylgalactose and bound conformational flexibility of sialylgalactose. The mutated polar amino acid residues Asn114 (S114N), Lys118 (G118K), and Arg118 (G118R) make direct and water mediated hydrogen bonds and hydrophobic interactions with sialylgalactose. An analysis of possible hydrogen bonds, hydrophobic interactions, total pair wise interaction energy between active site residues and sialylgalactose and MM-PBSA free energy calculation reveals the plausible binding modes and the role of water in stabilizing different binding modes. An interesting observation is that the binding specificity of mutated WGAs (cyborg lectin) towards sialylgalactose is found to be higher in double point mutation (WGA3). One of the substituted residues Arg118 plays a crucial role in sugar binding. Based on the interactions and energy calculations, it is concluded that the order of binding specificity of WGAs towards sialylgalactose is WGA3 > WGA1 > WGA2 > WGA. On comparing with the wild type, double point mutated WGA (WGA3) exhibits increased specificity towards sialylgalactose, and thus, it can be effectively used in targeted drug delivery and as biological cell marker in cancer therapeutics. Copyright © 2014 John Wiley & Sons, Ltd.

Biocompatible and biodegradable nanoparticles for enhancement of anti-cancer activities of phytochemicals

PubMed Central

Chuan, Li; Jia, Zhang; Yu-Jiao, Zu; Shu-Fang, Nie; Jun, Cao; Qian, Wang; Shao-Ping, Nie; Ze-Yuan, Deng; Ming-Yong, Xie; Shu, Wang

2017-01-01

Many phytochemicals show promise in cancer prevention and treatment, but their low aqueous solubility, poor stability, unfavorable bioavailability, and low target specificity make administering them at therapeutic doses unrealistic. This is particularly true for (–)-epigallocatechin gallate, curcumin, quercetin, resveratrol, and genistein. There is an increasing interest in developing novel delivery strategies for these natural products. Liposomes, micelles, nanoemulsions, solid lipid nanoparticles, nanostructured lipid carriers and poly (lactide-co-glycolide) nanoparticles are biocompatible and biodegradable nanoparticles. Those nanoparticles can increase the stability and solubility of phytochemicals, exhibit a sustained release property, enhance their absorption and bioavailability, protect them from premature enzymatic degradation or metabolism, prolong their circulation time, improve their target specificity to cancer cells or tumors via passive or targeted delivery, lower toxicity or side-effects to normal cells or tissues through preventing them from prematurely interacting with the biological environment, and enhance anti-cancer activities. Nanotechnology opens a door for developing phytochemical-loaded nanoparticles for prevention and treatment of cancer. PMID:26412423

Implementation of meiosis prophase I programme requires a conserved retinoid-independent stabilizer of meiotic transcripts

PubMed Central

Abby, Emilie; Tourpin, Sophie; Ribeiro, Jonathan; Daniel, Katrin; Messiaen, Sébastien; Moison, Delphine; Guerquin, Justine; Gaillard, Jean-Charles; Armengaud, Jean; Langa, Francina; Toth, Attila; Martini, Emmanuelle; Livera, Gabriel

2016-01-01

Sexual reproduction is crucially dependent on meiosis, a conserved, specialized cell division programme that is essential for the production of haploid gametes. Here we demonstrate that fertility and the implementation of the meiotic programme require a previously uncharacterized meiosis-specific protein, MEIOC. Meioc invalidation in mice induces early and pleiotropic meiotic defects in males and females. MEIOC prevents meiotic transcript degradation and interacts with an RNA helicase that binds numerous meiotic mRNAs. Our results indicate that proper engagement into meiosis necessitates the specific stabilization of meiotic transcripts, a previously little-appreciated feature in mammals. Remarkably, the upregulation of MEIOC at the onset of meiosis does not require retinoic acid and STRA8 signalling. Thus, we propose that the complete induction of the meiotic programme requires both retinoic acid-dependent and -independent mechanisms. The latter process involving post-transcriptional regulation likely represents an ancestral mechanism, given that MEIOC homologues are conserved throughout multicellular animals. PMID:26742488

HIM-8 binds to the X chromosome pairing center and mediates chromosome-specific meiotic synapsis.

PubMed

Phillips, Carolyn M; Wong, Chihunt; Bhalla, Needhi; Carlton, Peter M; Weiser, Pinky; Meneely, Philip M; Dernburg, Abby F

2005-12-16

The him-8 gene is essential for proper meiotic segregation of the X chromosomes in C. elegans. Here we show that loss of him-8 function causes profound X chromosome-specific defects in homolog pairing and synapsis. him-8 encodes a C2H2 zinc-finger protein that is expressed during meiosis and concentrates at a site on the X chromosome known as the meiotic pairing center (PC). A role for HIM-8 in PC function is supported by genetic interactions between PC lesions and him-8 mutations. HIM-8 bound chromosome sites associate with the nuclear envelope (NE) throughout meiotic prophase. Surprisingly, a point mutation in him-8 that retains both chromosome binding and NE localization fails to stabilize pairing or promote synapsis. These observations indicate that stabilization of homolog pairing is an active process in which the tethering of chromosome sites to the NE may be necessary but is not sufficient.

YAP controls retinal stem cell DNA replication timing and genomic stability

PubMed Central

Cabochette, Pauline; Vega-Lopez, Guillermo; Bitard, Juliette; Parain, Karine; Chemouny, Romain; Masson, Christel; Borday, Caroline; Hedderich, Marie; Henningfeld, Kristine A; Locker, Morgane; Bronchain, Odile; Perron, Muriel

2015-01-01

The adult frog retina retains a reservoir of active neural stem cells that contribute to continuous eye growth throughout life. We found that Yap, a downstream effector of the Hippo pathway, is specifically expressed in these stem cells. Yap knock-down leads to an accelerated S-phase and an abnormal progression of DNA replication, a phenotype likely mediated by upregulation of c-Myc. This is associated with an increased occurrence of DNA damage and eventually p53-p21 pathway-mediated cell death. Finally, we identified PKNOX1, a transcription factor involved in the maintenance of genomic stability, as a functional and physical interactant of YAP. Altogether, we propose that YAP is required in adult retinal stem cells to regulate the temporal firing of replication origins and quality control of replicated DNA. Our data reinforce the view that specific mechanisms dedicated to S-phase control are at work in stem cells to protect them from genomic instability. DOI: http://dx.doi.org/10.7554/eLife.08488.001 PMID:26393999

Stability in Real Food Webs: Weak Links in Long Loops

NASA Astrophysics Data System (ADS)

Neutel, Anje-Margriet; Heesterbeek, Johan A. P.; de Ruiter, Peter C.

2002-05-01

Increasing evidence that the strengths of interactions among populations in biological communities form patterns that are crucial for system stability requires clarification of the precise form of these patterns, how they come about, and why they influence stability. We show that in real food webs, interaction strengths are organized in trophic loops in such a way that long loops contain relatively many weak links. We show and explain mathematically that this patterning enhances stability, because it reduces maximum ``loop weight'' and thus reduces the amount of intraspecific interaction needed for matrix stability. The patterns are brought about by biomass pyramids, a feature common to most ecosystems. Incorporation of biomass pyramids in 104 food-web descriptions reveals that the low weight of the long loops stabilizes complex food webs. Loop-weight analysis could be a useful tool for exploring the structure and organization of complex communities.

The Functional Role of eL19 and eB12 Intersubunit Bridge in the Eukaryotic Ribosome.

PubMed

Kisly, Ivan; Gulay, Suna P; Mäeorg, Uno; Dinman, Jonathan D; Remme, Jaanus; Tamm, Tiina

2016-05-22

During translation, the two eukaryotic ribosomal subunits remain associated through 17 intersubunit bridges, five of which are eukaryote specific. These are mainly localized to the peripheral regions and are believed to stabilize the structure of the ribosome. The functional importance of these bridges remains largely unknown. Here, the essentiality of the eukaryote-specific bridge eB12 has been investigated. The main component of this bridge is ribosomal protein eL19 that is composed of an N-terminal globular domain, a middle region, and a long C-terminal α-helix. The analysis of deletion mutants demonstrated that the globular domain and middle region of eL19 are essential for cell viability, most likely functioning in ribosome assembly. The eB12 bridge, formed by contacts between the C-terminal α-helix of eL19 and 18S rRNA in concert with additional stabilizing interactions involving either eS7 or uS17, is dispensable for viability. Nevertheless, eL19 mutants impaired in eB12 bridge formation displayed slow growth phenotypes, altered sensitivity/resistance to translational inhibitors, and enhanced hyperosmotic stress tolerance. Biochemical analyses determined that the eB12 bridge contributes to the stability of ribosome subunit interactions in vitro. 60S subunits containing eL19 variants defective in eB12 bridge formation failed to form 80S ribosomes regardless of Mg(2+) concentration. The reassociation of 40S and mutant 60S subunits was markedly improved in the presence of deacetylated tRNA, emphasizing the importance of tRNAs during the subunit association. We propose that the eB12 bridge plays an important role in subunit joining and in optimizing ribosome functionality. Copyright © 2016 Elsevier Ltd. All rights reserved.

Lactoferrin denaturation induced by anionic surfactants: The role of the ferric ion in the protein stabilization.

PubMed

Ferreira, Gabriel Max Dias; Ferreira, Guilherme Max Dias; Agudelo, Álvaro Javier Patiño; Hudson, Eliara Acipreste; Dos Santos Pires, Ana Clarissa; da Silva, Luis Henrique Mendes

2018-05-11

Here, investigation was made of the interaction between Lactoferrin (Lf) and the anionic surfactants sodium dodecyl sulfate (SDS), sodium dodecylbenzene sulfonate (SDBS), and sodium decyl sulfate (DSS), using isothermal titration calorimetry, Nano differential scanning calorimetry (NanoDSC), and fluorescence spectroscopy. The Lf-surfactant interaction was enthalpically favorable (the integral enthalpy change ranged from -5.99 kJ mol -1 , for SDS at pH 3.0, to -0.61 kJ mol -1 , for DSS at pH 12.0) and promoted denaturation of the protein. The Lf denaturation efficiency followed the order DSS < SDS < SDBS. The extent of binding of the surfactants to Lf strongly depended on pH and the surfactant structure, reaching a maximum value of 505 SDBS molecules per gram of Lf at pH 3.0. The different efficiencies of the surfactants in denaturing Lf were attributed to the balance of hydrophobic and electrostatic interactions, which also depended on pH and the surfactant structure, highlighting the SDBS-tryptophan residue specific interaction, where SDBS acted as a quencher of fluorescence. Interestingly, the NanoDSC and fluorescence measurements showed that the ferric ion bound to Lf increased its stability against denaturation induced by the surfactants. The results have important implications for understanding the influence of surfactants on structural changes in metalloproteins. Copyright © 2017. Published by Elsevier B.V.

Functional Architecture of T7 RNA Polymerase Transcription Complexes

PubMed Central

Nayak, Dhananjaya; Guo, Qing; Sousa, Rui

2007-01-01

Summary T7 RNA polymerase is the best-characterized member of a widespread family of single-subunit RNA polymerases. Crystal structures of T7 RNA polymerase initiation and elongation complexes have provided a wealth of detailed information on RNA polymerase interactions with the promoter and transcription bubble, but the absence of DNA downstream of the melted region of the template in the initiation complex structure, and the absence of DNA upstream of the transcription bubble in the elongation complex structure means that our picture of the functional architecture of T7 RNA polymerase transcription complexes remains incomplete. Here we use the site-specifically tethered chemical nucleases and functional characterization of directed T7 RNAP mutants to both reveal the architecture of the duplex DNA that flanks the transcription bubble in the T7 RNAP initiation and elongation complexes, and to define the function of the interactions made by these duplex elements. We find that downstream duplex interactions made with a cluster of lysines (K711/K713/K714) are present during both elongation and initiation where they contribute to stabilizing a bend in the downstream DNA that is important for promoter opening. The upstream DNA in the elongation complex is also found to be sharply bent at the upstream edge of the transcription bubble, thereby allowing formation of upstream duplex:polymerase interactions that contribute to elongation complex stability. PMID:17580086

DNA base dimers are stabilized by hydrogen-bonding interactions including non-Watson-Crick pairing near graphite surfaces.

PubMed

Shankar, Akshaya; Jagota, Anand; Mittal, Jeetain

2012-10-11

Single- and double-stranded DNA are increasingly being paired with surfaces and nanoparticles for numerous applications, such as sensing, imaging, and drug delivery. Unlike the majority of DNA structures in bulk that are stabilized by canonical Watson-Crick pairing between Ade-Thy and Gua-Cyt, those adsorbed on surfaces are often stabilized by noncanonical base pairing, quartet formation, and base-surface stacking. Not much is known about these kinds of interactions. To build an understanding of the role of non-Watson-Crick pairing on DNA behavior near surfaces, one requires basic information on DNA base pair stacking and hydrogen-bonding interactions. All-atom molecular simulations of DNA bases in two cases--in bulk water and strongly adsorbed on a graphite surface--are conducted to study the relative strengths of stacking and hydrogen bond interactions for each of the 10 possible combinations of base pairs. The key information obtained from these simulations is the free energy as a function of distance between two bases in a pair. We find that stacking interactions exert the dominant influence on the stability of DNA base pairs in bulk water as expected. The strength of stability for these stacking interactions is found to decrease in the order Gua-Gua > Ade-Gua > Ade-Ade > Gua-Thy > Gua-Cyt > Ade-Thy > Ade-Cyt > Thy-Thy > Cyt-Thy > Cyt-Cyt. On the other hand, mutual interactions of surface-adsorbed base pairs are stabilized mostly by hydrogen-bonding interactions in the order Gua-Cyt > Ade-Gua > Ade-Thy > Ade-Ade > Cyt-Thy > Gua-Gua > Cyt-Cyt > Ade-Cyt > Thy-Thy > Gua-Thy. Interestingly, several non-Watson-Crick base pairings, which are commonly ignored, have similar stabilization free energies due to interbase hydrogen bonding as Watson-Crick pairs. This clearly highlights the importance of non-Watson-Crick base pairing in the development of secondary structures of oligonucleotides near surfaces.

Equilibrium denaturation and preferential interactions of an RNA tetraloop with urea

DOE PAGES

Miner, Jacob Carlson; García, Angel Enrique

2017-02-09

Urea is an important organic cosolute with implications in maintaining osmotic stress in cells and differentially stabilizing ensembles of folded biomolecules. We report an equilibrium study of urea-induced denaturation of a hyperstable RNA tetraloop through unbiased replica exchange molecular dynamics. We find that, in addition to destabilizing the folded state, urea smooths the RNA free energy landscape by destabilizing specific configurations, and forming favorable interactions with RNA nucleobases. A linear concentration-dependence of the free energy (m-value) is observed, in agreement with the results of other RNA hairpins and proteins. Additionally, analysis of the hydrogen-bonding and stacking interactions within RNA primarilymore » show temperature-dependence, while interactions between RNA and urea primarily show concentration-dependence. Lastly, our findings provide valuable insight into the effects of urea on RNA folding and describe the thermodynamics of a basic RNA hairpin as a function of solution chemistry.« less

The role of the peripheral anionic site and cation-pi interactions in the ligand penetration of the human AChE gorge.

PubMed

Branduardi, Davide; Gervasio, Francesco Luigi; Cavalli, Andrea; Recanatini, Maurizio; Parrinello, Michele

2005-06-29

We study the ligand (tetramethylammonium) recognition by the peripheral anionic site and its penetration of the human AChE gorge by using atomistic molecular dynamics simulations and our recently developed metadynamics method. The role of both the peripheral anionic site and the formation of cation-pi interactions in the ligand entrance are clearly shown. In particular, a simulation with the W286A mutant shows the fundamental role of this residue in anchoring the ligand at the peripheral anionic site of the enzyme and in positioning it prior to the gorge entrance. Once the ligand is properly oriented, the formation of specific and synchronized cation-pi interactions with W86, F295, and Y341 enables the gorge penetration. Eventually, the ligand is stabilized in a free energy basin by means of cation-pi interactions with W86.

Equilibrium Denaturation and Preferential Interactions of an RNA Tetraloop with Urea.

PubMed

Miner, Jacob C; García, Angel E

2017-04-20

Urea is an important organic cosolute with implications in maintaining osmotic stress in cells and differentially stabilizing ensembles of folded biomolecules. We report an equilibrium study of urea-induced denaturation of a hyperstable RNA tetraloop through unbiased replica exchange molecular dynamics. We find that, in addition to destabilizing the folded state, urea smooths the RNA free energy landscape by destabilizing specific configurations, and forming favorable interactions with RNA nucleobases. A linear concentration-dependence of the free energy (m-value) is observed, in agreement with the results of other RNA hairpins and proteins. Additionally, analysis of the hydrogen-bonding and stacking interactions within RNA primarily show temperature-dependence, while interactions between RNA and urea primarily show concentration-dependence. Our findings provide valuable insight into the effects of urea on RNA folding and describe the thermodynamics of a basic RNA hairpin as a function of solution chemistry.

Equilibrium denaturation and preferential interactions of an RNA tetraloop with urea

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

Miner, Jacob Carlson; García, Angel Enrique

Urea is an important organic cosolute with implications in maintaining osmotic stress in cells and differentially stabilizing ensembles of folded biomolecules. We report an equilibrium study of urea-induced denaturation of a hyperstable RNA tetraloop through unbiased replica exchange molecular dynamics. We find that, in addition to destabilizing the folded state, urea smooths the RNA free energy landscape by destabilizing specific configurations, and forming favorable interactions with RNA nucleobases. A linear concentration-dependence of the free energy (m-value) is observed, in agreement with the results of other RNA hairpins and proteins. Additionally, analysis of the hydrogen-bonding and stacking interactions within RNA primarilymore » show temperature-dependence, while interactions between RNA and urea primarily show concentration-dependence. Lastly, our findings provide valuable insight into the effects of urea on RNA folding and describe the thermodynamics of a basic RNA hairpin as a function of solution chemistry.« less

Targeting Allosteric Control Mechanisms in Heat Shock Protein 70 (Hsp70)

PubMed Central

Li, Xiaokai; Shao, Hao; Taylor, Isabelle R.; Gestwicki, Jason E.

2017-01-01

Heat shock protein 70 (Hsp70) is a molecular chaperone that plays critical roles in protein homeostasis. Hsp70’s chaperone activity is coordinated by intra-molecular interactions between its two domains, as well as inter-molecular interactions between Hsp70 and its co-chaperones. Each of these contacts represents a potential opportunity for the development of chemical inhibitors. To illustrate this concept, we review three classes of recently identified molecules that bind distinct pockets on Hsp70. Although all three compounds share the ability to interrupt core biochemical functions of Hsp70, they stabilize different conformers. Accordingly, each compound appears to interrupt a specific subset of inter- and intra-molecular interactions. Thus, an accurate definition of an Hsp70 inhibitor may require a particularly detailed understanding of the molecule’s binding site and its effects on protein-protein interactions. PMID:27072701

AFD: an application for bi-molecular interaction using axial frequency distribution.

PubMed

Raza, Saad; Azam, Syed Sikander

2018-03-06

Conformational flexibility and generalized structural features are responsible for specific phenomena existing in biological pathways. With advancements in computational chemistry, novel approaches and new methods are required to compare the dynamic nature of biomolecules, which are crucial not only to address dynamic functional relationships but also to gain detailed insights into the disturbance and positional fluctuation responsible for functional shifts. Keeping this in mind, axial frequency distribution (AFD) has been developed, designed, and implemented. AFD can profoundly represent distribution and density of ligand atom around a particular atom or set of atoms. It enabled us to obtain an explanation of local movements and rotations, which are not significantly highlighted by any other structural and dynamical parameters. AFD can be implemented on biological models representing ligand and protein interactions. It shows a comprehensive view of the binding pattern of ligand by exploring the distribution of atoms relative to the x-y plane of the system. By taking a relative centroid on protein or ligand, molecular interactions like hydrogen bonds, van der Waals, polar or ionic interaction can be analyzed to cater the ligand movement, stabilization or flexibility with respect to the protein. The AFD graph resulted in the residual depiction of bi-molecular interaction in gradient form which can yield specific information depending upon the system of interest.

Gonadotropin-Releasing Hormone (GnRH) Receptor Structure and GnRH Binding

PubMed Central

Flanagan, Colleen A.; Manilall, Ashmeetha

2017-01-01

Gonadotropin-releasing hormone (GnRH) regulates reproduction. The human GnRH receptor lacks a cytoplasmic carboxy-terminal tail but has amino acid sequence motifs characteristic of rhodopsin-like, class A, G protein-coupled receptors (GPCRs). This review will consider how recent descriptions of X-ray crystallographic structures of GPCRs in inactive and active conformations may contribute to understanding GnRH receptor structure, mechanism of activation and ligand binding. The structures confirmed that ligands bind to variable extracellular surfaces, whereas the seven membrane-spanning α-helices convey the activation signal to the cytoplasmic receptor surface, which binds and activates heterotrimeric G proteins. Forty non-covalent interactions that bridge topologically equivalent residues in different transmembrane (TM) helices are conserved in class A GPCR structures, regardless of activation state. Conformation-independent interhelical contacts account for a conserved receptor protein structure and their importance in the GnRH receptor structure is supported by decreased expression of receptors with mutations of residues in the network. Many of the GnRH receptor mutations associated with congenital hypogonadotropic hypogonadism, including the Glu2.53(90) Lys mutation, involve amino acids that constitute the conserved network. Half of the ~250 intramolecular interactions in GPCRs differ between inactive and active structures. Conformation-specific interhelical contacts depend on amino acids changing partners during activation. Conserved inactive conformation-specific contacts prevent receptor activation by stabilizing proximity of TM helices 3 and 6 and a closed G protein-binding site. Mutations of GnRH receptor residues involved in these interactions, such as Arg3.50(139) of the DRY/S motif or Tyr7.53(323) of the N/DPxxY motif, increase or decrease receptor expression and efficiency of receptor coupling to G protein signaling, consistent with the native residues stabilizing the inactive GnRH receptor structure. Active conformation-specific interhelical contacts stabilize an open G protein-binding site. Progress in defining the GnRH-binding site has recently slowed, with evidence that Tyr6.58(290) contacts Tyr5 of GnRH, whereas other residues affect recognition of Trp3 and Gly10NH2. The surprisingly consistent observations that GnRH receptor mutations that disrupt GnRH binding have less effect on “conformationally constrained” GnRH peptides may now be explained by crystal structures of agonist-bound peptide receptors. Analysis of GPCR structures provides insight into GnRH receptor function. PMID:29123501

Nanoparticle engineering of colloidal suspension behavior

NASA Astrophysics Data System (ADS)

Chan, Angel Thanda

We investigate the effects of highly charged nanoparticles on the phase behavior, structure, and assembly of colloidal microsphere suspensions. Specifically, by selectively tuning the electrostatic interactions between silica microspheres and polystyrene nanoparticles, we study the behavior of four key systems: (i) strongly repulsive, (ii) haloing, (iii) weakly attractive, and (iv) strongly attractive systems. In each system, a combination of nanoparticle adsorption, zeta potential, and confocal microscopy measurements are carried out to systematically study the effects of nanoparticle volume fraction, microsphere/nanoparticle size ratios, and interparticle interactions on their behavior. Our observations indicate that minimal adsorption of highly charged nanoparticles occurs on like-charged and negligibly-charged microspheres, whereas their extent of association increases dramatically with increasing microsphere-nanoparticle attraction. A rich phase behavior emerges in these systems based on whether the nanoparticle species serve as depletants, haloing, or bridging species. The phase transitions in the haloing system occur at constant nanoparticle volume fractions, φnano, over a broad range of microsphere volume fractions, φmicro . By contrast, the observed transitions in the weakly and strongly attractive mixtures occur at a constant number ratio of nanoparticles per microsphere, Nnano/Nmicro. Important structural differences emerge, which can be exploited in the assembly of colloidal gels for direct ink writing and colloidal crystals on epitaxially patterned substrates. Finally, for the first time, we explore nanoparticle haloing as a new route for stabilizing hydrophobic colloidal drugs in aqueous suspensions media for preparation of injectable pharmaceuticals. These microsphere suspensions exhibit improved stability relative to their surfactant-stabilized counterparts after autoclaving, a critical processing step for this target applications. This research opens up a new avenue for stabilization of hydrophobic particles, when surfactant additions alone do not provide sufficient stabilization.

Enhanced Conductivity and Electrochemical Performance of Electrode Material Based on Multifunctional Dye Doped Polypyrrole.

PubMed

Zang, Limin; Qiu, Jianhui; Yang, Chao; Sakai, Eiichi

2016-03-01

Polypyrrole were prepared via in-situ chemical oxidative polymerization in the presence of multisulfonate acid dye (acid violet 19). In this work, acid violet 19 could play the role as dopant, surfactant and physical cross-linker for pyrrole polymerization, and had impact on the morphology, dispersion stability, thermal stability, electrical conductivity and electrochemical behavior of the samples. The thermal stability of the dye doped polypyrrole was enhanced than pure polypyrrole due to the strong interactions between polypyrrole and acid violet 19. The dispersion stability of the samples in water was also improved by incorporating an appropriate amount of acid violet 19. The sample with 20% of acid violet 19 showed granular morphology with the smallest diameter of -50 nm and possessed the maximum electrical conductivity of 39.09 S/cm. The as-prepared multifunctional dye doped polypyrrole samples were used to fabricate electrodes and exhibited a mass specific capacitance of 379-206 F/g in the current density range of 0.2-1.0 A/g. The results indicated that the multifunctional dye could improve the performances of polypyrrole as electrode material for supercapacitors.

Stabilization of coiled-coil peptide domains by introduction of trifluoroleucine.

PubMed

Tang, Y; Ghirlanda, G; Vaidehi, N; Kua, J; Mainz, D T; Goddard III, W A; DeGrado, W F; Tirrell, D A

2001-03-06

Substitution of leucine residues by 5,5,5-trifluoroleucine at the d-positions of the leucine zipper peptide GCN4-p1d increases the thermal stability of the coiled-coil structure. The midpoint thermal unfolding temperature of the fluorinated peptide is elevated by 13 degrees C at 30 microM peptide concentration. The modified peptide is more resistant to chaotropic denaturants, and the free energy of folding of the fluorinated peptide is 0.5-1.2 kcal/mol larger than that of the hydrogenated form. A similarly fluorinated form of the DNA-binding peptide GCN4-bZip binds to target DNA sequences with affinity and specificity identical to those of the hydrogenated form, while demonstrating enhanced thermal stability. Molecular dynamics simulation on the fluorinated GCN4-p1d peptide using the Surface Generalized Born implicit solvation model revealed that the coiled-coil binding energy is 55% more favorable upon fluorination. These results suggest that fluorination of hydrophobic substructures in peptides and proteins may provide new means of increasing protein stability, enhancing protein assembly, and strengthening receptor-ligand interactions.

Using RNA as a tool to modify lipid nanoparticles

NASA Astrophysics Data System (ADS)

Wilner, Samantha E.

Lipid nanoparticles (LNPs) provide an attractive option for therapeutic applications because they can self-assemble and carry a diverse set of cargoes ranging from hydrophobic drugs to small interfering RNA (siRNA). Liposomes and micelles represent two classes of LNPs that have been developed for medicinal purposes; however, active targeting of LNPs to specific tissues and LNP stability in vivo remain significant challenges. We have exploited the structural characteristics and targeting ability of nucleic acids to address these obstacles. Specifically, we have introduced short nucleic acid targeting species, called aptamers, to the surface of stable nucleic acid lipid particles (SNALPs), a subset of liposomes used in siRNA delivery. In this manner, we have actively targeted SNALPs to cancer cells that overexpress the transferrin receptor (TfR). HeLa cells expressing enhanced green fluorescent protein (EGFP) were treated with SNALPs bearing an antiTfR aptamer (C2) that was identified in our lab. C2-conjugated SNALPs showed increased levels of uptake by cells by flow cytometry. More importantly, the enhanced uptake by C2-conjugated SNALPs translated to an increased level of gene knockdown when SNALPs were loaded with anti-EGFP siRNA or anti-Lamin NC siRNA. Expression of EGFP and Lamin NC decreased, respectively. These preliminary studies illustrate that aptamer-conjugated SNALPs can be designed to knock down both endogenous and exogenous genes in cancer cells with high specificity. We have also used nucleic acids to stabilize lipid micelles by introducing short quadruplex forming oligonucleotide sequences at the lipid headgroup. Micelle formation was confirmed via dynamic light scattering, transmission electron microscopy, and small angle X-ray scattering. Micelle stability was assessed using NMR and by a FRET-based assay in the presence of serum proteins. Quadruplex-stabilized micelles demonstrated enhanced stability suggesting that alterations to oligonucleotide headgroup interactions could be used to tune micelle stability. Therefore, we engineered stabilized micelles with an oligonucleotide extension and have shown that the addition of an antisense oligonucleotide leads to micelle destabilization and cargo release. The ability to control particle stability with antisense oligonucleotides represents a new paradigm in the design of programmed nanoscale devices, which we envision will find utility in the development of novel diagnostics and therapeutics.

Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF165 toward pharmaceutical applications.

PubMed

Kimoto, Michiko; Nakamura, Mana; Hirao, Ichiro

2016-09-06

A new technology, genetic alphabet expansion using artificial bases (unnatural bases), has created high-affinity DNA ligands (aptamers) that specifically bind to target proteins by ExSELEX (genetic alphabet Expansion for Systematic Evolution of Ligands by EXponential enrichment). We recently found that the unnatural-base DNA aptamers can be stabilized against nucleases, by introducing an extraordinarily stable, unique hairpin DNA (mini-hairpin DNA) and by reinforcing the stem region with G-C pairs. Here, to establish this aptamer generation method, we examined the stabilization of a high-affinity anti-VEGF165 unnatural-base DNA aptamer. The stabilized aptamers displayed significantly increased thermal and nuclease stabilities, and furthermore, exhibited higher affinity to the target. As compared to the well-known anti-VEGF165 RNA aptamer, pegaptanib (Macugen), our aptamers did not require calcium ions for binding to VEGF165 Biological experiments using cultured cells revealed that our stabilized aptamers efficiently inhibited the interaction between VEGF165 and its receptor, with the same or slightly higher efficiency than that of the pegaptanib RNA aptamer. The development of cost-effective and calcium ion-independent high-affinity anti-VEGF165 DNA aptamers encourages further progress in diagnostic and therapeutic applications. In addition, the stabilization process provided additional information about the key elements required for aptamer binding to VEGF165. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

2013 Early Career Achievement Award--Proteomics of muscle- and species-specificity in meat color stability.

PubMed

Suman, S P; Rentfrow, G; Nair, M N; Joseph, P

2014-03-01

Meat color is the most important quality trait influencing consumer purchase decisions. The interinfluential interactions between myoglobin and biomolecules govern color stability in meat. The advances in proteomics, such as high throughput analytical tools in mass spectrometry, 2-dimensional electrophoresis, and bioinformatics, offer themselves as robust techniques to characterize the proteome basis of muscle- and species-specific meat color phenomena. Differential abundance of chaperones and antioxidant proteins contributes to muscle-specific color stability in beef; the greater abundance of chaperones and antioxidant proteins in color-stable Longissimus lumborum than in color-labile Psoas major protects myoglobin and contributes to superior color stability of beef Longissimus steaks. Lipid oxidation-induced myoglobin oxidation is more critical to beef color than pork color due to the inherent differences in myoglobin chemistry; the number of nucleophilic histidine residues adducted by reactive aldehydes is greater in beef myoglobin than in pork myoglobin. Preferential adduction of secondary products of lipid oxidation to beef myoglobin accelerates metmyoglobin formation at a greater degree than in its pork counterpart. Mass spectrometric investigations revealed that although cherry-red carboxymyoglobin is more stable than oxymyoglobin, both redox forms undergo lipid oxidation-induced oxidation in model systems. The accuracy of mass spectrometry to detect the molecular mass of proteins has been applied to differentiate myoglobins from closely related meat animals, such as goats and sheep or emu and ostrich. In addition, this approach indicated that turkey myoglobin is 350 Da greater in molecular mass than beef myoglobin, and the unique biochemistry of turkey myoglobin could be responsible for its greater thermostability in model systems as well as the pink color defect observed in fully cooked uncured turkey products.

Development of Novel p16INK4a Mimetics as Anticancer Therapy

DTIC Science & Technology

2015-10-01

peptide (or substituted peptide) or the crystal structure of the relevant sequence from p16INK4 ( PDB 1BI7) was used as the starting structure . Model...small peptides that interact with CDK4/6. The specific aims are as follows. (1) Determine structure -function relationships of overlapping peptides...Determine structure -function relationships of overlapping peptides derived from p16 INK4a that inhibit the activity of CDK4/6 and identify stabilized

Urokinase receptor expression involves tyrosine phosphorylation of phosphoglycerate kinase.

PubMed

Shetty, Praveenkumar; Velusamy, Thirunavukkarasu; Bhandary, Yashodhar P; Liu, Ming C; Shetty, Sreerama

2010-02-01

The interaction of urokinase-type plasminogen activator (uPA) with its receptor, uPAR, plays a central role in several pathophysiological processes, including cancer. uPA induces its own cell surface receptor expression through stabilization of uPAR mRNA. The mechanism involves binding of a 51 nt uPAR mRNA coding sequence with phosphoglycerate kinase (PGK) to down regulate cell surface uPAR expression. Tyrosine phosphorylation of PGK mediated by uPA treatment enhances uPAR mRNA stabilization. In contrast, inhibition of tyrosine phosphorylation augments PGK binding to uPAR mRNA and attenuates uPA-induced uPAR expression. Mapping the specific peptide region of PGK indicated that its first quarter (amino acids 1-100) interacts with uPAR mRNA. To determine if uPAR expression by uPA is regulated through activation of tyrosine residues of PGK, we mutated the specific tyrosine residue and tested mutant PGK for its ability to interfere with uPAR expression. Inhibition of tyrosine phosphorylation by mutating Y76 residue abolished uPAR expression induced by uPA treatment. These findings collectively demonstrate that Y76 residue present in the first quarter of the PGK molecule is involved in lung epithelial cell surface uPAR expression. This region can effectively mimic the function of a whole PGK molecule in inhibiting tumor cell growth.

Ag, Co/graphene interactions and its effect on electrocatalytic oxygen reduction in alkaline media

NASA Astrophysics Data System (ADS)

Qaseem, Adnan; Chen, Fuyi; Wu, Xiaoqiang; Zhang, Nan; Xia, Zhenhai

2017-12-01

Silver nanoparticles supported on cobalt and nitrogen embedded reduced graphene oxide, Ag/Co-NGr, are synthesized by one-step hydrothermal route with remarkable catalytic activity for oxygen reduction reaction (ORR). As-synthesized electrocatalyst exhibits half-wave potential (0.82 V) comparable to commercial Pt/C (0.85 V), specific activity (0.45 mA cm-2) better than commercial Pt/C (0.35 mA cm-2) along with superior stability in alkaline environment (≈95% activity retention after 5000s compared to 80% for Pt/C). Moreover, Ag/Co-NGr is highly tolerant to methanol poisoning during ORR and delivers an excellent specific capacity of 789 mAh.g-1Zn with energy density of 947 Wh. kg-1 at a current density of 20 mA cm-2 in a Zn-air battery. For the first time, it is proven that particle size refinement and electronic perturbation of Ag nanoparticles take place due to metal-support interactions between Ag and Co/NGr. d-band center of Ag in Ag/Co-NGr upshifts toward the Fermi level with respect to the Ag/NGr as a result of charge transfer between Ag and Co/NGr. The superior catalytic activity and excellent stability of Ag/Co-NGr is attributed to the structural and electronic modification of Ag nanoparticles by Co and N elements on graphene.

Structure and stability of the ankyrin domain of the Drosophila Notch receptor.

PubMed

Zweifel, Mark E; Leahy, Daniel J; Hughson, Frederick M; Barrick, Doug

2003-11-01

The Notch receptor contains a conserved ankyrin repeat domain that is required for Notch-mediated signal transduction. The ankyrin domain of Drosophila Notch contains six ankyrin sequence repeats previously identified as closely matching the ankyrin repeat consensus sequence, and a putative seventh C-terminal sequence repeat that exhibits lower similarity to the consensus sequence. To better understand the role of the Notch ankyrin domain in Notch-mediated signaling and to examine how structure is distributed among the seven ankyrin sequence repeats, we have determined the crystal structure of this domain to 2.0 angstroms resolution. The seventh, C-terminal, ankyrin sequence repeat adopts a regular ankyrin fold, but the first, N-terminal ankyrin repeat, which contains a 15-residue insertion, appears to be largely disordered. The structure reveals a substantial interface between ankyrin polypeptides, showing a high degree of shape and charge complementarity, which may be related to homotypic interactions suggested from indirect studies. However, the Notch ankyrin domain remains largely monomeric in solution, demonstrating that this interface alone is not sufficient to promote tight association. Using the structure, we have classified reported mutations within the Notch ankyrin domain that are known to disrupt signaling into those that affect buried residues and those restricted to surface residues. We show that the buried substitutions greatly decrease protein stability, whereas the surface substitutions have only a marginal affect on stability. The surface substitutions are thus likely to interfere with Notch signaling by disrupting specific Notch-effector interactions and map the sites of these interactions.

Affinity Pulldown of Biotinylated RNA for Detection of Protein-RNA Complexes.

PubMed

Panda, Amaresh C; Martindale, Jennifer L; Gorospe, Myriam

2016-12-20

RNA-binding proteins (RBPs) have recently emerged as crucial players in the regulation of gene expression. The interactions of RBPs with target mRNAs control the levels of gene products by altering different regulatory steps, including pre-mRNA splicing and maturation, nuclear mRNA export, and mRNA stability and translation (Glisovic et al. , 2008). There are several methodologies available today to identify RNAs bound to specific RBPs; some detect only recombinant molecules in vitro , others detect recombinant and endogenous molecules, while others detect only endogenous molecules. Examples include systematic evolution of ligands by exponential enrichment (SELEX), biotinylated RNA pulldown assay, RNA immunoprecipitation (RIP) assay, electrophoretic mobility shift assay (EMSA), RNA footprinting analysis, and various UV crosslinking and immunoprecipitation (CLIP) methods such as CLIP, PAR-CLIP, and iCLIP (Popova et al. , 2015). Here, we describe a simple and informative method to study and identify the RNA region of interaction between an RBP and its target transcript (Panda et al. , 2014 and 2016). Its reproducibility and ease of use make this protocol a fast and useful method to identify interactions between RBPs and specific RNAs.

Differentially-Expressed Pseudogenes in HIV-1 Infection

PubMed Central

Gupta, Aditi; Brown, C. Titus; Zheng, Yong-Hui; Adami, Christoph

2015-01-01

Not all pseudogenes are transcriptionally silent as previously thought. Pseudogene transcripts, although not translated, contribute to the non-coding RNA pool of the cell that regulates the expression of other genes. Pseudogene transcripts can also directly compete with the parent gene transcripts for mRNA stability and other cell factors, modulating their expression levels. Tissue-specific and cancer-specific differential expression of these “functional” pseudogenes has been reported. To ascertain potential pseudogene:gene interactions in HIV-1 infection, we analyzed transcriptomes from infected and uninfected T-cells and found that 21 pseudogenes are differentially expressed in HIV-1 infection. This is interesting because parent genes of one-third of these differentially-expressed pseudogenes are implicated in HIV-1 life cycle, and parent genes of half of these pseudogenes are involved in different viral infections. Our bioinformatics analysis identifies candidate pseudogene:gene interactions that may be of significance in HIV-1 infection. Experimental validation of these interactions would establish that retroviruses exploit this newly-discovered layer of host gene expression regulation for their own benefit. PMID:26426037

Hyperbranched polyglycerols at the biointerface

NASA Astrophysics Data System (ADS)

Moore, Eli; Thissen, Helmut; Voelcker, Nicolas H.

2013-08-01

The control over biointerfacial interactions is the key to a broad range of biomedical applications, ranging from implantable devices to drug delivery and nanomedicine. In many of these applications, coatings are required that reduce or prevent non-specific interactions with the biological environment, while at the same time presenting specific bioactive signals. Whilst surface coatings based on polymers such as poly(ethylene glycol) (PEG) have been used successfully, many limitations persist in regard to the biocompatibility, stability and functionality of state-of-the-art polymer coatings. Most of these limitations are related to the fact that, typically, linear polymers are used with associated limited chemical functionality. Here, we examine the development of hyperbranched polyglycerols (HPGs) as promising candidates for the replacement of traditional linear polymers, such as the chemically analogous PEG, for the control of biointerfacial interactions. HPGs are highly branched globular molecules that exhibit a high valency, allow easy access to a variety of functionalities and can present biologically active signals. In this review, a comprehensive overview is provided with respect to the history, synthetic strategies, modifications and applications of HPGs.

Structural insights into Rhino-Deadlock complex for germline piRNA cluster specification.

PubMed

Yu, Bowen; Lin, Yu An; Parhad, Swapnil S; Jin, Zhaohui; Ma, Jinbiao; Theurkauf, William E; Zhang, Zz Zhao; Huang, Ying

2018-06-01

PIWI-interacting RNAs (piRNAs) silence transposons in germ cells to maintain genome stability and animal fertility. Rhino, a rapidly evolving heterochromatin protein 1 (HP1) family protein, binds Deadlock in a species-specific manner and so defines the piRNA-producing loci in the Drosophila genome. Here, we determine the crystal structures of Rhino-Deadlock complex in Drosophila melanogaster and simulans In both species, one Rhino binds the N-terminal helix-hairpin-helix motif of one Deadlock protein through a novel interface formed by the beta-sheet in the Rhino chromoshadow domain. Disrupting the interface leads to infertility and transposon hyperactivation in flies. Our structural and functional experiments indicate that electrostatic repulsion at the interaction interface causes cross-species incompatibility between the sibling species. By determining the molecular architecture of this piRNA-producing machinery, we discover a novel HP1-partner interacting mode that is crucial to piRNA biogenesis and transposon silencing. We thus explain the cross-species incompatibility of two sibling species at the molecular level. © 2018 The Authors.

High-affinity DNA-binding Domains of Replication Protein A (RPA) Direct SMARCAL1-dependent Replication Fork Remodeling*

PubMed Central

Bhat, Kamakoti P.; Bétous, Rémy; Cortez, David

2015-01-01

SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. PMID:25552480

High-affinity DNA-binding domains of replication protein A (RPA) direct SMARCAL1-dependent replication fork remodeling.

PubMed

Bhat, Kamakoti P; Bétous, Rémy; Cortez, David

2015-02-13

SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Understanding protein-nanoparticle interaction: a new gateway to disease therapeutics.

PubMed

Giri, Karuna; Shameer, Khader; Zimmermann, Michael T; Saha, Sounik; Chakraborty, Prabir K; Sharma, Anirudh; Arvizo, Rochelle R; Madden, Benjamin J; Mccormick, Daniel J; Kocher, Jean-Pierre A; Bhattacharya, Resham; Mukherjee, Priyabrata

2014-06-18

Molecular identification of protein molecules surrounding nanoparticles (NPs) may provide useful information that influences NP clearance, biodistribution, and toxicity. Hence, nanoproteomics provides specific information about the environment that NPs interact with and can therefore report on the changes in protein distribution that occurs during tumorigenesis. Therefore, we hypothesized that characterization and identification of protein molecules that interact with 20 nm AuNPs from cancer and noncancer cells may provide mechanistic insights into the biology of tumor growth and metastasis and identify new therapeutic targets in ovarian cancer. Hence, in the present study, we systematically examined the interaction of the protein molecules with 20 nm AuNPs from cancer and noncancerous cell lysates. Time-resolved proteomic profiles of NP-protein complexes demonstrated electrostatic interaction to be the governing factor in the initial time-points which are dominated by further stabilization interaction at longer time-points as determined by ultraviolet-visible spectroscopy (UV-vis), dynamic light scattering (DLS), ζ-potential measurements, transmission electron microscopy (TEM), and tandem mass spectrometry (MS/MS). Reduction in size, charge, and number of bound proteins were observed as the protein-NP complex stabilized over time. Interestingly, proteins related to mRNA processing were overwhelmingly represented on the NP-protein complex at all times. More importantly, comparative proteomic analyses revealed enrichment of a number of cancer-specific proteins on the AuNP surface. Network analyses of these proteins highlighted important hub nodes that could potentially be targeted for maximal therapeutic advantage in the treatment of ovarian cancer. The importance of this methodology and the biological significance of the network proteins were validated by a functional study of three hubs that exhibited variable connectivity, namely, PPA1, SMNDC1, and PI15. Western blot analysis revealed overexpression of these proteins in ovarian cancer cells when compared to normal cells. Silencing of PPA1, SMNDC1, and PI15 by the siRNA approach significantly inhibited proliferation of ovarian cancer cells and the effect correlated with the connectivity pattern obtained from our network analyses.

Molecular mechanics and dynamics studies on the interaction of gallic acid with collagen-like peptides

NASA Astrophysics Data System (ADS)

Madhan, B.; Thanikaivelan, P.; Subramanian, V.; Raghava Rao, J.; Unni Nair, Balachandran; Ramasami, T.

2001-10-01

Molecular modelling approaches have been used to understand the interaction of collagen-like peptides with gallic acid, which mimic vegetable tanning processes involved in protein stabilization. Several interaction sites have been identified and the binding energies of the complexes have been calculated. The calculated binding energies for various geometries are in the range 6-13 kcal/mol. It is found that some complexes exhibit hydrogen bonding, and electrostatic interaction plays a dominant role in the stabilization of the peptide by gallic acid. The π-OH type of interaction is also observed in the peptide stabilization. Molecular dynamics (MD) simulation for 600 ps revealed the possibility of hydrogen bonding between the collagen-like peptide and gallic acid.

Chlorophyll-Derivative Modulation of Rhodopsin Signaling Properties through Evolutionarily Conserved Interaction Pathways

PubMed Central

Woods, Kristina N.; Pfeffer, Jürgen; Klein-Seetharaman, Judith

2017-01-01

Retinal is the light-absorbing chromophore that is responsible for the activation of visual pigments and light-driven ion pumps. Evolutionary changes in the intermolecular interactions of the retinal with specific amino acids allow for adaptation of the spectral characteristics, referred to as spectral tuning. However, it has been proposed that a specific species of dragon fish has bypassed the adaptive evolutionary process of spectral tuning and replaced it with a single evolutionary event: photosensitization of rhodopsin by chlorophyll derivatives. Here, by using a combination of experimental measurements and computational modeling to probe retinal-receptor interactions in rhodopsin, we show how the binding of the chlorophyll derivative, chlorin-e6 (Ce6) in the intracellular domain (ICD) of the receptor allosterically excites G-protein coupled receptor class A (GPCR-A) conserved long-range correlated fluctuations that connect distant parts of the receptor. These long-range correlated motions are associated with regulating the dynamics and intermolecular interactions of specific amino acids in the retinal ligand-binding pocket that have been associated with shifts in the absorbance peak maximum (λmax) and hence, spectral sensitivity of the visual system. Moreover, the binding of Ce6 affects the overall global properties of the receptor. Specifically, we find that Ce6-induced dynamics alter the thermal stability of rhodopsin by adjusting hydrogen-bonding interactions near the receptor active-site that consequently also influences the intrinsic conformational equilibrium of the receptor. Due to the conservation of the ICD residues amongst different receptors in this class and the fact that all GPCR-A receptors share a common mechanism of activation, it is possible that the allosteric associations excited in rhodopsin with Ce6 binding are a common feature in all class A GPCRs. PMID:29312953

The biological acoustic sensor to record the interactions of the microbial cells with the phage antibodies in conducting suspensions.

PubMed

Guliy, О I; Zaitsev, B D; Borodina, I A; Shikhabudinov, А М; Teplykh, A A; Staroverov, S A; Fomin, A S

2018-02-01

The acoustic biological sensor for the analysis of the bacterial cells in conducting suspension was developed. The sensor represented the two channel delay line based on the piezoelectric plate of Y-X lithium niobate thick of 0.2mm. Two pairs of the interdigital transducers (IDT) for the excitation and reception of shear horizontal acoustic wave of zero order (SH 0 ) in each channel were deposited by the method of photolithography. One channel of the delay line was electrically shorted by the deposition of thin aluminum film between IDTs. The second channel remained as electrically open. The liquid container with the volume of 5ml was fixed on the plate surface between IDTs by the glue, which did not cause the additional insertion loss. For the first time the influence of the conductivity of the cell suspension on the registration of the specific and nonspecific interactions of the bacterial cells with phage-antibodies (phage-Abs) was studied by means of the developed sensor. The dependencies of the change in insertion loss and phase of the output signal on the conductivity of the buffer solution at specific/nonspecific interactions for the electrically open and shorted channels of the delay line were obtained. It was shown that the sensor successfully registered the interactions of microbial cells with phage-Abs in the range of the conductivity of 2-20 μS/cm on the model samples A. brasilense Sp245 - specific phage-Abs. The sensor in the time regime of the operation fast reacted on the specific/nonspecific interaction and the time of the stabilization of the output parameters did not exceed 10min. Copyright © 2017 Elsevier B.V. All rights reserved.

Stabilization of Large Generalized Lotka-Volterra Foodwebs By Evolutionary Feedback

NASA Astrophysics Data System (ADS)

Ackland, G. J.; Gallagher, I. D.

2004-10-01

Conventional ecological models show that complexity destabilizes foodwebs, suggesting that foodwebs should have neither large numbers of species nor a large number of interactions. However, in nature the opposite appears to be the case. Here we show that if the interactions between species are allowed to evolve within a generalized Lotka-Volterra model such stabilizing feedbacks and weak interactions emerge automatically. Moreover, we show that trophic levels also emerge spontaneously from the evolutionary approach, and the efficiency of the unperturbed ecosystem increases with time. The key to stability in large foodwebs appears to arise not from complexity perse but from evolution at the level of the ecosystem which favors stabilizing (negative) feedbacks.

Stabilization of large generalized Lotka-Volterra foodwebs by evolutionary feedback.

PubMed

Ackland, G J; Gallagher, I D

2004-10-08

Conventional ecological models show that complexity destabilizes foodwebs, suggesting that foodwebs should have neither large numbers of species nor a large number of interactions. However, in nature the opposite appears to be the case. Here we show that if the interactions between species are allowed to evolve within a generalized Lotka-Volterra model such stabilizing feedbacks and weak interactions emerge automatically. Moreover, we show that trophic levels also emerge spontaneously from the evolutionary approach, and the efficiency of the unperturbed ecosystem increases with time. The key to stability in large foodwebs appears to arise not from complexity per se but from evolution at the level of the ecosystem which favors stabilizing (negative) feedbacks.

Brain Region and Isoform-Specific Phosphorylation Alters Kalirin SH2 Domain Interaction Sites and Calpain Sensitivity

PubMed Central

Miller, Megan B.; Yan, Yan; Machida, Kazuya; Kiraly, Drew D.; Levy, Aaron D.; Wu, Yi I.; Lam, TuKiet T.; Abbott, Thomas; Koleske, Anthony J.; Eipper, Betty A.; Mains, Richard E.

2017-01-01

Kalirin7 (Kal7), a postsynaptic Rho GDP/GTP exchange factor (RhoGEF), plays a crucial role in long term potentiation and in the effects of cocaine on behavior and spine morphology. The KALRN gene has been linked to schizophrenia and other disorders of synaptic function. Mass spectrometry was used to quantify phosphorylation at 26 sites in Kal7 from individual adult rat nucleus accumbens and prefrontal cortex before and after exposure to acute or chronic cocaine. Region- and isoform-specific phosphorylation was observed along with region-specific effects of cocaine on Kal7 phosphorylation. Evaluation of the functional significance of multi-site phosphorylation in a complex protein like Kalirin is difficult. With the identification of five tyrosine phosphorylation (pY) sites, a panel of 71 SH2 domains was screened, identifying subsets that interacted with multiple pY sites in Kal7. In addition to this type of reversible interaction, endoproteolytic cleavage by calpain plays an essential role in long-term potentiation. Calpain cleaved Kal7 at two sites, separating the N-terminal domain, which affects spine length, and the PDZ binding motif from the GEF domain. Mutations preventing phosphorylation did not affect calpain sensitivity or GEF activity; phosphomimetic mutations at specific sites altered protein stability, increased calpain sensitivity and reduced GEF activity. PMID:28418645

A comparison of biophysical characterization techniques in predicting monoclonal antibody stability.

PubMed

Thiagarajan, Geetha; Semple, Andrew; James, Jose K; Cheung, Jason K; Shameem, Mohammed

2016-01-01

With the rapid growth of biopharmaceutical product development, knowledge of therapeutic protein stability has become increasingly important. We evaluated assays that measure solution-mediated interactions and key molecular characteristics of 9 formulated monoclonal antibody (mAb) therapeutics, to predict their stability behavior. Colloidal interactions, self-association propensity and conformational stability were measured using effective surface charge via zeta potential, diffusion interaction parameter (kD) and differential scanning calorimetry (DSC), respectively. The molecular features of all 9 mAbs were compared to their stability at accelerated (25°C and 40°C) and long-term storage conditions (2-8°C) as measured by size exclusion chromatography. At accelerated storage conditions, the majority of the mAbs in this study degraded via fragmentation rather than aggregation. Our results show that colloidal stability, self-association propensity and conformational characteristics (exposed tryptophan) provide reasonable prediction of accelerated stability, with limited predictive value at 2-8°C stability. While no correlations to stability behavior were observed with onset-of-melting temperatures or domain unfolding temperatures, by DSC, melting of the Fab domain with the CH2 domain suggests lower stability at stressed conditions. The relevance of identifying appropriate biophysical assays based on the primary degradation pathways is discussed.

Stability and phase transition of localized modes in Bose–Einstein condensates with both two- and three-body interactions

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

Bai, Xiao-Dong; Ai, Qing; Zhang, Mei

We investigate the stability and phase transition of localized modes in Bose–Einstein Condensates (BECs) in an optical lattice with the discrete nonlinear Schrödinger model by considering both two- and three-body interactions. We find that there are three types of localized modes, bright discrete breather (DB), discrete kink (DK), and multi-breather (MUB). Moreover, both two- and three-body on-site repulsive interactions can stabilize DB, while on-site attractive three-body interactions destabilize it. There is a critical value for the three-body interaction with which both DK and MUB become the most stable ones. We give analytically the energy thresholds for the destabilization of localizedmore » states and find that they are unstable (stable) when the total energy of the system is higher (lower) than the thresholds. The stability and dynamics characters of DB and MUB are general for extended lattice systems. Our result is useful for the blocking, filtering, and transfer of the norm in nonlinear lattices for BECs with both two- and three-body interactions.« less

MicroRNA-Dependent Transcriptional Silencing of Transposable Elements in Drosophila Follicle Cells.

PubMed

Mugat, Bruno; Akkouche, Abdou; Serrano, Vincent; Armenise, Claudia; Li, Blaise; Brun, Christine; Fulga, Tudor A; Van Vactor, David; Pélisson, Alain; Chambeyron, Séverine

2015-05-01

RNA interference-related silencing mechanisms concern very diverse and distinct biological processes, from gene regulation (via the microRNA pathway) to defense against molecular parasites (through the small interfering RNA and the Piwi-interacting RNA pathways). Small non-coding RNAs serve as specificity factors that guide effector proteins to ribonucleic acid targets via base-pairing interactions, to achieve transcriptional or post-transcriptional regulation. Because of the small sequence complementarity required for microRNA-dependent post-transcriptional regulation, thousands of microRNA (miRNA) putative targets have been annotated in Drosophila. In Drosophila somatic ovarian cells, genomic parasites, such as transposable elements (TEs), are transcriptionally repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs) that prevent them from invading the germinal genome. Here we show, for the first time, that a functional miRNA pathway is required for the piRNA-mediated transcriptional silencing of TEs in this tissue. Global miRNA depletion, caused by tissue- and stage-specific knock down of drosha (involved in miRNA biogenesis), AGO1 or gawky (both responsible for miRNA activity), resulted in loss of TE-derived piRNAs and chromatin-mediated transcriptional de-silencing of TEs. This specific TE de-repression was also observed upon individual titration (by expression of the complementary miRNA sponge) of two miRNAs (miR-14 and miR-34) as well as in a miR-14 loss-of-function mutant background. Interestingly, the miRNA defects differentially affected TE- and 3' UTR-derived piRNAs. To our knowledge, this is the first indication of possible differences in the biogenesis or stability of TE- and 3' UTR-derived piRNAs. This work is one of the examples of detectable phenotypes caused by loss of individual miRNAs in Drosophila and the first genetic evidence that miRNAs have a role in the maintenance of genome stability via piRNA-mediated TE repression.

Beyond Anchoring: the Expanding Role of the Hendra Virus Fusion Protein Transmembrane Domain in Protein Folding, Stability, and Function

PubMed Central

Smith, Everett Clinton; Culler, Megan R.; Hellman, Lance M.; Fried, Michael G.; Creamer, Trevor P.

2012-01-01

While work with viral fusion proteins has demonstrated that the transmembrane domain (TMD) can affect protein folding, stability, and membrane fusion promotion, the mechanism(s) remains poorly understood. TMDs could play a role in fusion promotion through direct TMD-TMD interactions, and we have recently shown that isolated TMDs from three paramyxovirus fusion (F) proteins interact as trimers using sedimentation equilibrium (SE) analysis (E. C. Smith, et al., submitted for publication). Immediately N-terminal to the TMD is heptad repeat B (HRB), which plays critical roles in fusion. Interestingly, addition of HRB decreased the stability of the trimeric TMD-TMD interactions. This result, combined with previous findings that HRB forms a trimeric coiled coil in the prefusion form of the whole protein though HRB peptides fail to stably associate in isolation, suggests that the trimeric TMD-TMD interactions work in concert with elements in the F ectodomain head to stabilize a weak HRB interaction. Thus, changes in TMD-TMD interactions could be important in regulating F triggering and refolding. Alanine insertions between the TMD and HRB demonstrated that spacing between these two regions is important for protein stability while not affecting TMD-TMD interactions. Additional mutagenesis of the C-terminal end of the TMD suggests that β-branched residues within the TMD play a role in membrane fusion, potentially through modulation of TMD-TMD interactions. Our results support a model whereby the C-terminal end of the Hendra virus F TMD is an important regulator of TMD-TMD interactions and show that these interactions help hold HRB in place prior to the triggering of membrane fusion. PMID:22238302

An overview of preparation and applications of stabilized zero-valent iron nanoparticles for soil and groundwater remediation.

PubMed

Zhao, Xiao; Liu, Wen; Cai, Zhengqing; Han, Bing; Qian, Tianwei; Zhao, Dongye

2016-09-01

Nano-scale zero-valent iron (nZVI) is one of the most intensively studied materials for environmental cleanup uses over the past 20 years or so. Freshly prepared nZVI is highly reactive due to its high specific surface area and strong reducing power. Over years, the classic borohydride reduction method for preparing nZVI has been modified by use of various stabilizers or surface modifiers to acquire more stable and soil deliverable nZVI for treatment of different organic and inorganic contaminants in water and soil. While most studies have been focused on testing nZVI for water treatment, the greater potential or advantage of nZVI appears to be for in situ remediation of contaminated soil and groundwater by directly delivering stabilized nZVI into the contaminated subsurface as it was proposed from the beginning. Compared to conventional remediation practices, the in situ remediation technique using stabilized nZVI offers some unique advantages. This work provides an update on the latest development of stabilized nZVI for various environmental cleanup uses, and overviews the evolution and environmental applications of stabilized nZVI. Commonly used stabilizers are compared and the stabilizing mechanisms are discussed. The effectiveness and constraints of the nZVI-based in situ remediation technology are summarized. This review also reveals some critical knowledge gaps and research needs, such as interactions between delivered nZVI and the local biogeochemical conditions. Copyright © 2016 Elsevier Ltd. All rights reserved.

A novel TPR–BEN domain interaction mediates PICH–BEND3 association

PubMed Central

Pitchai, Ganesha P.; Kaulich, Manuel; Mesa, Pablo; Yao, Qi; Sarlos, Kata; Streicher, Werner W.; Nigg, Erich A.

2017-01-01

Abstract PICH is a DNA translocase required for the maintenance of chromosome stability in human cells. Recent data indicate that PICH co-operates with topoisomerase IIα to suppress pathological chromosome missegregation through promoting the resolution of ultra-fine anaphase bridges (UFBs). Here, we identify the BEN domain-containing protein 3 (BEND3) as an interaction partner of PICH in human cells in mitosis. We have purified full length PICH and BEND3 and shown that they exhibit a functional biochemical interaction in vitro. We demonstrate that the PICH–BEND3 interaction occurs via a novel interface between a TPR domain in PICH and a BEN domain in BEND3, and have determined the crystal structure of this TPR–BEN complex at 2.2 Å resolution. Based on the structure, we identified amino acids important for the TPR–BEN domain interaction, and for the functional interaction of the full-length proteins. Our data reveal a proposed new function for BEND3 in association with PICH, and the first example of a specific protein–protein interaction mediated by a BEN domain. PMID:28977671

The effect of specific proline residues on the kinetic stability of the triosephosphate isomerases of two trypanosomes.

PubMed

Guzmán-Luna, Valeria; Quezada, Andrea G; Díaz-Salazar, A Jessica; Cabrera, Nallely; Pérez-Montfort, Ruy; Costas, Miguel

2017-04-01

The effect of specific residues on the kinetic stability of two closely related triosephosphate isomerases (from Trypanosoma cruzi, TcTIM and Trypanosoma brucei, TbTIM) has been studied. Based on a comparison of their β-turn occurrence, we engineered two chimerical enzymes where their super secondary β-loop-α motifs 2 ((βα) 2 ) were swapped. Differential scanning calorimetry (DSC) experiments showed that the (βα) 2 motif of TcTIM inserted into TbTIM (2Tc) increases the kinetic stability. On the other hand, the presence of the (βα) 2 motif of TbTIM inserted into TcTIM (2Tb) gave a chimerical protein difficult to purify in soluble form and with a significantly reduced kinetic stability. The comparison of the contact maps of the (βα) 2 of TbTIM and TcTIM showed differences in the contact pattern of residues 43 and 49. In TcTIM these residues are prolines, located at the N-terminal of loop-2 and the C-terminal of α-helix-2. Twelve mutants were engineered involving residues 43 and 49 to study the effect over the unfolding activation energy barrier (E A ). A systematic analysis of DSC data showed a large decrease on the E A of TcTIM (ΔE A ranging from 468 to 678 kJ/mol) when the single and double proline mutations are present. The relevance of Pro43 to the kinetic stability is also revealed by mutation S43P, which increased the free energy of the transition state of TbTIM by 17.7 kJ/mol. Overall, the results indicate that protein kinetic stability can be severely affected by punctual mutations, disturbing the complex network of interactions that, in concerted action, determine protein stability. Proteins 2017; 85:571-579. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

How plants connect pollination and herbivory networks and their contribution to community stability.

PubMed

Sauve, Alix M C; Thébault, Elisa; Pocock, Michael J O; Fontaine, Colin

2016-04-01

Pollination and herbivory networks have mainly been studied separately, highlighting their distinct structural characteristics and the related processes and dynamics. However, most plants interact with both pollinators and herbivores, and there is evidence that both types of interaction affect each other. Here we investigated the way plants connect these mutualistic and antagonistic networks together, and the consequences for community stability. Using an empirical data set, we show that the way plants connect pollination and herbivory networks is not random and promotes community stability. Analyses of the structure of binary and quantitative networks show different results: the plants' generalism with regard to pollinators is positively correlated to their generalism with regard to herbivores when considering binary interactions, but not when considering quantitative interactions. We also show that plants that share the same pollinators do not share the same herbivores. However, the way plants connect pollination and herbivory networks promotes stability for both binary and quantitative networks. Our results highlight the relevance of considering the diversity of interaction types in ecological communities, and stress the need to better quantify the costs and benefits of interactions, as well as to develop new metrics characterizing the way different interaction types are combined within ecological networks.

A force-based, parallel assay for the quantification of protein-DNA interactions.

PubMed

Limmer, Katja; Pippig, Diana A; Aschenbrenner, Daniela; Gaub, Hermann E

2014-01-01

Analysis of transcription factor binding to DNA sequences is of utmost importance to understand the intricate regulatory mechanisms that underlie gene expression. Several techniques exist that quantify DNA-protein affinity, but they are either very time-consuming or suffer from possible misinterpretation due to complicated algorithms or approximations like many high-throughput techniques. We present a more direct method to quantify DNA-protein interaction in a force-based assay. In contrast to single-molecule force spectroscopy, our technique, the Molecular Force Assay (MFA), parallelizes force measurements so that it can test one or multiple proteins against several DNA sequences in a single experiment. The interaction strength is quantified by comparison to the well-defined rupture stability of different DNA duplexes. As a proof-of-principle, we measured the interaction of the zinc finger construct Zif268/NRE against six different DNA constructs. We could show the specificity of our approach and quantify the strength of the protein-DNA interaction.

Genome-driven evolutionary game theory helps understand the rise of metabolic interdependencies in microbial communities.

PubMed

Zomorrodi, Ali R; Segrè, Daniel

2017-11-16

Metabolite exchanges in microbial communities give rise to ecological interactions that govern ecosystem diversity and stability. It is unclear, however, how the rise of these interactions varies across metabolites and organisms. Here we address this question by integrating genome-scale models of metabolism with evolutionary game theory. Specifically, we use microbial fitness values estimated by metabolic models to infer evolutionarily stable interactions in multi-species microbial "games". We first validate our approach using a well-characterized yeast cheater-cooperator system. We next perform over 80,000 in silico experiments to infer how metabolic interdependencies mediated by amino acid leakage in Escherichia coli vary across 189 amino acid pairs. While most pairs display shared patterns of inter-species interactions, multiple deviations are caused by pleiotropy and epistasis in metabolism. Furthermore, simulated invasion experiments reveal possible paths to obligate cross-feeding. Our study provides genomically driven insight into the rise of ecological interactions, with implications for microbiome research and synthetic ecology.

Thermodynamics-hydration relationships within loops that affect G-quadruplexes under molecular crowding conditions.

PubMed

Fujimoto, Takeshi; Nakano, Shu-ichi; Sugimoto, Naoki; Miyoshi, Daisuke

2013-01-31

We systematically investigated the effects of loop length on the conformation, thermodynamic stability, and hydration of DNA G-quadruplexes under dilute and molecular crowding conditions in the presence of Na(+). Structural analysis showed that molecular crowding induced conformational switches of oligonucleotides with the longer guanine stretch and the shorter thymine loop. Thermodynamic parameters further demonstrated that the thermodynamic stability of G-quadruplexes increased by increasing the loop length from two to four, whereas it decreased by increasing the loop length from four to six. Interestingly, we found by osmotic pressure analysis that the number of water molecules released from the G-quadruplex decreased with increasing thermodynamic stability. We assumed that base-stacking interactions within the loops not only stabilized the whole G-quadruplex structure but also created hydration sites by accumulating nucleotide functional groups. The molecular crowding effects on the stability of G-quadruplexes composed of abasic sites, which reduce the stacking interactions at the loops, further demonstrated that G-quadruplexes with fewer stacking interactions within the loops released a larger number of water molecules upon folding. These results showed that the stacking interactions within the loops determined the thermodynamic stability and hydration of the whole G-quadruplex.

Interaction of zanamivir with DNA and RNA: Models for drug DNA and drug RNA bindings

NASA Astrophysics Data System (ADS)

Nafisi, Shohreh; Kahangi, Fatemeh Ghoreyshi; Azizi, Ebrahim; Zebarjad, Nader; Tajmir-Riahi, Heidar-Ali

2007-03-01

Zanamivir (ZAN) is the first of a new generation of influenza virus-specific drugs known as neuraminidase inhibitors, which acts by interfering with life cycles of influenza viruses A and B. It prevents the virus spreading infection to other cells by blocking the neuraminidase enzyme present on the surface of the virus. The aim of this study was to examine the stability and structural features of calf thymus DNA and yeast RNA complexes with zanamivir in aqueous solution, using constant DNA or RNA concentration (12.5 mM) and various zanamivir/polynucleotide ( P) ratios of 1/20, 1/10, 1/4, and 1/2. FTIR and UV-visible spectroscopy are used to determine the drug external binding modes, the binding constant and the stability of zanamivir-DNA and RNA complexes in aqueous solution. Structural analysis showed major interaction of zanamivir with G-C (major groove) and A-T (minor groove) base pairs and minor perturbations of the backbone PO 2 group with overall binding constants of Kzanamivir-DNA = 1.30 × 10 4 M -1 and Kzanamivir-RNA = 1.38 × 10 4 M -1. The drug interaction induces a partial B to A-DNA transition, while RNA remains in A-conformation.

Attributions and self-efficacy for physical activity in multiple sclerosis.

PubMed

Nickel, D; Spink, K; Andersen, M; Knox, K

2014-01-01

Self-efficacy is an important predictor of health-related physical activity in multiple sclerosis (MS). While past experiences are believed to influence efficacy beliefs, the explanations individuals provide for these experiences also may be critical. Our objective was to test the hypothesis that perceived success or failure to accumulate 150 min of physical activity in the previous week would moderate the relationship between the attributional dimension of stability and self-efficacy to exercise in the future. Forty-two adults with MS participated in this cross-sectional descriptive study. Participants completed questions assessing physical activity, perceived outcome for meeting the recommended level of endurance activity, attributions for the outcome, and exercise self-efficacy. Results from hierarchical multiple regression revealed a significant main effect for perceived outcome predicting self-efficacy that was qualified by a significant interaction. The final model, which included perceived outcome, stability, and the interaction term, predicted 37% of the variance in exercise self-efficacy, F (3, 38) = 7.27, p = .001. Our findings suggest that the best prediction of self-efficacy in the MS population may include the interaction of specific attributional dimensions with success/failure at meeting the recommended physical activity dose. Attributions may be another target for interventions aimed at increasing the physical activity in MS.

Alginate/sodium caseinate aqueous-core capsules: a pH-responsive matrix.

PubMed

Ben Messaoud, Ghazi; Sánchez-González, Laura; Jacquot, Adrien; Probst, Laurent; Desobry, Stéphane

2015-02-15

Alginate capsules have several applications. Their functionality depends considerably on their permeability, chemical and mechanical stability. Consequently, the creation of composite system by addition of further components is expected to control mechanical and release properties of alginate capsules. Alginate and alginate-sodium caseinate composite liquid-core capsules were prepared by a simple extrusion. The influence of the preparation pH and sodium caseinate concentration on capsules physico-chemical properties was investigated. Results showed that sodium caseinate influenced significantly capsules properties. As regards to the membrane mechanical stability, composite capsules prepared at pH below the isoelectric point of sodium caseinate exhibited the highest surface Young's modulus, increasing with protein content, explained by potential electrostatic interactions between sodium caseinate amino-groups and alginate carboxylic group. The kinetic of cochineal red A release changed significantly for composite capsules and showed a pH-responsive release. Sodium caseinate-dye mixture studied by absorbance and fluorescence spectroscopy confirmed complex formation at pH 2 by electrostatic interactions between sodium caseinate tryptophan residues and cochineal red sulfonate-groups. Consequently, the release mechanism was explained by membrane adsorption process. This global approach is useful to control release mechanism from macro and micro-capsules by incorporating guest molecules which can interact with the entrapped molecule under specific conditions. Copyright © 2014 Elsevier Inc. All rights reserved.

The polymeric stability of the Escherichia coli F4 (K88) fimbriae enhances its mucosal immunogenicity following oral immunization.

PubMed

Verdonck, Frank; Joensuu, Jussi Joonas; Stuyven, Edith; De Meyer, Julie; Muilu, Mikko; Pirhonen, Minna; Goddeeris, Bruno Maria; Mast, Jan; Niklander-Teeri, Viola; Cox, Eric

2008-10-23

Only a few vaccines are commercially available against intestinal infections since the induction of a protective intestinal immune response is difficult to achieve. For instance, oral administration of most proteins results in oral tolerance instead of an antigen-specific immune response. We have shown before that as a result of oral immunization of piglets with F4 fimbriae purified from pathogenic enterotoxigenic Escherichia coli (ETEC), the fimbriae bind to the F4 receptor (F4R) in the intestine and induce a protective F4-specific immune response. F4 fimbriae are very stable polymeric structures composed of some minor subunits and a major subunit FaeG that is also the fimbrial adhesin. In the present study, the mutagenesis experiments identified FaeG amino acids 97 (N to K) and 201 (I to V) as determinants for F4 polymeric stability. The interaction between the FaeG subunits in mutant F4 fimbriae is reduced but both mutant and wild type fimbriae behaved identically in F4R binding and showed equal stability in the gastro-intestinal lumen. Oral immunization experiments indicated that a higher degree of polymerisation of the fimbriae in the intestine was correlated with a better F4-specific mucosal immunogenicity. These data suggest that the mucosal immunogenicity of soluble virulence factors can be increased by the construction of stable polymeric structures and therefore help in the development of effective mucosal vaccines.

Ubiquitin-like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway.

PubMed

Oka, Yasuyoshi; Bekker-Jensen, Simon; Mailand, Niels

2015-05-12

Ubiquitin and ubiquitin-like proteins (UBLs) function in a wide array of cellular processes. UBL5 is an atypical UBL that does not form covalent conjugates with cellular proteins and which has a known role in modulating pre-mRNA splicing. Here, we report an unexpected involvement of human UBL5 in promoting the function of the Fanconi anemia (FA) pathway for repair of DNA interstrand crosslinks (ICLs), mediated by a specific interaction with the central FA pathway component FANCI. UBL5-deficient cells display spliceosome-independent reduction of FANCI protein stability, defective FANCI function in response to DNA damage and hypersensitivity to ICLs. By mapping the sequence determinants underlying UBL5-FANCI binding, we generated separation-of-function mutants to demonstrate that key aspects of FA pathway function, including FANCI-FANCD2 heterodimerization, FANCD2 and FANCI monoubiquitylation and maintenance of chromosome stability after ICLs, are compromised when the UBL5-FANCI interaction is selectively inhibited by mutations in either protein. Together, our findings establish UBL5 as a factor that promotes the functionality of the FA DNA repair pathway. © 2015 The Authors.

Head formation requires Dishevelled degradation that is mediated by March2 in concert with Dapper1.

PubMed

Lee, Hyeyoon; Cheong, Seong-Moon; Han, Wonhee; Koo, Youngmu; Jo, Saet-Byeol; Cho, Gun-Sik; Yang, Jae-Seong; Kim, Sanguk; Han, Jin-Kwan

2018-04-10

Dishevelled (Dvl/Dsh) is a key scaffold protein that propagates Wnt signaling essential for embryogenesis and homeostasis. However, whether the antagonism of Wnt signaling that is necessary for vertebrate head formation can be achieved through regulation of Dsh protein stability is unclear. Here, we show that membrane-associated RING-CH2 (March2), a RING-type E3 ubiquitin ligase, antagonizes Wnt signaling by regulating the turnover of Dsh protein via ubiquitin-mediated lysosomal degradation in the prospective head region of Xenopus We further found that March2 acquires regional and functional specificities for head formation from the Dsh-interacting protein Dapper1 (Dpr1). Dpr1 stabilizes the interaction between March2 and Dsh in order to mediate ubiquitylation and the subsequent degradation of Dsh protein only in the dorso-animal region of Xenopus embryo. These results suggest that March2 restricts cytosolic pools of Dsh protein and reduces the need for Wnt signaling in precise vertebrate head development. © 2018. Published by The Company of Biologists Ltd.

Cu doped diamond: Effect of charge state and defect aggregation on spin interactions in a 3d transition metal doped wide band-gap semiconductor

NASA Astrophysics Data System (ADS)

Benecha, E. M.; Lombardi, E. B.

2018-05-01

We present a first principles study of Cu in diamond using DFT+U electronic structure methods, by carefully considering the impact of co-doping, charge state, and Fermi level position on its stability, lattice location, spin states, and electronic properties. We show that the energetic stability and spin states of Cu are strongly dependent on the Fermi level position and the type of diamond co-doping, with Cu being energetically more favorable in n-type or p-type co-doped diamond compared to intrinsic diamond. Since Cu has been predicted to order magnetically in a number of other wide band-gap semiconductors, we have also evaluated this possibility for Cu doped diamond. We show that while Cu exhibits strong spin interactions at specific interatomic separations in diamond, a detailed consideration of the impact of Fermi level position and Cu aggregation precludes magnetic ordering, with Cu forming non-magnetic, antiferromagnetic, or paramagnetic clusters. These results have important implications in the understanding of the properties of transition metal dopants in diamond for device applications.

In vivo ultrasound visualization of non-occlusive blood clots with thrombin-sensitive contrast agents.

PubMed

Nakatsuka, Matthew A; Barback, Christopher V; Fitch, Kirsten R; Farwell, Alexander R; Esener, Sadik C; Mattrey, Robert F; Cha, Jennifer N; Goodwin, Andrew P

2013-12-01

The use of microbubbles as ultrasound contrast agents is one of the primary methods to diagnose deep venous thrombosis. However, current microbubble imaging strategies require either a clot sufficiently large to produce a circulation filling defect or a clot with sufficient vascularization to allow for targeted accumulation of contrast agents. Previously, we reported the design of a microbubble formulation that modulated its ability to generate ultrasound contrast from interaction with thrombin through incorporation of aptamer-containing DNA crosslinks in the encapsulating shell, enabling the measurement of a local chemical environment by changes in acoustic activity. However, this contrast agent lacked sufficient stability and lifetime in blood to be used as a diagnostic tool. Here we describe a PEG-stabilized, thrombin-activated microbubble (PSTA-MB) with sufficient stability to be used in vivo in circulation with no change in biomarker sensitivity. In the presence of actively clotting blood, PSTA-MBs showed a 5-fold increase in acoustic activity. Specificity for the presence of thrombin and stability under constant shear flow were demonstrated in a home-built in vitro model. Finally, PSTA-MBs were able to detect the presence of an active clot within the vena cava of a rabbit sufficiently small as to not be visible by current non-specific contrast agents. By activating in non-occlusive environments, these contrast agents will be able to detect clots not diagnosable by current contrast agents. Copyright © 2013 Elsevier Ltd. All rights reserved.

ZFP36L1 and ZFP36L2 control LDLR mRNA stability via the ERK-RSK pathway.

PubMed

Adachi, Shungo; Homoto, Masae; Tanaka, Rikou; Hioki, Yusaku; Murakami, Hiroshi; Suga, Hiroaki; Matsumoto, Masaki; Nakayama, Keiichi I; Hatta, Tomohisa; Iemura, Shun-ichiro; Natsume, Tohru

2014-09-01

Low-density lipoprotein receptor (LDLR) mRNA is unstable, but is stabilized upon extracellular signal-regulated kinase (ERK) activation, possibly through the binding of certain proteins to the LDLR mRNA 3'-untranslated region (UTR), although the detailed mechanism underlying this stability control is unclear. Here, using a proteomic approach, we show that proteins ZFP36L1 and ZFP36L2 specifically bind to the 3'-UTR of LDLR mRNA and recruit the CCR4-NOT-deadenylase complex, resulting in mRNA destabilization. We also show that the C-terminal regions of ZFP36L1 and ZFP36L2 are directly phosphorylated by p90 ribosomal S6 kinase, a kinase downstream of ERK, resulting in dissociation of the CCR4-NOT-deadenylase complex and stabilization of LDLR mRNA. We further demonstrate that targeted disruption of the interaction between LDLR mRNA and ZFP36L1 and ZFP36L2 using antisense oligonucleotides results in upregulation of LDLR mRNA and protein. These results indicate that ZFP36L1 and ZFP36L2 regulate LDLR protein levels downstream of ERK. Our results also show the usefulness of our method for identifying critical regulators of specific RNAs and the potency of antisense oligonucleotide-based therapeutics. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

The denaturation and degradation of stable enzymes at high temperatures.

PubMed Central

Daniel, R M; Dines, M; Petach, H H

1996-01-01

Now that enzymes are available that are stable above 100 degrees C it is possible to investigate conformational stability at this temperature, and also the effect of high-temperature degradative reactions in functioning enzymes and the inter-relationship between degradation and denaturation. The conformational stability of proteins depends upon stabilizing forces arising from a large number of weak interactions, which are opposed by an almost equally large destabilizing force due mostly to conformational entropy. The difference between these, the net free energy of stabilization, is relatively small, equivalent to a few interactions. The enhanced stability of very stable proteins can be achieved by an additional stabilizing force which is again equivalent to only a few stabilizing interactions. There is currently no strong evidence that any particular interaction (e.g. hydrogen bonds, hydrophobic interactions) plays a more important role in proteins that are stable at 100 degrees C than in those stable at 50 degrees C, or that the structures of very stable proteins are systematically different from those of less stable proteins. The major degradative mechanisms are deamidation of asparagine and glutamine, and succinamide formation at aspartate and glutamate leading to peptide bond hydrolysis. In addition to being temperature-dependent, these reactions are strongly dependent upon the conformational freedom of the susceptible amino acid residues. Evidence is accumulating which suggests that even at 100 degrees C deamidation and succinamide formation proceed slowly or not at all in conformationally intact (native) enzymes. Whether this is the case at higher temperatures is not yet clear, so it is not known whether denaturation of degradation will set the upper limit of stability for enzymes. PMID:8694749

QM/MM Investigation of Substrate and Product Specificities of Suv4-20h2: How Does This Enzyme Generate Dimethylated H4K20 from Monomethylated Substrate?

PubMed

Qian, Ping; Guo, Haobo; Wang, Liang; Guo, Hong

2017-06-13

Protein lysine methyltransferases (PKMTs) catalyze the methylation of lysine residues on histone proteins in the regulation of chromatin structure and gene expression. In contrast to many other PKMTs for which unmodified lysine is the methylation target, the enzymes in the Suv4-20 family are able to generate dimethylated product (H4K20me2) based exclusively on the monomethylated H4K20 substrate (H4K20me1). The origin of such substrate/product specificity is still not clear. Here, molecular dynamics (MD) and free energy (potential of mean force) simulations are undertaken using quantum mechanical/molecular mechanical (QM/MM) potentials to understand the substrate/product specificities of Suv4-20h2, a member of the Suv4-20 family. The free energy barriers for mono-, di-, and trimethylation in Suv4-20h2 obtained from the simulations are found to be well correlated with the specificities observed experimentally with the allowed dimethylation based on the H4K20me1 substrate and prohibited monomethylation and trimethylation based on H4K20 and H4K20me2, respectively. It is demonstrated that the reason for the relatively efficient dimethylation is an effective transition state (TS) stabilization through strengthening the CH···O interactions as well as the presence of a cation-π interaction at the transition state. The simulations also show that the failures of Suv4-20h2 to catalyze monomethylation and trimethylation are due, respectively, to a less effective TS stabilization and inability of the reactant complex containing H4K20me2 to adopt a reactive (near attack) configuration for methyl transfer. The results suggest that care must be exercised in the prediction of the substrate specificity based only on the existence of near attack configurations in substrate complexes.

A Study on the Effect of Surface Lysine to Arginine Mutagenesis on Protein Stability and Structure Using Green Fluorescent Protein

PubMed Central

Sokalingam, Sriram; Raghunathan, Govindan; Soundrarajan, Nagasundarapandian; Lee, Sun-Gu

2012-01-01

Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP) was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering. PMID:22792305

Physical stability of micronized powders produced by spray-freezing into liquid (SFL) to enhance the dissolution of an insoluble drug.

PubMed

Rogers, True L; Johnston, Keith P; Williams, Robert O

2003-01-01

The objective of this study was to investigate the physical stability of micronized powders produced by the spray-freezing into liquid (SFL) particle engineeringtechnology. Danazol was formulated with polyvinyl alcohol (MW 22,000), poloxamer 407, and polyvinylpyrrolidone K-15 to form a cosolvent solution that was SFL processed. The dried micronized SFL powders were sealed in glass vials with desiccant and exposed to 25 degrees C/60% RH for 3 and 6 mo, 40 degrees C/75% RH for 1, 2, 3, and 6 mo, and conditions where the temperature was cycled between -5 and +40 degrees C (6 cycles/24 hr) with constant 75% RH for 1, 2, 3 and 4 wk. The samples were characterized by using Karl-Fisher titration, differential scanning calorimetry, x-ray diffraction, specific surface area, scanning electron microscopy, and dissolution testing. Micronized SFL powders consisting of porous aggregates with small-particle domains were characterized as having high surface areas and consisted of amorphous danazol embedded within a hydrophilic excipient matrix. Karl-Fischer titration revealed no moisture absorption over the duration of the stability studies. Differential scanning calorimetry studies demonstrated high degrees of molecular interactions between danazol, PVA, poloxamer, and PVP. Scanning electron microscopy studies confirmed these interactions, especially those between danazol and poloxamer. These interactions facilitated API dissolution in the aqueous media. Powder surface area remained constant during storage at the various stability conditions, and danazol recrystallization did not occur during the entirety of the stability studies. Micronized SFL powders containing danazol dissolved rapidly and completely within 5 min in aqueous media. No differences were observed in the enhanced dissolution profiles of danazol after exposure to the storage conditions investigated. Physically stable micronized powders produced by the SFL particle engineering technology were produced for the purpose of enhancing the dissolution of an insoluble drug. The potential of the SFL particle-engineering technology as a micronization technique for enhancing the dissolution of hydrophobic drugs was demonstrated in this study. The robustness of the micronized SFL powders to withstand stressed storage conditions was shown.

Peripheral stator of the yeast V-ATPase: stoichiometry and specificity of interaction between the EG complex and subunits C and H.

PubMed

Féthière, James; Venzke, David; Madden, Dean R; Böttcher, Bettina

2005-12-06

V-ATPases are multisubunit membrane protein complexes that use the energy provided by ATP hydrolysis to generate a proton gradient across various intracellular and plasma membranes. In doing so, they maintain an acidic pH in the lumen of intracellular organelles and acidify extracellular milieu to support specific cellular functions. V-ATPases are structurally similar to the F1F0-ATP synthase, with an intrinsic membrane domain (V0) and an extrinsic peripheral domain (V1) joined by several connecting elements. To gain a clear functional understanding of the catalytic mechanism, and of the stability requirements for regulatory processes in the enzyme, a clear topology of the enzyme has to be established. In particular, the composition and arrangement of the peripheral stator subunits must be firmly settled, as these play specific roles in catalysis and regulation. We have designed a strategy allowing us to coexpress different combinations of these subunits to delineate specific interactions. In this study, we report the interaction between the peripheral stator EG complex and subunits C and H of the V-ATPase from the yeast Saccharomyces cerevisae. A combination of analytical gel filtration, native gel electrophoresis, and ultracentrifugation analysis allowed us to ascertain the homogeneity and molar mass of the purified EGC complex as well as of the EG complex, supporting the formation of 1:1(:1) stoichiometric complexes. The EGC complex can be formed in vitro by combining equimolar amounts of subunit C and the EG subcomplex and results most likely from the initial interaction between subunits E and C.

Stepwise Adaptations to Low Temperature as Revealed by Multiple Mutants of Psychrophilic α-Amylase from Antarctic Bacterium*

PubMed Central

Cipolla, Alexandre; D'Amico, Salvino; Barumandzadeh, Roya; Matagne, André; Feller, Georges

2011-01-01

The mutants Mut5 and Mut5CC from a psychrophilic α-amylase bear representative stabilizing interactions found in the heat-stable porcine pancreatic α-amylase but lacking in the cold-active enzyme from an Antarctic bacterium. From an evolutionary perspective, these mutants can be regarded as structural intermediates between the psychrophilic and the mesophilic enzymes. We found that these engineered interactions improve all the investigated parameters related to protein stability as follows: compactness; kinetically driven stability; thermodynamic stability; resistance toward chemical denaturation, and the kinetics of unfolding/refolding. Concomitantly to this improved stability, both mutants have lost the kinetic optimization to low temperature activity displayed by the parent psychrophilic enzyme. These results provide strong experimental support to the hypothesis assuming that the disappearance of stabilizing interactions in psychrophilic enzymes increases the amplitude of concerted motions required by catalysis and the dynamics of active site residues at low temperature, leading to a higher activity. PMID:21900238

Centrobin–tubulin interaction is required for centriole elongation and stability

PubMed Central

Gudi, Radhika; Zou, Chaozhong; Li, Jun

2011-01-01

Centrobin is a daughter centriole protein that is essential for centrosome duplication. However, the molecular mechanism by which centrobin functions during centriole duplication remains undefined. In this study, we show that centrobin interacts with tubulin directly, and centrobin–tubulin interaction is pivotal for the function of centrobin during centriole duplication. We found that centrobin is recruited to the centriole biogenesis site via its interaction with tubulins during the early stage of centriole biogenesis, and its recruitment is dependent on hSAS-6 but not centrosomal P4.1–associated protein (CPAP) and CP110. The function of centrobin is also required for the elongation of centrioles, which is likely mediated by its interaction with tubulin. Furthermore, disruption of centrobin–tubulin interaction led to destabilization of existing centrioles and the preformed procentriole-like structures induced by CPAP expression, indicating that centrobin–tubulin interaction is critical for the stability of centrioles. Together, our study demonstrates that centrobin facilitates the elongation and stability of centrioles via its interaction with tubulins. PMID:21576394

Biomimetic DNA emulsions: specific, thermo-reversible and adjustable binding from a liquid-like DNA layer

NASA Astrophysics Data System (ADS)

Pontani, Lea-Laetitia; Feng, Lang; Dreyfus, Remi; Seeman, Nadrian; Chaikin, Paul; Brujic, Jasna

2013-03-01

We develop micron-sized emulsions coated with specific DNA sequences and complementary sticky ends. The emulsions are stabilized with phospholipids on which the DNA strands are grafted through biotin-streptavidin interactions, which allows the DNA to diffuse freely on the surface. We produce two complementary emulsions: one is functionalized with S sticky ends and dyed with red streptavidin, the other displays the complementary S' sticky ends and green streptavidin. Mixing those emulsions reveals specific adhesion between them due to the short-range S-S' hybridization. As expected this interaction is thermo-reversible: the red-green adhesive droplets dissociate upon heating and reassemble after cooling. Here the fluid phospholipids layer also leads to diffusive adhesion patches, which allows the bound droplets to rearrange throughout the packing structure. We quantify the adhesion strength between two droplets and build a theoretical framework that captures the observed trends through parameters such as the size of the droplets, the DNA surface density, the various DNA constructs or the temperature. This colloidal-scale, specific, thermo-reversible biomimetic emulsion offers a new versatile and powerful tool for the development of complex self-assembled materials.

[Identification of C(2)M interacting proteins by yeast two-hybrid screening].

PubMed

Yue, Shan-shan; Xia, Lai-xin

2015-11-01

The synaptonemal complex (SC) is a huge structure which assembles between the homologous chromosomes during meiotic prophase I. Drosophila germ cell-specific nucleoprotein C(2)M clustering at chromosomes can induce SC formation. To further study the molecular function and mechanism of C(2)M in meiosis, we constructed a bait vector for C(2)M and used the yeast two-hybrid system to identify C(2)M interacting proteins. Forty interacting proteins were obtained, including many DNA and histone binding proteins, ATP synthases and transcription factors. Gene silencing assays in Drosophila showed that two genes, wech and Psf1, may delay the disappearance of SC. These results indicate that Wech and Psf1 may form a complex with C(2)M to participate in the formation or stabilization of the SC complex.

Ménage à trois: the complex relationships between mitogen-activated protein kinases, WRKY transcription factors, and VQ-motif-containing proteins.

PubMed

Weyhe, Martin; Eschen-Lippold, Lennart; Pecher, Pascal; Scheel, Dierk; Lee, Justin

2014-01-01

Out of the 34 members of the VQ-motif-containing protein (VQP) family, 10 are phosphorylated by the mitogen-activated protein kinases (MAPKs), MPK3 and MPK6. Most of these MPK3/6-targeted VQPs (MVQs) interacted with specific sub-groups of WRKY transcription factors in a VQ-motif-dependent manner. In some cases, the MAPK appears to phosphorylate either the MVQ or the WRKY, while in other cases, both proteins have been reported to act as MAPK substrates. We propose a network of dynamic interactions between members from the MAPK, MVQ and WRKY families - either as binary or as tripartite interactions. The compositions of the WRKY-MVQ transcriptional protein complexes may change - for instance, through MPK3/6-mediated modulation of protein stability - and therefore control defense gene transcription.

All-Atom Molecular Dynamics-Based Analysis of Membrane-Stabilizing Copolymer Interactions with Lipid Bilayers Probed under Constant Surface Tensions.

PubMed

Houang, Evelyne M; Bates, Frank S; Sham, Yuk Y; Metzger, Joseph M

2017-11-30

An all-atom phospholipid bilayer and triblock copolymer model was developed for molecular dynamics (MD) studies. These were performed to investigate the mechanism of interaction between membrane-stabilizing triblock copolymer P188 and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayers under applied lateral surface tension (γ) to model membrane mechanical stress. Results showed that P188 insertion is driven by the hydrophobic poly(propylene oxide) (PPO) core and dependent on bilayer area per lipid. Moreover, insertion of P188 increased the bilayer's resistance to mechanical rupture, as observed by a significant increase in the absolute lateral pressure required to disrupt the bilayer. To further investigate the specific chemical features of P188 underlying membrane stabilizer function, a series of MD simulations with triblock copolymers of the same class as P188 but of varying chemical composition and sizes were performed. Results showed that triblock copolymer insertion into the lipid bilayer is dependent on overall copolymer hydrophobicity, with higher copolymer hydrophobicity requiring a reduced bilayer area per lipid ratio for insertion. Further analysis revealed that the effect of copolymer insertion on membrane mechanical integrity was also dependent on hydrophobicity. Here, P188 insertion significantly increased the absolute apparent lateral pressure required to rupture the POPC bilayer, thereby protecting the membrane against mechanical stress. In marked contrast, highly hydrophobic copolymers decreased the lateral pressure necessary for membrane rupture and thus rendering the membrane significantly more susceptible to mechanical stress. These new in silico findings align with recent experimental findings using synthetic lipid bilayers and in muscle cells in vitro and mouse models in vivo. Collectively, these data underscore the importance of PEO-PPO-PEO copolymer chemical composition in copolymer-based muscle membrane stabilization in vitro and in vivo. All-atom modeling with MD simulations holds promise for investigating novel copolymers with enhanced membrane interacting properties.

Stabilization of perturbed Boolean network attractors through compensatory interactions

PubMed Central

2014-01-01

Background Understanding and ameliorating the effects of network damage are of significant interest, due in part to the variety of applications in which network damage is relevant. For example, the effects of genetic mutations can cascade through within-cell signaling and regulatory networks and alter the behavior of cells, possibly leading to a wide variety of diseases. The typical approach to mitigating network perturbations is to consider the compensatory activation or deactivation of system components. Here, we propose a complementary approach wherein interactions are instead modified to alter key regulatory functions and prevent the network damage from triggering a deregulatory cascade. Results We implement this approach in a Boolean dynamic framework, which has been shown to effectively model the behavior of biological regulatory and signaling networks. We show that the method can stabilize any single state (e.g., fixed point attractors or time-averaged representations of multi-state attractors) to be an attractor of the repaired network. We show that the approach is minimalistic in that few modifications are required to provide stability to a chosen attractor and specific in that interventions do not have undesired effects on the attractor. We apply the approach to random Boolean networks, and further show that the method can in some cases successfully repair synchronous limit cycles. We also apply the methodology to case studies from drought-induced signaling in plants and T-LGL leukemia and find that it is successful in both stabilizing desired behavior and in eliminating undesired outcomes. Code is made freely available through the software package BooleanNet. Conclusions The methodology introduced in this report offers a complementary way to manipulating node expression levels. A comprehensive approach to evaluating network manipulation should take an "all of the above" perspective; we anticipate that theoretical studies of interaction modification, coupled with empirical advances, will ultimately provide researchers with greater flexibility in influencing system behavior. PMID:24885780

A theoretical study of complexes formed between cations and curved aromatic systems: electrostatics does not always control cation-π interaction.

PubMed

Carrazana-García, Jorge A; Cabaleiro-Lago, Enrique M; Rodríguez-Otero, Jesús

2017-04-19

The present work studies the interaction of two extended curved π-systems (corannulene and sumanene) with various cations (sodium, potassium, ammonium, tetramethylammonium, guanidinium and imidazolium). Polyatomic cations are models of groups found in important biomolecules in which cation-π interaction plays a fundamental role. The results indicate an important size effect: with extended π systems and cations of the size of potassium and larger, dispersion is much more important than has been generally recognized for cation-π interactions. In most of the systems studied here, the stability of the cation-π complexes is the result of a balanced combination of electrostatic, induction and dispersion contributions. None of the systems studied here owes its stability to the electrostatic interaction more than 42%. Induction dominates stabilization in complexes with sodium, and in some of the potassium and ammonium complexes. In complexes with large cations and with flat cations dispersion is the major stabilizing contribution and can provide more than 50% of the stabilization energy. This implies that theoretical studies of the cation-π interaction involving large or even medium-size fragments require a level of calculation capable of properly modelling dispersion. The separation between the cation and the π system is another important factor to take into account, especially when the fragments of the cation-π complex are bound (for example, to a protein backbone) and cannot interact at the most favourable distance.

Assimilable organic carbon (AOC) variation in reclaimed water: Insight on biological stability evaluation and control for sustainable water reuse.

PubMed

Chen, Zhuo; Yu, Tong; Ngo, Huu Hao; Lu, Yun; Li, Guoqiang; Wu, Qianyuan; Li, Kuixiao; Bai, Yu; Liu, Shuming; Hu, Hong-Ying

2018-04-01

This review highlights the importance of conducting biological stability evaluation due to water reuse progression. Specifically, assimilable organic carbon (AOC) has been identified as a practical indicator for microbial occurrence and regrowth which ultimately influence biological stability. Newly modified AOC bioassays aimed for reclaimed water are introduced. Since elevated AOC levels are often detected after tertiary treatment, the review emphasizes that actions can be taken to either limit AOC levels prior to disinfection or conduct post-treatment (e.g. biological filtration) as a supplement to chemical oxidation based approaches (e.g. ozonation and chlorine disinfection). During subsequent distribution and storage, microbial community and possible microbial regrowth caused by complex interactions are discussed. It is suggested that microbial surveillance, AOC threshold values, real-time field applications and surrogate parameters could provide additional information. This review can be used to formulate regulatory plans and strategies, and to aid in deriving relevant control, management and operational guidance. Copyright © 2018 Elsevier Ltd. All rights reserved.

In-tank precipitation facility (ITP) and H-Tank Farm (HTF) geotechnical report, WSRC-TR-95-0057, Revision 0, Volume 5

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

NONE

A geotechnical study has been completed in H-Area for the In-Tank Precipitation Facility (ITP) and the balance of the H-Area Tank Farm (HTF) at the Savannah River Site (SRS) in South Carolina. The study consisted of subsurface field exploration, field and laboratory testing, and engineering analyses. The purpose of these investigations is to evaluate the overall stability of the H-Area tanks under static and dynamic conditions. The objectives of the study are to define the site-specific geological conditions at ITP and HTF, obtain engineering properties for the assessment of the stability of the native soils and embankment under static andmore » dynamic loads (i.e., slope stability, liquefaction potential, and potential settlements), and derive properties for soil-structure interaction studies. This document (Volume 5) contains the laboratory test results for the In-Tank Precipitation Facility (ITP) and H-Tank Farm (HTF) Geotechnical Report.« less

A native interactor scaffolds and stabilizes toxic ATAXIN-1 oligomers in SCA1

PubMed Central

Lasagna-Reeves, Cristian A; Rousseaux, Maxime WC; Guerrero-Muñoz, Marcos J; Park, Jeehye; Jafar-Nejad, Paymaan; Richman, Ronald; Lu, Nan; Sengupta, Urmi; Litvinchuk, Alexandra; Orr, Harry T; Kayed, Rakez; Zoghbi, Huda Y

2015-01-01

Recent studies indicate that soluble oligomers drive pathogenesis in several neurodegenerative proteinopathies, including Alzheimer and Parkinson disease. Curiously, the same conformational antibody recognizes different disease-related oligomers, despite the variations in clinical presentation and brain regions affected, suggesting that the oligomer structure might be responsible for toxicity. We investigated whether polyglutamine-expanded ATAXIN-1, the protein that underlies spinocerebellar ataxia type 1, forms toxic oligomers and, if so, what underlies their toxicity. We found that mutant ATXN1 does form oligomers and that oligomer levels correlate with disease progression in the Atxn1154Q/+ mice. Moreover, oligomeric toxicity, stabilization and seeding require interaction with Capicua, which is expressed at greater ratios with respect to ATXN1 in the cerebellum than in less vulnerable brain regions. Thus, specific interactors, not merely oligomeric structure, drive pathogenesis and contribute to regional vulnerability. Identifying interactors that stabilize toxic oligomeric complexes could answer longstanding questions about the pathogenesis of other proteinopathies. DOI: http://dx.doi.org/10.7554/eLife.07558.001 PMID:25988806

Kinesin expands and stabilizes the GDP-microtubule lattice

NASA Astrophysics Data System (ADS)

Peet, Daniel R.; Burroughs, Nigel J.; Cross, Robert A.

2018-05-01

Kinesin-1 is a nanoscale molecular motor that walks towards the fast-growing (plus) ends of microtubules, hauling molecular cargo to specific reaction sites in cells. Kinesin-driven transport is central to the self-organization of eukaryotic cells and shows great promise as a tool for nano-engineering1. Recent work hints that kinesin may also play a role in modulating the stability of its microtubule track, both in vitro2,3 and in vivo4, but the results are conflicting5-7 and the mechanisms are unclear. Here, we report a new dimension to the kinesin-microtubule interaction, whereby strong-binding state (adenosine triphosphate (ATP)-bound and apo) kinesin-1 motor domains inhibit the shrinkage of guanosine diphosphate (GDP) microtubules by up to two orders of magnitude and expand their lattice spacing by 1.6%. Our data reveal an unexpected mechanism by which the mechanochemical cycles of kinesin and tubulin interlock, and so allow motile kinesins to influence the structure, stability and mechanics of their microtubule track.

US-RERTR Advanced Fuel Development Plans : 1999.

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

Meyer, M. K.

1998-10-22

Twelve fuel alloys were included in the very-high-density RERTR-1 and RERTR-2 microplate irradiation experiments. Experience gained during fabrication and results from the post-irradiation examination of these fuels has allowed us to narrow the focus of our fuel development efforts in preparation for the next set of irradiation experiments. Specific technical problems in both the areas of fuel fabrication and irradiation performance remain to be addressed. Examples of these are powder fabrication, fuel phase gamma stability versus density, and fuel-matrix interaction. In order to more efficiently address metal alloy fuel performance issues, work will continue on establishing a theoretical basis formore » alloy stability and metal alloy dispersion fuel irradiation performance. Plans to address these fuel development issues in the coming year will be presented.« less

Role of Hydrophobic Clusters and Long-Range Contact Networks in the Folding of (α/β)8 Barrel Proteins

PubMed Central

Selvaraj, S.; Gromiha, M. Michael

2003-01-01

Analysis on the three dimensional structures of (α/β)8 barrel proteins provides ample light to understand the factors that are responsible for directing and maintaining their common fold. In this work, the hydrophobically enriched clusters are identified in 92% of the considered (α/β)8 barrel proteins. The residue segments with hydrophobic clusters have high thermal stability. Further, these clusters are formed and stabilized through long-range interactions. Specifically, a network of long-range contacts connects adjacent β-strands of the (α/β)8 barrel domain and the hydrophobic clusters. The implications of hydrophobic clusters and long-range networks in providing a feasible common mechanism for the folding of (α/β)8 barrel proteins are proposed. PMID:12609894

Special methods for aerodynamic-moment calculations from parachute FSI modeling

NASA Astrophysics Data System (ADS)

Takizawa, Kenji; Tezduyar, Tayfun E.; Boswell, Cody; Tsutsui, Yuki; Montel, Kenneth

2015-06-01

The space-time fluid-structure interaction (STFSI) methods for 3D parachute modeling are now at a level where they can bring reliable, practical analysis to some of the most complex parachute systems, such as spacecraft parachutes. The methods include the Deforming-Spatial-Domain/Stabilized ST method as the core computational technology, and a good number of special FSI methods targeting parachutes. Evaluating the stability characteristics of a parachute based on how the aerodynamic moment varies as a function of the angle of attack is one of the practical analyses that reliable parachute FSI modeling can deliver. We describe the special FSI methods we developed for this specific purpose and present the aerodynamic-moment data obtained from FSI modeling of NASA Orion spacecraft parachutes and Japan Aerospace Exploration Agency (JAXA) subscale parachutes.

The arrestin-1 finger loop interacts with two distinct conformations of active rhodopsin.

PubMed

Elgeti, Matthias; Kazmin, Roman; Rose, Alexander S; Szczepek, Michal; Hildebrand, Peter W; Bartl, Franz J; Scheerer, Patrick; Hofmann, Klaus Peter

2018-03-23

Signaling of the prototypical G protein-coupled receptor (GPCR) rhodopsin through its cognate G protein transducin (G t ) is quenched when arrestin binds to the activated receptor. Although the overall architecture of the rhodopsin/arrestin complex is known, many questions regarding its specificity remain unresolved. Here, using FTIR difference spectroscopy and a dual pH/peptide titration assay, we show that rhodopsin maintains certain flexibility upon binding the "finger loop" of visual arrestin (prepared as synthetic peptide ArrFL-1). We found that two distinct complexes can be stabilized depending on the protonation state of E3.49 in the conserved (D)ERY motif. Both complexes exhibit different interaction modes and affinities of ArrFL-1 binding. The plasticity of the receptor within the rhodopsin/ArrFL-1 complex stands in contrast to the complex with the C terminus of the G t α-subunit (GαCT), which stabilizes only one specific substate out of the conformational ensemble. However, G t α-subunit binding and both ArrFL-1-binding modes involve a direct interaction to conserved R3.50, as determined by site-directed mutagenesis. Our findings highlight the importance of receptor conformational flexibility and cytoplasmic proton uptake for modulation of rhodopsin signaling and thereby extend the picture provided by crystal structures of the rhodopsin/arrestin and rhodopsin/ArrFL-1 complexes. Furthermore, the two binding modes of ArrFL-1 identified here involve motifs of conserved amino acids, which indicates that our results may have elucidated a common modulation mechanism of class A GPCR-G protein/-arrestin signaling. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Interaction between Wnt/β-catenin and RAS-ERK pathways and an anti-cancer strategy via degradations of β-catenin and RAS by targeting the Wnt/β-catenin pathway.

PubMed

Jeong, Woo-Jeong; Ro, Eun Ji; Choi, Kang-Yell

2018-01-01

Aberrant activation of the Wnt/β-catenin and RAS-extracellular signal-regulated kinase (ERK) pathways play important roles in the tumorigenesis of many different types of cancer, most notably colorectal cancer (CRC). Genes for these two pathways, such as adenomatous polyposis coli ( APC ) and KRAS are frequently mutated in human CRC, and involved in the initiation and progression of the tumorigenesis, respectively. Moreover, recent studies revealed interaction of APC and KRAS mutations in the various stages of colorectal tumorigenesis and even in metastasis accompanying activation of the cancer stem cells (CSCs). A key event in the synergistic cooperation between Wnt/β-catenin and RAS-ERK pathways is a stabilization of both β-catenin and RAS especially mutant KRAS by APC loss, and pathological significance of this was indicated by correlation of increased β-catenin and RAS levels in human CRC where APC mutations occur as high as 90% of CRC patients. Together with the notion of the protein activity reduction by lowering its level, inhibition of both β-catenin and RAS especially by degradation could be a new ideal strategy for development of anti-cancer drugs for CRC. In this review, we will discuss interaction between the Wnt/β-catenin and RAS-ERK pathways in the colorectal tumorigenesis by providing the mechanism of RAS stabilization by aberrant activation of Wnt/β-catenin. We will also discuss our small molecular anti-cancer approach controlling CRC by induction of specific degradations of both β-catenin and RAS via targeting Wnt/β-catenin pathway especially for the KYA1797K, a small molecule specifically binding at the regulator of G-protein signaling (RGS)-domain of Axin.

Carboxyl Terminus of HSC70-interacting Protein (CHIP) Down-regulates NF-κB-inducing Kinase (NIK) and Suppresses NIK-induced Liver Injury*

PubMed Central

Jiang, Bijie; Shen, Hong; Chen, Zheng; Yin, Lei; Zan, Linsen; Rui, Liangyou

2015-01-01

Ser/Thr kinase NIK (NF-κB-inducing kinase) mediates the activation of the noncanonical NF-κB2 pathway, and it plays an important role in regulating immune cell development and liver homeostasis. NIK levels are extremely low in quiescent cells due to ubiquitin/proteasome-mediated degradation, and cytokines stimulate NIK activation through increasing NIK stability; however, regulation of NIK stability is not fully understood. Here we identified CHIP (carboxyl terminus of HSC70-interacting protein) as a new negative regulator of NIK. CHIP contains three N-terminal tetratricopeptide repeats (TPRs), a middle dimerization domain, and a C-terminal U-box. The U-box domain contains ubiquitin E3 ligase activity that promotes ubiquitination of CHIP-bound partners. We observed that CHIP bound to NIK via its TPR domain. In both HEK293 and primary hepatocytes, overexpression of CHIP markedly decreased NIK levels at least in part through increasing ubiquitination and degradation of NIK. Accordingly, CHIP suppressed NIK-induced activation of the noncanonical NF-κB2 pathway. CHIP also bound to TRAF3, and CHIP and TRAF3 acted coordinately to efficiently promote NIK degradation. The TPR but not the U-box domain was required for CHIP to promote NIK degradation. In mice, hepatocyte-specific overexpression of NIK resulted in liver inflammation and injury, leading to death, and liver-specific expression of CHIP reversed the detrimental effects of hepatic NIK. Our data suggest that CHIP/TRAF3/NIK interactions recruit NIK to E3 ligase complexes for ubiquitination and degradation, thus maintaining NIK at low levels. Defects in CHIP regulation of NIK may result in aberrant NIK activation in the liver, contributing to live injury, inflammation, and disease. PMID:25792747

Exploring the specific features of interfacial enzymology based on lipase studies.

PubMed

Aloulou, Ahmed; Rodriguez, Jorge A; Fernandez, Sylvie; van Oosterhout, Dirk; Puccinelli, Delphine; Carrière, Frédéric

2006-09-01

Many enzymes are active at interfaces in the living world (such as in the signaling processes at the surface of cell membranes, digestion of dietary lipids, starch and cellulose degradation, etc.), but fundamental enzymology remains largely focused on the interactions between enzymes and soluble substrates. The biochemical and kinetic characterization of lipolytic enzymes has opened up new paths of research in the field of interfacial enzymology. Lipases are water-soluble enzymes hydrolyzing insoluble triglyceride substrates, and studies on these enzymes have led to the development of specific interfacial kinetic models. Structure-function studies on lipases have thrown light on the interfacial recognition sites present in the molecular structure of these enzymes, the conformational changes occurring in the presence of lipids and amphiphiles, and the stability of the enzymes present at interfaces. The pH-dependent activity, substrate specificity and inhibition of these enzymes can all result from both "classical" interactions between a substrate or inhibitor and the active site, as well as from the adsorption of the enzymes at the surface of aggregated substrate particles such as oil drops, lipid bilayers or monomolecular lipid films. The adsorption step can provide an alternative target for improving substrate specificity and developing specific enzyme inhibitors. Several data obtained with gastric lipase, classical pancreatic lipase, pancreatic lipase-related protein 2 and phosphatidylserine-specific phospholipase A1 were chosen here to illustrate these specific features of interfacial enzymology.

Molecular biomimetics: GEPI-based biological routes to technology.

PubMed

Tamerler, Candan; Khatayevich, Dmitriy; Gungormus, Mustafa; Kacar, Turgay; Oren, E Emre; Hnilova, Marketa; Sarikaya, Mehmet

2010-01-01

In nature, the viability of biological systems is sustained via specific interactions among the tens of thousands of proteins, the major building blocks of organisms from the simplest single-celled to the most complex multicellular species. Biomolecule-material interaction is accomplished with molecular specificity and efficiency leading to the formation of controlled structures and functions at all scales of dimensional hierarchy. Through evolution, Mother Nature developed molecular recognition by successive cycles of mutation and selection. Molecular specificity of probe-target interactions, e.g., ligand-receptor, antigen-antibody, is always based on specific peptide molecular recognition. Using biology as a guide, we can now understand, engineer, and control peptide-material interactions and exploit them as a new design tool for novel materials and systems. We adapted the protocols of combinatorially designed peptide libraries, via both cell surface or phage display methods; using these we select short peptides with specificity to a variety of practical materials. These genetically engineered peptides for inorganics (GEPI) are then studied experimentally to establish their binding kinetics and surface stability. The bound peptide structure and conformations are interrogated both experimentally and via modeling, and self-assembly characteristics are tested via atomic force microscopy. We further engineer the peptide binding and assembly characteristics using a computational biomimetics approach where bioinformatics based peptide-sequence similarity analysis is developed to design higher generation function-specific peptides. The molecular biomimetic approach opens up new avenues for the design and utilization of multifunctional molecular systems in a wide-range of applications from tissue engineering, disease diagnostics, and therapeutics to various areas of nanotechnology where integration is required among inorganic, organic and biological materials. Here, we describe lessons from biology with examples of protein-mediated functional biological materials, explain how novel peptides can be designed with specific affinity to inorganic solids using evolutionary engineering approaches, give examples of their potential utilizations in technology and medicine, and, finally, provide a summary of challenges and future prospects. (c) 2010 Wiley Periodicals, Inc.

Evolution of RNA-Protein Interactions: Non-Specific Binding Led to RNA Splicing Activity of Fungal Mitochondrial Tyrosyl-tRNA Synthetases

PubMed Central

Lamech, Lilian T.; Mallam, Anna L.; Lambowitz, Alan M.

2014-01-01

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mtTyrRS; CYT-18 protein) evolved a new function as a group I intron splicing factor by acquiring the ability to bind group I intron RNAs and stabilize their catalytically active RNA structure. Previous studies showed: (i) CYT-18 binds group I introns by using both its N-terminal catalytic domain and flexibly attached C-terminal anticodon-binding domain (CTD); and (ii) the catalytic domain binds group I introns specifically via multiple structural adaptations that occurred during or after the divergence of Peziomycotina and Saccharomycotina. However, the function of the CTD and how it contributed to the evolution of splicing activity have been unclear. Here, small angle X-ray scattering analysis of CYT-18 shows that both CTDs of the homodimeric protein extend outward from the catalytic domain, but move inward to bind opposite ends of a group I intron RNA. Biochemical assays show that the isolated CTD of CYT-18 binds RNAs non-specifically, possibly contributing to its interaction with the structurally different ends of the intron RNA. Finally, we find that the yeast mtTyrRS, which diverged from Pezizomycotina fungal mtTyrRSs prior to the evolution of splicing activity, binds group I intron and other RNAs non-specifically via its CTD, but lacks further adaptations needed for group I intron splicing. Our results suggest a scenario of constructive neutral (i.e., pre-adaptive) evolution in which an initial non-specific interaction between the CTD of an ancestral fungal mtTyrRS and a self-splicing group I intron was “fixed” by an intron RNA mutation that resulted in protein-dependent splicing. Once fixed, this interaction could be elaborated by further adaptive mutations in both the catalytic domain and CTD that enabled specific binding of group I introns. Our results highlight a role for non-specific RNA binding in the evolution of RNA-binding proteins. PMID:25536042

Evolution of RNA-protein interactions: non-specific binding led to RNA splicing activity of fungal mitochondrial tyrosyl-tRNA synthetases.

PubMed

Lamech, Lilian T; Mallam, Anna L; Lambowitz, Alan M

2014-12-01

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mtTyrRS; CYT-18 protein) evolved a new function as a group I intron splicing factor by acquiring the ability to bind group I intron RNAs and stabilize their catalytically active RNA structure. Previous studies showed: (i) CYT-18 binds group I introns by using both its N-terminal catalytic domain and flexibly attached C-terminal anticodon-binding domain (CTD); and (ii) the catalytic domain binds group I introns specifically via multiple structural adaptations that occurred during or after the divergence of Peziomycotina and Saccharomycotina. However, the function of the CTD and how it contributed to the evolution of splicing activity have been unclear. Here, small angle X-ray scattering analysis of CYT-18 shows that both CTDs of the homodimeric protein extend outward from the catalytic domain, but move inward to bind opposite ends of a group I intron RNA. Biochemical assays show that the isolated CTD of CYT-18 binds RNAs non-specifically, possibly contributing to its interaction with the structurally different ends of the intron RNA. Finally, we find that the yeast mtTyrRS, which diverged from Pezizomycotina fungal mtTyrRSs prior to the evolution of splicing activity, binds group I intron and other RNAs non-specifically via its CTD, but lacks further adaptations needed for group I intron splicing. Our results suggest a scenario of constructive neutral (i.e., pre-adaptive) evolution in which an initial non-specific interaction between the CTD of an ancestral fungal mtTyrRS and a self-splicing group I intron was "fixed" by an intron RNA mutation that resulted in protein-dependent splicing. Once fixed, this interaction could be elaborated by further adaptive mutations in both the catalytic domain and CTD that enabled specific binding of group I introns. Our results highlight a role for non-specific RNA binding in the evolution of RNA-binding proteins.

Interdisciplinary optimum design. [of aerospace structures

NASA Technical Reports Server (NTRS)

Sobieszczanski-Sobieski, Jaroslaw; Haftka, Raphael T.

1986-01-01

Problems related to interdisciplinary interactions in the design of a complex engineering systems are examined with reference to aerospace applications. The interdisciplinary optimization problems examined include those dealing with controls and structures, materials and structures, control and stability, structure and aerodynamics, and structure and thermodynamics. The discussion is illustrated by the following specific applications: integrated aerodynamic/structural optimization of glider wing; optimization of an antenna parabolic dish structure for minimum weight and prescribed emitted signal gain; and a multilevel optimization study of a transport aircraft.

The concept of coupling impedance in the self-consistent plasma wake field excitation

NASA Astrophysics Data System (ADS)

Fedele, R.; Akhter, T.; De Nicola, S.; Migliorati, M.; Marocchino, A.; Massimo, F.; Palumbo, L.

2016-09-01

Within the framework of the Vlasov-Maxwell system of equations, we describe the self-consistent interaction of a relativistic charged-particle beam with the surroundings while propagating through a plasma-based acceleration device. This is done in terms of the concept of coupling (longitudinal) impedance in full analogy with the conventional accelerators. It is shown that also here the coupling impedance is a very useful tool for the Nyquist-type stability analysis. Examples of specific physical situations are finally illustrated.

Genomic control of patterning

PubMed Central

Peter, Isabelle S.; Davidson, Eric H.

2014-01-01

The development of multicellular organisms involves the partitioning of the organism into territories of cells of specific structure and function. The information for spatial patterning processes is directly encoded in the genome. The genome determines its own usage depending on stage and position, by means of interactions that constitute gene regulatory networks (GRNs). The GRN driving endomesoderm development in sea urchin embryos illustrates different regulatory strategies by which developmental programs are initiated, orchestrated, stabilized or excluded to define the pattern of specified territories in the developing embryo. PMID:19378258

[Intermolecular hydrogen bond between protein and flavonoid and its contribution to the stability of the flavonoids].

PubMed

Fang, Ru; Leng, Xiao-jing; Wu, Xia; Li, Qi; Hao, Rui-fang; Ren, Fa-zheng; Jing, Hao

2012-01-01

The interactions between three proteins (BSA, lysozyme and myoglobin) and three flavonoids (quercetin, kaempferol and rutin) were analyzed, using three-dimensional fluorescence spectrometry in combination with UV-Vis spectrometry and Fourier transform infrared (FTIR) spectroscopy. The stabilities of unbound flavonoids and protein-bound flavonoids were compared. The correlation between the interaction and stability was analyzed. The results showed that the hydrophobic interaction was the main binding code in all proteins and flavonoids systems. However, the hydrogen bond has been involved merely in the BSA system. The stability of all three flavonoids (quercetin, kaempferol and rutin) was improved by BSA. There was a great correlation between the hydrogen bonding and the stability of the flavonoids in the presence of BSA. It suggested that the protection of BSA on the flavonoids was due to the intermolecular hydrogen bonding between BSA and flavonoid, and the stronger hydrogen bonding resulted in more protection.

Direct colorimetric detection of unamplified pathogen DNA by dextrin-capped gold nanoparticles.

PubMed

Baetsen-Young, Amy M; Vasher, Matthew; Matta, Leann L; Colgan, Phil; Alocilja, Evangelyn C; Day, Brad

2018-03-15

The interaction between gold nanoparticles (AuNPs) and nucleic acids has facilitated a variety of diagnostic applications, with further diversification of synthesis match bio-applications while reducing biotoxicity. However, DNA interactions with unique surface capping agents have not been fully defined. Using dextrin-capped AuNPs (d-AuNPs), we have developed a novel unamplified genomic DNA (gDNA) nanosensor, exploiting dispersion and aggregation characteristics of d-AuNPs, in the presence of gDNA, for sequence-specific detection. We demonstrate that d-AuNPs are stable in a five-fold greater salt concentration than citrate-capped AuNPs and the d-AuNPs were stabilized by single stranded DNA probe (ssDNAp). However, in the elevated salt concentrations of the DNA detection assay, the target reactions were surprisingly further stabilized by the formation of a ssDNAp-target gDNA complex. The results presented herein lead us to propose a mechanism whereby genomic ssDNA secondary structure formation during ssDNAp-to-target gDNA binding enables d-AuNP stabilization in elevated ionic environments. Using the assay described herein, we were successful in detecting as little as 2.94 fM of pathogen DNA, and using crude extractions of a pathogen matrix, as few as 18 spores/µL. Copyright © 2017 Elsevier B.V. All rights reserved.

A diffuse interface model of grain boundary faceting

NASA Astrophysics Data System (ADS)

Abdeljawad, F.; Medlin, D. L.; Zimmerman, J. A.; Hattar, K.; Foiles, S. M.

2016-06-01

Interfaces, free or internal, greatly influence the physical properties and stability of materials microstructures. Of particular interest are the processes that occur due to anisotropic interfacial properties. In the case of grain boundaries (GBs) in metals, several experimental observations revealed that an initially flat GB may facet into hill-and-valley structures with well defined planes and corners/edges connecting them. Herein, we present a diffuse interface model that is capable of accounting for strongly anisotropic GB properties and capturing the formation of hill-and-valley morphologies. The hallmark of our approach is the ability to independently examine the various factors affecting GB faceting and subsequent facet coarsening. More specifically, our formulation incorporates higher order expansions to account for the excess energy due to facet junctions and their non-local interactions. As a demonstration of the modeling capability, we consider the Σ5 <001 > tilt GB in body-centered-cubic iron, where faceting along the {210} and {310} planes was experimentally observed. Atomistic calculations were utilized to determine the inclination-dependent GB energy, which was then used as an input in our model. Linear stability analysis and simulation results highlight the role of junction energy and associated non-local interactions on the resulting facet length scales. Broadly speaking, our modeling approach provides a general framework to examine the microstructural stability of polycrystalline systems with highly anisotropic GBs.

High Mobility Group A2 protects cancer cells against telomere dysfunction

PubMed Central

Natarajan, Suchitra; Begum, Farhana; Gim, Jeonga; Wark, Landon; Henderson, Dana; Davie, James R.

2016-01-01

The non-histone chromatin binding protein High Mobility Group AT-hook protein 2 (HMGA2) plays important roles in the repair and protection of genomic DNA in embryonic stem cells and cancer cells. Here we show that HMGA2 localizes to mammalian telomeres and enhances telomere stability in cancer cells. We present a novel interaction of HMGA2 with the key shelterin protein TRF2. We found that the linker (L1) region of HMGA2 contributes to this interaction but the ATI-L1-ATII molecular region of HMGA2 is required for strong interaction with TRF2. This interaction was independent of HMGA2 DNA-binding and did not require the TRF2 interacting partner RAP1 but involved the homodimerization and hinge regions of TRF2. HMGA2 retained TRF2 at telomeres and reduced telomere-dysfunction despite induced telomere stress. Silencing of HMGA2 resulted in (i) reduced binding of TRF2 to telomere DNA as observed by ChIP, (ii) increased telomere instability and (iii) the formation of telomere dysfunction-induced foci (TIF). This resulted in increased telomere aggregation, anaphase bridges and micronuclei. HMGA2 prevented ATM-dependent pTRF2T188 phosphorylation and attenuated signaling via the telomere specific ATM-CHK2-CDC25C DNA damage signaling axis. In summary, our data demonstrate a unique and novel role of HMGA2 in telomere protection and promoting telomere stability in cancer cells. This identifies HMGA2 as a new therapeutic target for the destabilization of telomeres in HMGA2+ cancer cells. PMID:26799419

Syntenin-1 Is a New Component of Tetraspanin-Enriched Microdomains: Mechanisms and Consequences of the Interaction of Syntenin-1 with CD63▿

PubMed Central

Latysheva, Nadya; Muratov, Gairat; Rajesh, Sundaresan; Padgett, Matthew; Hotchin, Neil A.; Overduin, Michael; Berditchevski, Fedor

2006-01-01

Tetraspanins are clustered in specific microdomains (named tetraspanin-enriched microdomains, or TERM) in the plasma membrane and regulate the functions of associated transmembrane receptors, including integrins and receptor tyrosine kinases. We have identified syntenin-1, a PDZ domain-containing protein, as a new component of TERM and show that syntenin-1 specifically interacts with the tetraspanin CD63. Detailed biochemical and heteronuclear magnetic resonance spectroscopy (NMR) studies have demonstrated that the interaction is mediated by the C-terminal cytoplasmic region of the tetraspanin and the PDZ domains of syntenin-1. Upon interaction, NMR chemical shift perturbations were predominantly localized to residues around the binding pocket of PDZ1, indicating a specific mode of recognition of the cytoplasmic tail of CD63. In addition, the C terminus of syntenin-1 has a stabilizing role in the CD63-syntenin-1 association, as deletion of the last 17 amino acids abolished the interaction. The CD63-syntenin-1 complex is abundant on the plasma membrane, and the elevated expression of the wild-type syntenin-1 slows down constitutive internalization of the tetraspanin. Furthermore, internalization of CD63 was completely blocked in cells expressing a syntenin-1 mutant lacking the first 100 amino acids. Previous results have shown that CD63 is internalized via AP-2-dependent mechanisms. Hence, our data indicate that syntenin-1 can counteract the AP-2-dependent internalization and identify this tandem PDZ protein as a new regulator of endocytosis. PMID:16908530

Characterization of the Staphylococcal enterotoxin A: Vβ receptor interaction using human receptor fragments engineered for high affinity.

PubMed

Sharma, P; Postel, S; Sundberg, E J; Kranz, D M

2013-12-01

Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection.

Characterization of the Staphylococcal enterotoxin A: Vβ receptor interaction using human receptor fragments engineered for high affinity

PubMed Central

Sharma, P.; Postel, S.; Sundberg, E.J.; Kranz, D.M.

2013-01-01

Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection. PMID:24167300

Modified nucleotides reveal the indirect role of the central base pairs in stabilizing the lac repressor-operator complex.

PubMed Central

Zhang, X; Gottlieb, P A

1995-01-01

Guanine residues in the lac operator were replaced by 2-aminopurine or purine analogues, pairing the modified nucleotides with C. The observed equilibrium dissociation constants for lac repressor binding to substituted operators were measured in 10 mM Tris, 150 mM KCl, 0.1 mM EDTA, 0.1 mM DTE, pH 7.6 at 25 degrees C. These measurements revealed five positions that destabilized the complex when substituted with either analogue. Two positions, which are related by a 2-fold symmetry, are in the major groove of the operator thought to directly interact with the protein. Three sites were in the central region of the operator. A purine analogue at a sixth site perturbed the local DNA structure and destabilized the complex. Alkylation interference experiments of the 2-aminopurine substituted operators demonstrated that, of the five affected, two substitutions displayed altered phosphate interference patterns at the phosphate adjacent to the substituted base. For these operators, complex formation was measured in different concentrations of KCl to assess the contribution of counterion release to the bimolecular process. The results indicated that both complexes were similar to wild-type, although minor changes were observed. The Kobs of the complex was then measured when 2-aminopurine or purine analogues were paired with uracil nucleotide, a base pair that serves to stabilize the DNA. The introduction of the new base pairs revealed two effects on the bimolecular interaction. For those operator sites that are thought to perturb the interaction directly, the affinity of the complex was weakened to levels observed for the singly-substituted operators. In contrast, the nucleotides of 2-aminopurine paired with uracil positioned in the central region of the operator served to enhance the stability of the complex. The purine-uracil base pair substitution on the other hand had a significant destabilizing effect on the interaction. We propose that the central base pairs modulate binding of the complex by altering the intrinsic properties of the DNA. Two specific attributes are required to achieve the lowest free energy of interaction. The DNA must have two interstrand hydrogen bonds to stabilize the duplex and it must have properties associated with directional bending or unwinding. This analysis does not rule out contributions by direct interactions between the protein and the central region of the operator but underscores how indirect effects play a major role in complex formation in this system. Images PMID:7784203

Uncovering Specific Electrostatic Interactions in the Denatured States of Proteins

PubMed Central

Shen, Jana K.

2010-01-01

The stability and folding of proteins are modulated by energetically significant interactions in the denatured state that is in equilibrium with the native state. These interactions remain largely invisible to current experimental techniques, however, due to the sparse population and conformational heterogeneity of the denatured-state ensemble under folding conditions. Molecular dynamics simulations using physics-based force fields can in principle offer atomistic details of the denatured state. However, practical applications are plagued with the lack of rigorous means to validate microscopic information and deficiencies in force fields and solvent models. This study presents a method based on coupled titration and molecular dynamics sampling of the denatured state starting from the extended sequence under native conditions. The resulting denatured-state pKas allow for the prediction of experimental observables such as pH- and mutation-induced stability changes. I show the capability and use of the method by investigating the electrostatic interactions in the denatured states of wild-type and K12M mutant of NTL9 protein. This study shows that the major errors in electrostatics can be identified by validating the titration properties of the fragment peptides derived from the sequence of the intact protein. Consistent with experimental evidence, our simulations show a significantly depressed pKa for Asp8 in the denatured state of wild-type, which is due to a nonnative interaction between Asp8 and Lys12. Interestingly, the simulation also shows a nonnative interaction between Asp8 and Glu48 in the denatured state of the mutant. I believe the presented method is general and can be applied to extract and validate microscopic electrostatics of the entire folding energy landscape. PMID:20682271

Cryo-EM structure of the spinach chloroplast ribosome reveals the location of plastid-specific ribosomal proteins and extensions

PubMed Central

Graf, Michael; Arenz, Stefan; Huter, Paul; Dönhöfer, Alexandra; Nováček, Jiří

2017-01-01

Abstract Ribosomes are the protein synthesizing machines of the cell. Recent advances in cryo-EM have led to the determination of structures from a variety of species, including bacterial 70S and eukaryotic 80S ribosomes as well as mitoribosomes from eukaryotic mitochondria, however, to date high resolution structures of plastid 70S ribosomes have been lacking. Here we present a cryo-EM structure of the spinach chloroplast 70S ribosome, with an average resolution of 5.4 Å for the small 30S subunit and 3.6 Å for the large 50S ribosomal subunit. The structure reveals the location of the plastid-specific ribosomal proteins (RPs) PSRP1, PSRP4, PSRP5 and PSRP6 as well as the numerous plastid-specific extensions of the RPs. We discover many features by which the plastid-specific extensions stabilize the ribosome via establishing additional interactions with surrounding ribosomal RNA and RPs. Moreover, we identify a large conglomerate of plastid-specific protein mass adjacent to the tunnel exit site that could facilitate interaction of the chloroplast ribosome with the thylakoid membrane and the protein-targeting machinery. Comparing the Escherichia coli 70S ribosome with that of the spinach chloroplast ribosome provides detailed insight into the co-evolution of RP and rRNA. PMID:27986857

DbPTM 3.0: an informative resource for investigating substrate site specificity and functional association of protein post-translational modifications.

PubMed

Lu, Cheng-Tsung; Huang, Kai-Yao; Su, Min-Gang; Lee, Tzong-Yi; Bretaña, Neil Arvin; Chang, Wen-Chi; Chen, Yi-Ju; Chen, Yu-Ju; Huang, Hsien-Da

2013-01-01

Protein modification is an extremely important post-translational regulation that adjusts the physical and chemical properties, conformation, stability and activity of a protein; thus altering protein function. Due to the high throughput of mass spectrometry (MS)-based methods in identifying site-specific post-translational modifications (PTMs), dbPTM (http://dbPTM.mbc.nctu.edu.tw/) is updated to integrate experimental PTMs obtained from public resources as well as manually curated MS/MS peptides associated with PTMs from research articles. Version 3.0 of dbPTM aims to be an informative resource for investigating the substrate specificity of PTM sites and functional association of PTMs between substrates and their interacting proteins. In order to investigate the substrate specificity for modification sites, a newly developed statistical method has been applied to identify the significant substrate motifs for each type of PTMs containing sufficient experimental data. According to the data statistics in dbPTM, >60% of PTM sites are located in the functional domains of proteins. It is known that most PTMs can create binding sites for specific protein-interaction domains that work together for cellular function. Thus, this update integrates protein-protein interaction and domain-domain interaction to determine the functional association of PTM sites located in protein-interacting domains. Additionally, the information of structural topologies on transmembrane (TM) proteins is integrated in dbPTM in order to delineate the structural correlation between the reported PTM sites and TM topologies. To facilitate the investigation of PTMs on TM proteins, the PTM substrate sites and the structural topology are graphically represented. Also, literature information related to PTMs, orthologous conservations and substrate motifs of PTMs are also provided in the resource. Finally, this version features an improved web interface to facilitate convenient access to the resource.

Microstructure and rheology of particle stabilized emulsions: Effects of particle shape and inter-particle interactions.

PubMed

Katepalli, Hari; John, Vijay T; Tripathi, Anubhav; Bose, Arijit

2017-01-01

Using fumed and spherical silica particles of similar hydrodynamic size, we investigated the effects of particle shape and inter-particle interactions on the formation, stability and rheology of bromohexadecane-in-water Pickering emulsions. The interparticle interactions were varied from repulsive to attractive by modifying the salt concentration in the aqueous phase. Optical microscope images revealed smaller droplet sizes for the fumed silica stabilized emulsions. All the emulsions remained stable for several weeks. Cryo-SEM images of the emulsion droplets showed a hexagonally packed single layer of particles at oil-water interfaces in emulsions stabilized with silica spheres, irrespective of the nature of the inter-particle interactions. Thus, entropic, excluded volume interactions dominate the fate of spherical particles at oil-water interfaces. On the other hand, closely packed layers of particles were observed at oil-water interfaces for the fumed silica stabilized emulsions for both attractive and repulsive interparticle interactions. At the high salt concentrations, attractive inter-particles interactions led to aggregation of fumed silica particles, and multiple layers of these particles were then observed on the droplet surfaces. A network of fumed silica particles was also observed between the emulsion droplets, suggesting that enthalpic interactions are responsible for the determining particle configurations at oil-water interfaces as well as in the aqueous phase. Steady shear viscosity measurements over a range of shear stresses, as well as oscillatory shear measurements at 1Hz confirm the presence of a network in fumed silica suspensions and emulsions, and the lack of such a network when spherical particles are used. The fractal structure of fumed silica leads to several contact points and particle interlocking in the water as well as on the bromohexadecane-water interfaces, with corresponding effects on the structure and rheology of the emulsions. The attenuation of droplet motion due to the formation of a particle network can be exploited for stabilizing emulsions and for modulating their rheology. Copyright © 2016 Elsevier Inc. All rights reserved.

Friends or foes? Emerging insights from fungal interactions with plants.

PubMed

Zeilinger, Susanne; Gupta, Vijai K; Dahms, Tanya E S; Silva, Roberto N; Singh, Harikesh B; Upadhyay, Ram S; Gomes, Eriston Vieira; Tsui, Clement Kin-Ming; Nayak S, Chandra

2016-03-01

Fungi interact with plants in various ways, with each interaction giving rise to different alterations in both partners. While fungal pathogens have detrimental effects on plant physiology, mutualistic fungi augment host defence responses to pathogens and/or improve plant nutrient uptake. Tropic growth towards plant roots or stomata, mediated by chemical and topographical signals, has been described for several fungi, with evidence of species-specific signals and sensing mechanisms. Fungal partners secrete bioactive molecules such as small peptide effectors, enzymes and secondary metabolites which facilitate colonization and contribute to both symbiotic and pathogenic relationships. There has been tremendous advancement in fungal molecular biology, omics sciences and microscopy in recent years, opening up new possibilities for the identification of key molecular mechanisms in plant-fungal interactions, the power of which is often borne out in their combination. Our fragmentary knowledge on the interactions between plants and fungi must be made whole to understand the potential of fungi in preventing plant diseases, improving plant productivity and understanding ecosystem stability. Here, we review innovative methods and the associated new insights into plant-fungal interactions. © FEMS 2015.

Friends or foes? Emerging insights from fungal interactions with plants

PubMed Central

Zeilinger, Susanne; Gupta, Vijai K.; Dahms, Tanya E. S.; Silva, Roberto N.; Singh, Harikesh B.; Upadhyay, Ram S.; Gomes, Eriston Vieira; Tsui, Clement Kin-Ming; Nayak S, Chandra

2015-01-01

Fungi interact with plants in various ways, with each interaction giving rise to different alterations in both partners. While fungal pathogens have detrimental effects on plant physiology, mutualistic fungi augment host defence responses to pathogens and/or improve plant nutrient uptake. Tropic growth towards plant roots or stomata, mediated by chemical and topographical signals, has been described for several fungi, with evidence of species-specific signals and sensing mechanisms. Fungal partners secrete bioactive molecules such as small peptide effectors, enzymes and secondary metabolites which facilitate colonization and contribute to both symbiotic and pathogenic relationships. There has been tremendous advancement in fungal molecular biology, omics sciences and microscopy in recent years, opening up new possibilities for the identification of key molecular mechanisms in plant–fungal interactions, the power of which is often borne out in their combination. Our fragmentary knowledge on the interactions between plants and fungi must be made whole to understand the potential of fungi in preventing plant diseases, improving plant productivity and understanding ecosystem stability. Here, we review innovative methods and the associated new insights into plant–fungal interactions. PMID:26591004

Interactions between drugs and polymers influencing hot melt extrusion.

PubMed

Li, Yongcheng; Pang, Huishi; Guo, Zhefei; Lin, Ling; Dong, Yixuan; Li, Ge; Lu, Ming; Wu, Chuangbin

2014-02-01

Hot melt extrusion (HME) as a technique for producing amorphous solid dispersion (ASD) has been widely used in pharmaceutical research. The biggest challenge for the application of HME is the thermal degradation of drug, poor physical stability of ASD and precipitation of drug during dissolution. Interactions between drugs and polymers may play an important role in overcoming these barriers. In this review, influence of drug-polymer interactions on HME and the methods for characterizing the drug-polymer interactions were reviewed. Strong drug-polymer interactions, especially ionic interactions and hydrogen bonds, are helpful to improving the thermal stability of drug during HME, enhancing the physical stability of ASD during storage and maintaining supersaturated solution after dissolution in gastrointestinal tract. The interactions can be quantitatively and qualitatively characterized by many analysing methods. As many factors collectively determine the properties of HME products, drug-polymer interactions play an extremely important role. However, the action mechanisms of drug-polymer interactions need intensive investigation to provide more useful information for optimizing the formulation and the process parameters of HME. © 2013 Royal Pharmaceutical Society.

Stabilizing liquid drops of arbitrary shape by the interfacial jamming of nanoparticles

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

Russell, Thomas P.; Cui, Mengmeng; Emrick, Todd

A stabilized assembly including a first liquid phase of non-spherical droplets in a second liquid phase, wherein the second liquid phase is immiscible with the first phase, and nanoparticle surfactants assembled at an interface of the non-spherical droplets and the second phase is disclosed. The nanoparticle surfactants include nanoparticles and end-functionalized polymers that can interact through ligand type interactions, and the first phase is stabilized by a disordered, jammed layer of nanoparticle surfactants. A method of preparing a stabilized assembly is also disclosed.

Laboratory sample stability. Is it possible to define a consensus stability function? An example of five blood magnitudes.

PubMed

Gómez Rioja, Rubén; Martínez Espartosa, Débora; Segovia, Marta; Ibarz, Mercedes; Llopis, María Antonia; Bauça, Josep Miquel; Marzana, Itziar; Barba, Nuria; Ventura, Monserrat; García Del Pino, Isabel; Puente, Juan José; Caballero, Andrea; Gómez, Carolina; García Álvarez, Ana; Alsina, María Jesús; Álvarez, Virtudes

2018-05-05

The stability limit of an analyte in a biological sample can be defined as the time required until a measured property acquires a bias higher than a defined specification. Many studies assessing stability and presenting recommendations of stability limits are available, but differences among them are frequent. The aim of this study was to classify and to grade a set of bibliographic studies on the stability of five common blood measurands and subsequently generate a consensus stability function. First, a bibliographic search was made for stability studies for five analytes in blood: alanine aminotransferase (ALT), glucose, phosphorus, potassium and prostate specific antigen (PSA). The quality of every study was evaluated using an in-house grading tool. Second, the different conditions of stability were uniformly defined and the percent deviation (PD%) over time for each analyte and condition were scattered while unifying studies with similar conditions. From the 37 articles considered as valid, up to 130 experiments were evaluated and 629 PD% data were included (106 for ALT, 180 for glucose, 113 for phosphorus, 145 for potassium and 85 for PSA). Consensus stability equations were established for glucose, potassium, phosphorus and PSA, but not for ALT. Time is the main variable affecting stability in medical laboratory samples. Bibliographic studies differ in recommedations of stability limits mainly because of different specifications for maximum allowable error. Definition of a consensus stability function in specific conditions can help laboratories define stability limits using their own quality specifications.

Recent Methods for Purification and Structure Determination of Oligonucleotides.

PubMed

Zhang, Qiulong; Lv, Huanhuan; Wang, Lili; Chen, Man; Li, Fangfei; Liang, Chao; Yu, Yuanyuan; Jiang, Feng; Lu, Aiping; Zhang, Ge

2016-12-18

Aptamers are single-stranded DNA or RNA oligonucleotides that can interact with target molecules through specific three-dimensional structures. The excellent features, such as high specificity and affinity for target proteins, small size, chemical stability, low immunogenicity, facile chemical synthesis, versatility in structural design and engineering, and accessible for site-specific modifications with functional moieties, make aptamers attractive molecules in the fields of clinical diagnostics and biopharmaceutical therapeutics. However, difficulties in purification and structural identification of aptamers remain a major impediment to their broad clinical application. In this mini-review, we present the recently attractive developments regarding the purification and identification of aptamers. We also discuss the advantages, limitations, and prospects for the major methods applied in purifying and identifying aptamers, which could facilitate the application of aptamers.

Enhanced stability of a chimeric hepatitis B core antigen virus-like-particle (HBcAg-VLP) by a C-terminal linker-hexahistidine-peptide.

PubMed

Schumacher, Jens; Bacic, Tijana; Staritzbichler, René; Daneschdar, Matin; Klamp, Thorsten; Arnold, Philipp; Jägle, Sabrina; Türeci, Özlem; Markl, Jürgen; Sahin, Ugur

2018-04-13

Virus-like-particles (VLPs) are attractive nanoparticulate scaffolds for broad applications in material/biological sciences and medicine. Prior their functionalization, specific adaptations have to be carried out. These adjustments frequently lead to disordered particles, but the particle integrity is an essential factor for the VLP suitability. Therefore, major requirements for particle stabilization exist. The objective of this study was to evaluate novel stabilizing elements for functionalized chimeric hepatitis B virus core antigen virus-like particles (HBcAg-VLP), with beneficial characteristics for vaccine development, imaging or delivery. The effects of a carboxy-terminal polyhistidine-peptide and an intradimer disulfide-bridge on the stability of preclinically approved chimeric HBcAg-VLPs were assessed. We purified recombinant chimeric HBcAg-VLPs bearing different modified C-termini and compared their physical and chemical particle stability by quantitative protein-biochemical and biophysical techniques. We observed lower chemical resistance of T = 3- compared to T = 4-VLP (triangulation number) capsids and profound impairment of accessibility of hexahistidine-peptides in assembled VLPs. Histidines attached to the C-terminus were associated with superior mechanical and/or chemical particle stability depending on the number of histidine moieties. A molecular modeling approach based on cryo-electron microscopy and biolayer interferometry revealed the underlying structural mechanism for the strengthening of the integrity of VLPs. Interactions triggering capsid stabilization occur on a highly conserved residue on the basis of HBcAg-monomers as well as on hexahistidine-peptides of adjacent monomers. This new stabilization mechanism appears to mimic an evolutionary conserved stabilization concept for hepadnavirus core proteins. These findings establish the genetically simply transferable C-terminal polyhistidine-peptide as a general stabilizing element for chimeric HBcAg-VLPs to increase their suitability.

Synthesis of water soluble glycine capped silver nanoparticles and their surface selective interaction

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

Agasti, Nityananda, E-mail: nnagasti@gmail.com; Singh, Vinay K.; Kaushik, N.K.

Highlights: • Synthesis of water soluble silver nanoparticles at ambient reaction conditions. • Glycine as stabilizing agent for silver nanoparticles. • Surface selective interaction of glycine with silver nanoparticles. • Glycine concentration influences crystalinity and optical property of silver nanoparticles. - Abstract: Synthesis of biocompatible metal nanoparticles has been an area of significant interest because of their wide range of applications. In the present study, we have successfully synthesized water soluble silver nanoparticles assisted by small amino acid glycine. The method is primarily based on reduction of AgNO{sub 3} with NaBH{sub 4} in aqueous solution under atmospheric air in themore » presence of glycine. UV–vis spectroscopy, transmission electron microscopy (TEM), X–ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, thermogravimetry (TG) and differential thermal analysis (DTA) techniques used for characterization of resulting silver nanoparticles demonstrated that, glycine is an effective capping agent to stabilize silver nanoparticles. Surface selective interaction of glycine on (1 1 1) face of silver nanoparticles has been investigated. The optical property and crystalline behavior of silver nanoparticles were found to be sensitive to concentration of glycine. X–ray diffraction studies ascertained the phase specific interaction of glycine on silver nanoparticles. Silver nanoparticles synthesized were of diameter 60 nm. We thus demonstrated an efficient synthetic method for synthesis of water soluble silver nanoparticles capped by amino acid under mild reaction conditions with excellent reproducibility.« less

Agglomeration, colloidal stability, and magnetic separation of magnetic nanoparticles: collective influences on environmental engineering applications

NASA Astrophysics Data System (ADS)

Yeap, Swee Pin; Lim, JitKang; Ooi, Boon Seng; Ahmad, Abdul Latif

2017-11-01

Magnetic nanoparticles (MNPs) which exhibit magnetic and catalytic bifunctionalities have been widely accepted as one of the most promising nanoagents used in water purification processes. However, due to the magnetic dipole-dipole interaction, MNPs can easily lose their colloidal stability and tend to agglomerate. Thus, it is necessary to enhance their colloidal stability in order to maintain the desired high specific surface area. Meanwhile, in order to successfully utilize MNPs for environmental engineering applications, an effective magnetic separation technology has to be developed. This step is to ensure the MNPs that have been used for pollutant removal can be fully reharvested back. Unfortunately, it was recently highlighted that there exists a conflicting role between colloidal stability and magnetic separability of the MNPs, whereby the more colloidally stable the particle is, the harder for it to be magnetically separated. In other words, attaining a win-win scenario in which the MNPs possess both good colloidal stability and fast magnetic separation rate becomes challenging. Such phenomenon has to be thoroughly understood as the colloidal stability and the magnetic separability of MNPs play a pivotal role on affecting their effective implementation in water purification processes. Accordingly, it is the aim of this paper to provide reviews on (i) the colloidal stability and (ii) the magnetic separation of MNPs, as well as to provide insights on (iii) their conflicting relationship based on recent research findings. [Figure not available: see fulltext.

Epistasis in protein evolution

PubMed Central

Starr, Tyler N.

2016-01-01

Abstract The structure, function, and evolution of proteins depend on physical and genetic interactions among amino acids. Recent studies have used new strategies to explore the prevalence, biochemical mechanisms, and evolutionary implications of these interactions—called epistasis—within proteins. Here we describe an emerging picture of pervasive epistasis in which the physical and biological effects of mutations change over the course of evolution in a lineage‐specific fashion. Epistasis can restrict the trajectories available to an evolving protein or open new paths to sequences and functions that would otherwise have been inaccessible. We describe two broad classes of epistatic interactions, which arise from different physical mechanisms and have different effects on evolutionary processes. Specific epistasis—in which one mutation influences the phenotypic effect of few other mutations—is caused by direct and indirect physical interactions between mutations, which nonadditively change the protein's physical properties, such as conformation, stability, or affinity for ligands. In contrast, nonspecific epistasis describes mutations that modify the effect of many others; these typically behave additively with respect to the physical properties of a protein but exhibit epistasis because of a nonlinear relationship between the physical properties and their biological effects, such as function or fitness. Both types of interaction are rampant, but specific epistasis has stronger effects on the rate and outcomes of evolution, because it imposes stricter constraints and modulates evolutionary potential more dramatically; it therefore makes evolution more contingent on low‐probability historical events and leaves stronger marks on the sequences, structures, and functions of protein families. PMID:26833806

Centromere Protein (CENP)-W Interacts with Heterogeneous Nuclear Ribonucleoprotein (hnRNP) U and May Contribute to Kinetochore-Microtubule Attachment in Mitotic Cells

PubMed Central

Chun, Younghwa; Kim, Raehyung; Lee, Soojin

2016-01-01

Background Recent studies have shown that heterogeneous nuclear ribonucleoprotein U (hnRNP U), a component of the hnRNP complex, contributes to stabilize the kinetochore-microtubule interaction during mitosis. CENP-W was identified as an inner centromere component that plays crucial roles in the formation of a functional kinetochore complex. Results We report that hnRNP U interacts with CENP-W, and the interaction between hnRNP U and CENP-W mutually increased each other’s protein stability by inhibiting the proteasome-mediated degradation. Further, their co-localization was observed chiefly in the nuclear matrix region and at the microtubule-kinetochore interface during interphase and mitosis, respectively. Both microtubule-stabilizing and microtubule-destabilizing agents significantly decreased the protein stability of CENP-W. Furthermore, loss of microtubules and defects in microtubule organization were observed in CENP-W-depleted cells. Conclusion Our data imply that CENP-W plays an important role in the attachment and interaction between microtubules and kinetochore during mitosis. PMID:26881882

Entropy and enthalpy of interaction between amino acid side chains in nanopores

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

Vaitheeswaran, S., E-mail: vaithee05@gmail.com; Thirumalai, D.; Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742

2014-12-14

Understanding the stabilities of proteins in nanopores requires a quantitative description of confinement induced interactions between amino acid side chains. We use molecular dynamics simulations to study the nature of interactions between the side chain pairs ALA-PHE, SER-ASN, and LYS-GLU in bulk water and in water-filled nanopores. The temperature dependence of the bulk solvent potentials of mean force and the interaction free energies in cylindrical and spherical nanopores is used to identify the corresponding entropic and enthalpic components. The entropically stabilized hydrophobic interaction between ALA and PHE in bulk water is enthalpically dominated upon confinement depending on the relative orientationsmore » between the side chains. In the case of SER-ASN, hydrogen bonded configurations that are similar in bulk water are thermodynamically distinct in a cylindrical pore, thus making rotamer distributions different from those in the bulk. Remarkably, salt bridge formation between LYS-GLU is stabilized by entropy in contrast to the bulk. Implications of our findings for confinement-induced alterations in protein stability are briefly outlined.« less

Specific anions effects of on the stability of azurin in ice.

PubMed

Strambini, Giovanni B; Gonnelli, Margherita

2008-08-21

This investigation represents a first attempt to gain a quantitative estimate of the effects of the anions sulfate, citrate, acetate, chloride and thiocyanate on the thermodynamic stability (DeltaG degrees) of a model globular protein in ice at -15 degrees C. The method, based on guanidinium chloride denaturation of the azurin mutant C112S from Pseudomonas aeruginosa, distinguishes between the effects of cooling to subfreezing temperatures from those induced specifically by the formation of a solid ice phase. The results confirm that, both in liquid and frozen states, kosmotropes (sulfate, citrate and acetate) increase significantly protein stability, relative to chloride, whereas the chaotrope thiocyanate decreases it. Throughout, their stabilizing efficacy was found to rank according to the Hofmeister series, sulfate>citrate>acetate>chloride>thiocyanate, although the magnitude of Delta(DeltaG degrees) exhibited a distinct sensitivity among the anions to low temperature and to ice formation. In the liquid state, lowering the temperature from +20 to -15 degreesC weakens considerably the stabilizing efficacy of the organic anions citrate and acetate. Among the anions sulfate stands out as the only strong stabilizer at subfreezing temperatures while SCN- becomes an even stronger denaturant. Freezing of the solution in the presence the "neutral" salt NaCl destabilizes the protein, DeltaG degrees progressively decreasing up to 3-4 kcal/mol as the fraction of liquid water in equilibrium with ice (VL) is reduced to less than 1%. Kosmotropes do attenuate the decrease in protein stability in ice although in the case of citrate and acetate, their efficacy diminishes sharply as the liquid fraction shrinks to below 2.7%. On the contrary, sulfate is remarkable for it maintains constantly high the stability of azurin in liquid and frozen solutions, down to the smallest VL (0.5%) examined. Throughout, the reduction in DeltaG degrees caused by the solidification of water correlates with the decrease in the denaturant m value, an indirect indication that protein-ice interactions generally lead to partial unfolding of the native state. It is proposed that binding of the kosmotropes to the ice interface may inhibit protein adsorption to the solid phase and thereby counter the ice perturbation.

Kinetic Contributions to Gating by Interactions Unique to N-methyl-d-aspartate (NMDA) Receptors*

PubMed Central

Borschel, William F.; Cummings, Kirstie A.; Tindell, LeeAnn K.; Popescu, Gabriela K.

2015-01-01

Among glutamate-gated channels, NMDA receptors produce currents that subside with unusually slow kinetics, and this feature is essential to the physiology of central excitatory synapses. Relative to the homologous AMPA and kainate receptors, NMDA receptors have additional intersubunit contacts in the ligand binding domain that occur at both conserved and non-conserved sites. We examined GluN1/GluN2A single-channel currents with kinetic analyses and modeling to probe these class-specific intersubunit interactions for their role in glutamate binding and receptor gating. We found that substitutions that eliminate such interactions at non-conserved sites reduced stationary gating, accelerated deactivation, and imparted sensitivity to aniracetam, an AMPA receptor-selective positive modulator. Abolishing unique contacts at conserved sites also reduced stationary gating and accelerated deactivation. These results show that contacts specific to NMDA receptors, which brace the heterodimer interface within the ligand binding domain, stabilize actively gating receptor conformations and result in longer bursts and slower deactivations. They support the view that the strength of the heterodimer interface modulates gating in both NMDA and non-NMDA receptors and that unique interactions at this interface are responsible in part for basic differences between the kinetics of NMDA and non-NMDA currents at glutamatergic synapses. PMID:26370091

User's manual for MMLE3, a general FORTRAN program for maximum likelihood parameter estimation

NASA Technical Reports Server (NTRS)

Maine, R. E.; Iliff, K. W.

1980-01-01

A user's manual for the FORTRAN IV computer program MMLE3 is described. It is a maximum likelihood parameter estimation program capable of handling general bilinear dynamic equations of arbitrary order with measurement noise and/or state noise (process noise). The theory and use of the program is described. The basic MMLE3 program is quite general and, therefore, applicable to a wide variety of problems. The basic program can interact with a set of user written problem specific routines to simplify the use of the program on specific systems. A set of user routines for the aircraft stability and control derivative estimation problem is provided with the program.

Contribution of cation-π interactions to the stability of Sm/LSm oligomeric assemblies.

PubMed

Mucić, Ivana D; Nikolić, Milan R; Stojanović, Srđan Đ

2015-07-01

In this work, we have analyzed the influence of cation-π interactions to the stability of Sm/LSm assemblies and their environmental preferences. The number of interactions formed by arginine is higher than lysine in the cationic group, while histidine is comparatively higher than phenylalanine and tyrosine in the π group. Arg-Tyr interactions are predominant among the various pairs analyzed. The furcation level of multiple cation-π interactions is much higher than that of single cation-π interactions in Sm/LSm interfaces. We have found hot spot residues forming cation-π interactions, and hot spot composition is similar for all aromatic residues. The Arg-Phe pair has the strongest interaction energy of -8.81 kcal mol(-1) among all the possible pairs of amino acids. The extent of burial of the residue side-chain correlates with the ΔΔG of binding for residues in the core and also for hot spot residues cation-π bonded across the interface. Secondary structure of the cation-π residues shows that Arg and Lys preferred to be in strand. Among the π residues, His prefers to be in helix, Phe prefers to be in turn, and Tyr prefers to be in strand. Stabilization centers for these proteins showed that all the five residues found in cation-π interactions are important in locating one or more of such centers. More than 50 % of the cation-π interacting residues are highly conserved. It is likely that the cation-π interactions contribute significantly to the overall stability of Sm/LSm proteins.

Interaction between drilled shaft and mechanically stabilized earth (MSE) wall : technical report.

DOT National Transportation Integrated Search

2017-04-01

Drilled shafts are being constructed within the reinforced zone of mechanically stabilized earth (MSE) walls especially in the case of overpass bridges where the drilled shafts carry the bridge deck or traffic signs. The interaction between the drill...

Magic tilt angle for stabilizing two-dimensional solitons by dipole-dipole interactions

NASA Astrophysics Data System (ADS)

Chen, Xing-You; Chuang, You-Lin; Lin, Chun-Yan; Wu, Chien-Ming; Li, Yongyao; Malomed, Boris A.; Lee, Ray-Kuang

2017-10-01

In the framework of the Gross-Pitaevskii equation, we study the formation and stability of effectively two-dimensional solitons in dipolar Bose-Einstein condensates (BECs), with dipole moments polarized at an arbitrary angle θ relative to the direction normal to the system's plane. Using numerical methods and the variational approximation, we demonstrate that unstable Townes solitons, created by the contact attractive interaction, may be completely stabilized (with an anisotropic shape) by the dipole-dipole interaction (DDI), in the interval θcr<θ ≤π /2 . The stability boundary θcr weakly depends on the relative strength of the DDI, remaining close to the magic angle θm=arccos(1 /√{3 }) . The results suggest that DDIs provide a generic mechanism for the creation of stable BEC solitons in higher dimensions.

Function of ubiquitin (Ub) specific protease 15 (USP15) in HIV-1 replication and viral protein degradation.

PubMed

Pyeon, Dohun; Timani, Khalid Amine; Gulraiz, Fahad; He, Johnny J; Park, In-Woo

2016-09-02

HIV-1 Nef is necessary and may be sufficient for HIV-1-associated AIDS pathogenicity, in that knockout of Nef alone can protect HIV-infected patients from AIDS. We therefore investigated the feasibility of physical knockout of Nef, using the host ubiquitin proteasome system in HIV-1-infected cells. Our co-immunoprecipitation analysis demonstrated that Nef interacted with ubiquitin specific protease 15 (USP15), and that USP15, which is known to stabilize cellular proteins, degraded Nef. Nef could also cause decay of USP15, although Nef-mediated degradation of USP15 was weaker than USP15-mediated Nef degradation. Direct interaction between Nef and USP15 was essential for the observed reciprocal decay of the proteins. Further, USP15 degraded not only Nef but also HIV-1 structural protein, Gag, thereby substantially inhibiting HIV-1 replication. However, Gag did not degrade USP15, indicating that the Nef and USP15 complex, in distinction to other viral proteins, play an integral role in coordinating viral protein degradation and hence HIV-1 replication. Moreover, Nef and USP15 globally suppressed ubiquitylation of cellular proteins, indicating that these proteins are major determinants for the stability of cellular as well as viral proteins. Taken together, these data indicate that Nef and USP15 are vital in regulating degradation of viral and cellular proteins and thus HIV-1 replication, and specific degradation of viral, not cellular proteins, by USP15 points to USP15 as a candidate therapeutic agent to combat AIDS by eliminating viral proteins from the infected cells via USP15-mediated proteosomal degradation. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Small Molecule Interactome Mapping by Photoaffinity Labeling Reveals Binding Site Hotspots for the NSAIDs.

PubMed

Gao, Jinxu; Mfuh, Adelphe; Amako, Yuka; Woo, Christina M

2018-03-28

Many therapeutics elicit cell-type specific polypharmacology that is executed by a network of molecular recognition events between a small molecule and the whole proteome. However, measurement of the structures that underpin the molecular associations between the proteome and even common therapeutics, such as the nonsteroidal anti-inflammatory drugs (NSAIDs), is limited by the inability to map the small molecule interactome. To address this gap, we developed a platform termed small molecule interactome mapping by photoaffinity labeling (SIM-PAL) and applied it to the in cellulo direct characterization of specific NSAID binding sites. SIM-PAL uses (1) photochemical conjugation of NSAID derivatives in the whole proteome and (2) enrichment and isotope-recoding of the conjugated peptides for (3) targeted mass spectrometry-based assignment. Using SIM-PAL, we identified the NSAID interactome consisting of over 1000 significantly enriched proteins and directly characterized nearly 200 conjugated peptides representing direct binding sites of the photo-NSAIDs with proteins from Jurkat and K562 cells. The enriched proteins were often identified as parts of complexes, including known targets of NSAID activity (e.g., NF-κB) and novel interactions (e.g., AP-2, proteasome). The conjugated peptides revealed direct NSAID binding sites from the cell surface to the nucleus and a specific binding site hotspot for the three photo-NSAIDs on histones H2A and H2B. NSAID binding stabilized COX-2 and histone H2A by cellular thermal shift assay. Since small molecule stabilization of protein complexes is a gain of function regulatory mechanism, it is conceivable that NSAIDs affect biological processes through these broader proteomic interactions. SIM-PAL enabled characterization of NSAID binding site hotspots and is amenable to map global binding sites for virtually any molecule of interest.

Rare sugar D-allose induces specific up-regulation of TXNIP and subsequent G1 cell cycle arrest in hepatocellular carcinoma cells by stabilization of p27kip1.

PubMed

Yamaguchi, Fuminori; Takata, Maki; Kamitori, Kazuyo; Nonaka, Machiko; Dong, Youyi; Sui, Li; Tokuda, Masaaki

2008-02-01

'Rare sugars' are defined as monosaccharides that exist in nature but are only present in limited quantities. The development of mass production method of rare sugars revealed some interesting physiological effects of these on animal cells, but the mechanisms have not been well studied. We examined the effect of D-allose on the proliferation of cancer cells and the underlying molecular mechanism of the action. The HuH-7 hepatocellular carcinoma cells were treated with various monosaccharides for 48 h and D-allose was shown to inhibit cell growth by 40% in a dose-dependent manner. D-allose induced G1 cell cycle arrest but not apoptosis. The microarray analysis revealed that D-allose significantly up-regulated thioredoxin interacting protein (TXNIP) gene expression, which is often suppressed in tumor cells and western blot analysis confirmed its increase at protein level. The overexpression of TXNIP also induced G1 cell cycle arrest. Analysis of cell cycle regulatory genes showed p27kip1, a key regulator of G1/S cell cycle transition, to be increased at the protein but not the transcriptional level. Protein interaction between TXNIP and jab1, and p27kip1 and jab1, was observed, suggesting stabilization of p27kip1 protein by the competitive inhibition of jab1-mediated nuclear export of p27kip1 by TXNIP. In addition, increased interaction and nuclear localization of TXNIP and p27kip1 were apparent after D-allose treatment. Our findings surprisingly suggest that D-allose, a simple monosaccharide, may act as a novel anticancer agent via unique TXNIP induction and p27kip1 protein stabilization.

Incorporation of Pentraxin 3 into Hyaluronan Matrices Is Tightly Regulated and Promotes Matrix Cross-linking

PubMed Central

Baranova, Natalia S.; Inforzato, Antonio; Briggs, David C.; Tilakaratna, Viranga; Enghild, Jan J.; Thakar, Dhruv; Milner, Caroline M.; Day, Anthony J.; Richter, Ralf P.

2014-01-01

Mammalian oocytes are surrounded by a highly hydrated hyaluronan (HA)-rich extracellular matrix with embedded cumulus cells, forming the cumulus cell·oocyte complex (COC) matrix. The correct assembly, stability, and mechanical properties of this matrix, which are crucial for successful ovulation, transport of the COC to the oviduct, and its fertilization, depend on the interaction between HA and specific HA-organizing proteins. Although the proteins inter-α-inhibitor (IαI), pentraxin 3 (PTX3), and TNF-stimulated gene-6 (TSG-6) have been identified as being critical for COC matrix formation, its supramolecular organization and the molecular mechanism of COC matrix stabilization remain unknown. Here we used films of end-grafted HA as a model system to investigate the molecular interactions involved in the formation and stabilization of HA matrices containing TSG-6, IαI, and PTX3. We found that PTX3 binds neither to HA alone nor to HA films containing TSG-6. This long pentraxin also failed to bind to products of the interaction between IαI, TSG-6, and HA, among which are the covalent heavy chain (HC)·HA and HC·TSG-6 complexes, despite the fact that both IαI and TSG-6 are ligands of PTX3. Interestingly, prior encounter with IαI was required for effective incorporation of PTX3 into TSG-6-loaded HA films. Moreover, we demonstrated that this ternary protein mixture made of IαI, PTX3, and TSG-6 is sufficient to promote formation of a stable (i.e. cross-linked) yet highly hydrated HA matrix. We propose that this mechanism is essential for correct assembly of the COC matrix and may also have general implications in other inflammatory processes that are associated with HA cross-linking. PMID:25190808

Importance of a Lys113–Glu195 Intermonomer Ionic Bond in F-actin Stabilization and Regulation by Yeast Formins Bni1p and Bnr1p*

PubMed Central

Wen, Kuo-Kuang; McKane, Melissa; Rubenstein, Peter A.

2013-01-01

Proper actin cytoskeletal function requires actin's ability to generate a stable filament and requires that this reaction be regulated by actin-binding proteins via allosteric effects on the actin. A proposed ionic interaction in the actin filament interior between Lys113 of one monomer and Glu195 of a monomer in the apposing strand potentially fosters cross-strand stabilization and allosteric communication between the filament interior and exterior. We interrupted the potential interaction by creating either K113E or E195K actin. By combining the two, we also reversed the interaction with a K113E/E195K (E/K) mutant. In all cases, we isolated viable cells expressing only the mutant actin. Either single mutant cell displays significantly decreased growth in YPD medium. This deficit is rescued in the double mutant. All three mutants display abnormal phalloidin cytoskeletal staining. K113E actin exhibits a critical concentration of polymerization 4 times higher than WT actin, nucleates more poorly, and forms shorter filaments. Restoration of the ionic bond, E/K, eliminates most of these problems. E195K actin behaves much more like WT actin, indicating accommodation of the neighboring lysines. Both Bni1 and Bnr1 formin FH1-FH2 fragment accelerate polymerization of WT, E/K, and to a lesser extent E195K actin. Bni1p FH1-FH2 dramatically inhibits K113E actin polymerization, consistent with barbed end capping. However, Bnr1p FH1-FH2 restores K113E actin polymerization, forming single filaments. In summary, the proposed ionic interaction plays an important role in filament stabilization and in the propagation of allosteric changes affecting formin regulation in an isoform-specific fashion. PMID:23653364

Solution structure of a lipid transfer protein extracted from rice seeds. Comparison with homologous proteins.

PubMed

Poznanski, J; Sodano, P; Suh, S W; Lee, J Y; Ptak, M; Vovelle, F

1999-02-01

Nuclear magnetic resonance (NMR) spectroscopy was used to determine the three dimensional structure of rice nonspecific lipid transfer protein (ns-LTP), a 91 amino acid residue protein belonging to the broad family of plant ns-LTP. Sequence specific assignment was obtained for all but three HN backbone 1H resonances and for more than 95% of the 1H side-chain resonances using a combination of 1H 2D NOESY; TOCSY and COSY experiments at 293 K. The structure was calculated on the basis of four disulfide bridge restraints, 1259 distance constraints derived from 1H-1H Overhauser effects, 72 phi angle restraints and 32 hydrogen-bond restraints. The final solution structure involves four helices (H1: Cys3-Arg18, H2: Ala25-Ala37, H3: Thr41-Ala54 and H4: Ala66-Cys73) followed by a long C-terminal tail (T) with no observable regular structure. N-capping residues (Thr2, Ser24, Thr40), whose side-chain oxygen atoms are involved in hydrogen bonds with i + 3 amide proton additionally stabilize the N termini of the first three helices. The fourth helix involving Pro residues display a mixture of alpha and 3(10) conformation. The rms deviation of 14 final structures with respect to the average structure is 1.14 +/- 0.16 A for all heavy atoms (C, N, O and S) and 0.72 +/- 0.01 A for the backbone atoms. The global fold of rice ns-LTP is close to the previously published structures of wheat, barley and maize ns-LTPs exhibiting nearly identical pattern of the numerous sequence specific interactions. As reported previously for different four-helix topology proteins, hydrophobic, hydrogen bonding and electrostatic mechanisms of fold stabilization were found for the rice ns-LTP. The sequential alignment of 36 ns-LTP primary structures strongly suggests that there is a uniform pattern of specific long-range interactions (in terms of sequence), which stabilize the fold of all plant ns-LTPs.

Nucleic acid aptamers as stabilizers of proteins: the stability of tetanus toxoid.

PubMed

Jain, Nishant Kumar; Jetani, Hardik C; Roy, Ipsita

2013-07-01

Exposure of tetanus toxoid to moisture leads to its aggregation and reduction of potency. The aim of this work was to use SELEX (systematic evolution of ligands by exponential enrichment) protocol and select aptamers which recognize tetanus toxoid (Mr ~150 kDa) with high affinity. Colyophilized preparations of tetanus toxoid and specific aptamers were encapsulated in PLGA microspheres and sustained release of the antigen was observed up to 55 days using different techniques. The total protein released was between 40-55% (24-45% residual antigenicity) in the presence of the aptamers as compared to 25% (11% residual antigenicity) for the antigen alone. We show that instead of inhibiting absorption of moisture, the aptamers blocked the protein unfolding upon absorption of moisture, inhibiting the initiation of aggregation. When exposed to accelerated storage conditions, some of the RNA sequences were able to inhibit moisture-induced aggregation in vitro and retain antigenicity of tetanus toxoid. Nucleic acid aptamers represent a novel class of protein stabilizers which stabilize the protein by interacting directly with it. This mechanism is unlike that of small molecules which alter the medium properties and hence depend on the stress condition a protein is exposed to.

WGM Temperature Tracker

NASA Technical Reports Server (NTRS)

Strekalov, Dmitry V.

2012-01-01

This software implements digital control of a WGM (whispering-gallerymode) resonator temperature based on the dual-mode approach. It comprises one acquisition (dual-channel) and three control modules. The interaction of the proportional-integral loops is designed in the original way, preventing the loops from fighting. The data processing is organized in parallel with the acquisition, which allows the computational overhead time to be suppressed or often completely avoided. WGM resonators potentially provide excellent optical references for metrology, clocks, spectroscopy, and other applications. However, extremely accurate (below micro-Kelvin) temperature stabilization is required. This software allows one specifically advantageous method of such stabilization to be implemented, which is immune to a variety of effects that mask the temperature variation. WGM Temperature Tracker 2.3 (see figure) is a LabVIEW code developed for dual-mode temperature stabilization of WGM resonators. It has allowed for the temperature stabilization at the level of 200 nK with one-second integration time, and 6 nK with 10,000-second integration time, with the above room-temperature set point. This software, in conjunction with the appropriate hardware, can be used as a noncryogenic temperature sensor/ controller with sub-micro-Kelvin sensitivity, which at the time of this reporting considerably outperforms the state of the art.

Spatial Cues Provided by Sound Improve Postural Stabilization: Evidence of a Spatial Auditory Map?

PubMed Central

Gandemer, Lennie; Parseihian, Gaetan; Kronland-Martinet, Richard; Bourdin, Christophe

2017-01-01

It has long been suggested that sound plays a role in the postural control process. Few studies however have explored sound and posture interactions. The present paper focuses on the specific impact of audition on posture, seeking to determine the attributes of sound that may be useful for postural purposes. We investigated the postural sway of young, healthy blindfolded subjects in two experiments involving different static auditory environments. In the first experiment, we compared effect on sway in a simple environment built from three static sound sources in two different rooms: a normal vs. an anechoic room. In the second experiment, the same auditory environment was enriched in various ways, including the ambisonics synthesis of a immersive environment, and subjects stood on two different surfaces: a foam vs. a normal surface. The results of both experiments suggest that the spatial cues provided by sound can be used to improve postural stability. The richer the auditory environment, the better this stabilization. We interpret these results by invoking the “spatial hearing map” theory: listeners build their own mental representation of their surrounding environment, which provides them with spatial landmarks that help them to better stabilize. PMID:28694770

Effect of polyethylene glycol conjugation on conformational and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab').

PubMed

Roque, Cristopher; Sheung, Anthony; Rahman, Nausheen; Ausar, S Fernando

2015-02-02

We have investigated the effects of site specific "hinge" polyethylene glycol conjugation (PEGylation) on thermal, pH, and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab') using a variety of biophysical techniques. The results obtained by circular dichroism (CD), ultraviolet (UV) absorbance, and fluorescence spectroscopy suggested that the physical stability of the Fab' is maximized at pH 6-7 with no apparent differences due to PEGylation. Temperature-induced aggregation experiments revealed that PEGylation was able to increase the transition temperature, as well as prevent the formation of visible and subvisible aggregates. Statistical comparison of the three-index empirical phase diagram (EPD) revealed significant differences in thermal and pH stability signatures between Fab' and PEG-Fab'. Upon mechanical stress, micro-flow imaging (MFI) and measurement of the optical density at 360 nm showed that the PEG-Fab' had significantly higher resistance to surface-induced aggregation compared to the Fab'. Analysis of the interaction parameter, kD, indicated repulsive intermolecular forces for PEG-Fab' and attractive forces for Fab'. In conclusion, PEGylation appears to protect Fab' against thermal and mechanical stress-induced aggregation, likely due to a steric hindrance mechanism.

Emerging directions in the study of the ecology and evolution of plant-animal mutualistic networks: a review.

PubMed

Gu, Hao; Goodale, Eben; Chen, Jin

2015-03-18

The study of mutualistic plant and animal networks is an emerging field of ecological research. We reviewed progress in this field over the past 30 years. While earlier studies mostly focused on network structure, stability, and biodiversity maintenance, recent studies have investigated the conservation implications of mutualistic networks, specifically the influence of invasive species and how networks respond to habitat loss. Current research has also focused on evolutionary questions including phylogenetic signal in networks, impact of networks on the coevolution of interacting partners, and network influences on the evolution of interacting species. We outline some directions for future research, particularly the evolution of specialization in mutualistic networks, and provide concrete recommendations for environmental managers.

Biocompatible and biodegradable nanoparticles for enhancement of anti-cancer activities of phytochemicals.

PubMed

Li, Chuan; Zhang, Jia; Zu, Yu-Jiao; Nie, Shu-Fang; Cao, Jun; Wang, Qian; Nie, Shao-Ping; Deng, Ze-Yuan; Xie, Ming-Yong; Wang, Shu

2015-09-01

Many phytochemicals show promise in cancer prevention and treatment, but their low aqueous solubility, poor stability, unfavorable bioavailability, and low target specificity make administering them at therapeutic doses unrealistic. This is particularly true for (-)-epigallocatechin gallate, curcumin, quercetin, resveratrol, and genistein. There is an increasing interest in developing novel delivery strategies for these natural products. Liposomes, micelles, nanoemulsions, solid lipid nanoparticles, nanostructured lipid carriers and poly (lactide-co-glycolide) nanoparticles are biocompatible and biodegradable nanoparticles. Those nanoparticles can increase the stability and solubility of phytochemicals, exhibit a sustained release property, enhance their absorption and bioavailability, protect them from premature enzymatic degradation or metabolism, prolong their circulation time, improve their target specificity to cancer cells or tumors via passive or targeted delivery, lower toxicity or side-effects to normal cells or tissues through preventing them from prematurely interacting with the biological environment, and enhance anti-cancer activities. Nanotechnology opens a door for developing phytochemical-loaded nanoparticles for prevention and treatment of cancer. Copyright © 2015 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Adaptability and phenotypic stability of common bean genotypes through Bayesian inference.

PubMed

Corrêa, A M; Teodoro, P E; Gonçalves, M C; Barroso, L M A; Nascimento, M; Santos, A; Torres, F E

2016-04-27

This study used Bayesian inference to investigate the genotype x environment interaction in common bean grown in Mato Grosso do Sul State, and it also evaluated the efficiency of using informative and minimally informative a priori distributions. Six trials were conducted in randomized blocks, and the grain yield of 13 common bean genotypes was assessed. To represent the minimally informative a priori distributions, a probability distribution with high variance was used, and a meta-analysis concept was adopted to represent the informative a priori distributions. Bayes factors were used to conduct comparisons between the a priori distributions. The Bayesian inference was effective for the selection of upright common bean genotypes with high adaptability and phenotypic stability using the Eberhart and Russell method. Bayes factors indicated that the use of informative a priori distributions provided more accurate results than minimally informative a priori distributions. According to Bayesian inference, the EMGOPA-201, BAMBUÍ, CNF 4999, CNF 4129 A 54, and CNFv 8025 genotypes had specific adaptability to favorable environments, while the IAPAR 14 and IAC CARIOCA ETE genotypes had specific adaptability to unfavorable environments.

Genotype-environment interaction and stability in ten-year height growth of Norway spruce Clones (Picea abies Karst.).

Treesearch

J.B. St. Clair; J. Kleinschmit

1986-01-01

Norway spruce cuttings of 40 clones were tested on seven contrasting sites in northern Germany. Analysis of variance for ten-year height growth indicate a highly significant clone x site interaction. This interaction may be reduced by selection of stable clones. Several measures of stability were calculated and discussed. Characterization of sites by the method of...

On the context-dependent scaling of consumer feeding rates.

PubMed

Barrios-O'Neill, Daniel; Kelly, Ruth; Dick, Jaimie T A; Ricciardi, Anthony; MacIsaac, Hugh J; Emmerson, Mark C

2016-06-01

The stability of consumer-resource systems can depend on the form of feeding interactions (i.e. functional responses). Size-based models predict interactions - and thus stability - based on consumer-resource size ratios. However, little is known about how interaction contexts (e.g. simple or complex habitats) might alter scaling relationships. Addressing this, we experimentally measured interactions between a large size range of aquatic predators (4-6400 mg over 1347 feeding trials) and an invasive prey that transitions among habitats: from the water column (3D interactions) to simple and complex benthic substrates (2D interactions). Simple and complex substrates mediated successive reductions in capture rates - particularly around the unimodal optimum - and promoted prey population stability in model simulations. Many real consumer-resource systems transition between 2D and 3D interactions, and along complexity gradients. Thus, Context-Dependent Scaling (CDS) of feeding interactions could represent an unrecognised aspect of food webs, and quantifying the extent of CDS might enhance predictive ecology. © The Authors. Ecology Letters published by CNRS and John Wiley & Sons Ltd.

Mechanisms of protein stabilization and prevention of protein aggregation by glycerol.

PubMed

Vagenende, Vincent; Yap, Miranda G S; Trout, Bernhardt L

2009-11-24

The stability of proteins in aqueous solution is routinely enhanced by cosolvents such as glycerol. Glycerol is known to shift the native protein ensemble to more compact states. Glycerol also inhibits protein aggregation during the refolding of many proteins. However, mechanistic insight into protein stabilization and prevention of protein aggregation by glycerol is still lacking. In this study, we derive mechanisms of glycerol-induced protein stabilization by combining the thermodynamic framework of preferential interactions with molecular-level insight into solvent-protein interactions gained from molecular simulations. Contrary to the common conception that preferential hydration of proteins in polyol/water mixtures is determined by the molecular size of the polyol and the surface area of the protein, we present evidence that preferential hydration of proteins in glycerol/water mixtures mainly originates from electrostatic interactions that induce orientations of glycerol molecules at the protein surface such that glycerol is further excluded. These interactions shift the native protein toward more compact conformations. Moreover, glycerol preferentially interacts with large patches of contiguous hydrophobicity where glycerol acts as an amphiphilic interface between the hydrophobic surface and the polar solvent. Accordingly, we propose that glycerol prevents protein aggregation by inhibiting protein unfolding and by stabilizing aggregation-prone intermediates through preferential interactions with hydrophobic surface regions that favor amphiphilic interface orientations of glycerol. These mechanisms agree well with experimental data available in the literature, and we discuss the extent to which these mechanisms apply to other cosolvents, including polyols, arginine, and urea.

Stability and adaptability of soybean cultivars in Minas Gerais.

PubMed

Soares, I O; Bruzi, A T; Zambiazzi, E V; Guilherme, S R; Bianchi, M C; Silva, K B; Fronza, V; Teixeira, C M

2017-08-17

Genotypes x environment (G x E) interaction consists of different behavior of genotypes cultivated in different environments. This interaction occurs due to the performance variation of each genotype in different environments. To reduce the effect of the interaction in soybean crops, some studies have been reported in the literature to study their adaptability and stability. However, these studies are still scarce in Minas Gerais State. Thus, the aim of this study was to verify the adaptability and stability of soybean cultivars and identify the cultivars that contribute least to the G x E interaction in Minas Gerais. Six soybean cultivars were evaluated in 9 different environments. The plots were composed of 4 rows of 5 m with a spacing of 0.5 m between rows, and only the two central rows were harvested. The inoculation with Bradyrhizobium japonicum was performed through sowing furrow. The fertilization followed the recommendations of the Soil Fertility Commission of Minas Gerais. Grain yield was evaluated in kg/ha after conversion to 13% moisture. After individual analysis, the joint analysis was performed by grouping the phenotypic means by the Scott and Knott (1974) test. Wricke's ecovalence methodologies and the Annicchiarico confidence index were applied for the adaptability and stability analysis. The interaction was decomposed into a simple and a complex part. The cultivars BRSMG 820RR and BRSMG 760SRR have wide adaptability and stability. The first one presents a better index of confidence and a small contribution to the interaction.

Use of polyclonal and monoclonal antibodies to study hCG-receptor interactions

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

Milius, R.P.

1985-01-01

Although the glycoprotein hormones lutropin (LH), follitropin (FSH), and thyrotropin (TSH) bind to different receptors, each contains an identical alpha subunit. Specificity is somehow endowed by theta subunits which are distinct for each hormone. Human choriogonadotropin (hCG) is a natural LH analog that contains a beta subunit nearly identical to that of LH. The roles of these subunits in the recognition and high affinity binding of hCG to receptor was examined. Polyclonal and monoclonal antibodies specific for the individual subunits of hCG were used to probe the hormone-receptor interaction. Conformation-specific and sequence-specific antibodies were examined for their abilities to bindmore » Triton X-100-solubilized /sup 125/I-hCG-receptor complex and to inhibit hormone binding to crude rat ovarian membranes containing receptor. Even though the immunoreactive sites are not located on the receptor binding surface of the beta subunit, most, but not all, of these polyclonal and monoclonal antibodies were able to inhibit /sup 125/I-hCG binding to receptor. Although the inhibition of binding may be due to steric interference due to the size of the antibody molecules, a two-step model for hCG binding to receptor is presented that also explains these results. In this model, the beta subunit initially binds with the receptor with a highly specific but low affinity interaction. This activates a site for the high affinity binding of the alpha subunit and stabilization of the complex. This is an attractive model as it may be applied to other glycoprotein hormones sharing an alpha subunit.« less

46 CFR 170.160 - Specific applicability.

Code of Federal Regulations, 2012 CFR

2012-10-01

...) A vessel that performs one of the simplified stability proof tests described in subpart C of part... COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Intact Stability Criteria § 170.160 Specific applicability. (a) Except...

46 CFR 170.160 - Specific applicability.

Code of Federal Regulations, 2011 CFR

2011-10-01

...) A vessel that performs one of the simplified stability proof tests described in subpart C of part... COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Intact Stability Criteria § 170.160 Specific applicability. (a) Except...

46 CFR 170.160 - Specific applicability.

Code of Federal Regulations, 2014 CFR

2014-10-01

...) A vessel that performs one of the simplified stability proof tests described in subpart C of part... COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Intact Stability Criteria § 170.160 Specific applicability. (a) Except...

46 CFR 170.160 - Specific applicability.

Code of Federal Regulations, 2013 CFR

2013-10-01

...) A vessel that performs one of the simplified stability proof tests described in subpart C of part... COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY STABILITY REQUIREMENTS FOR ALL INSPECTED VESSELS Intact Stability Criteria § 170.160 Specific applicability. (a) Except...

Redundancy of primary RNA-binding functions of the bacterial transcription terminator Rho

PubMed Central

Shashni, Rajesh; Qayyum, M. Zuhaib; Vishalini, V.; Dey, Debashish; Sen, Ranjan

2014-01-01

The bacterial transcription terminator, Rho, terminates transcription at half of the operons. According to the classical model derived from in vitro assays on a few terminators, Rho is recruited to the transcription elongation complex (EC) by recognizing specific sites (rut) on the nascent RNA. Here, we explored the mode of in vivo recruitment process of Rho. We show that sequence specific recognition of the rut site, in majority of the Rho-dependent terminators, can be compromised to a great extent without seriously affecting the genome-wide termination function as well as the viability of Escherichia coli. These terminators function optimally only through a NusG-assisted recruitment and activation of Rho. Our data also indicate that at these terminators, Rho-EC-bound NusG interaction facilitates the isomerization of Rho into a translocase-competent form by stabilizing the interactions of mRNA with the secondary RNA binding site, thereby overcoming the defects of the primary RNA binding functions. PMID:25081210

Thermodynamics of protein destabilization in live cells

PubMed Central

Danielsson, Jens; Mu, Xin; Lang, Lisa; Wang, Huabing; Binolfi, Andres; Theillet, François-Xavier; Bekei, Beata; Logan, Derek T.; Selenko, Philipp; Wennerström, Håkan; Oliveberg, Mikael

2015-01-01

Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein’s interplay with the functionally optimized “interaction landscape” of the cellular interior. PMID:26392565

Interaction of an anticancer peptide fragment of azurin with p53 and its isolated domains studied by atomic force spectroscopy.

PubMed

Bizzarri, Anna Rita; Santini, Simona; Coppari, Emilia; Bucciantini, Monica; Di Agostino, Silvia; Yamada, Tohru; Beattie, Craig W; Cannistraro, Salvatore

2011-01-01

p28 is a 28-amino acid peptide fragment of the cupredoxin azurin derived from Pseudomonas aeruginosa that preferentially penetrates cancerous cells and arrests their proliferation in vitro and in vivo. Its antitumor activity reportedly arises from post-translational stabilization of the tumor suppressor p53 normally downregulated by the binding of several ubiquitin ligases. This would require p28 to specifically bind to p53 to inhibit specific ligases from initiating proteosome-mediated degradation. In this study, atomic force spectroscopy, a nanotechnological approach, was used to investigate the interaction of p28 with full-length p53 and its isolated domains at the single molecule level. Analysis of the unbinding forces and the dissociation rate constant suggest that p28 forms a stable complex with the DNA-binding domain of p53, inhibiting the binding of ubiquitin ligases other than Mdm2 to reduce proteasomal degradation of p53.

Interaction of an anticancer peptide fragment of azurin with p53 and its isolated domains studied by atomic force spectroscopy

PubMed Central

Bizzarri, Anna Rita; Santini, Simona; Coppari, Emilia; Bucciantini, Monica; Di Agostino, Silvia; Yamada, Tohru; Beattie, Craig W; Cannistraro, Salvatore

2011-01-01

p28 is a 28-amino acid peptide fragment of the cupredoxin azurin derived from Pseudomonas aeruginosa that preferentially penetrates cancerous cells and arrests their proliferation in vitro and in vivo. Its antitumor activity reportedly arises from post-translational stabilization of the tumor suppressor p53 normally downregulated by the binding of several ubiquitin ligases. This would require p28 to specifically bind to p53 to inhibit specific ligases from initiating proteosome-mediated degradation. In this study, atomic force spectroscopy, a nanotechnological approach, was used to investigate the interaction of p28 with full-length p53 and its isolated domains at the single molecule level. Analysis of the unbinding forces and the dissociation rate constant suggest that p28 forms a stable complex with the DNA-binding domain of p53, inhibiting the binding of ubiquitin ligases other than Mdm2 to reduce proteasomal degradation of p53. PMID:22162658

MKP-7, a JNK phosphatase, blocks ERK-dependent gene activation by anchoring phosphorylated ERK in the cytoplasm

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

Masuda, Kouhei; Katagiri, Chiaki; Division of Biochemical Oncology and Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo

2010-03-05

MAPK phosphatase-7 (MKP-7) was identified as a JNK-specific phosphatase. However, despite its high specificity for JNK, MKP-7 interacts also with ERK. We previously showed that as a physiological consequence of their interaction, activated ERK phosphorylates MKP-7 at Ser-446, and stabilizing MKP-7. In the present study, we analyzed MKP-7 function in activation of ERK. A time-course experiment showed that both MKP-7 and its phosphatase-dead mutant prolonged mitogen-induced ERK phosphorylation, suggesting that MKP-7 functions as a scaffold for ERK. An important immunohistological finding was that nuclear translocation of phospho-ERK following PMA stimulation was blocked by co-expressed MKP-7 and, moreover, that phospho-ERK co-localizedmore » with MKP-7 in the cytoplasm. Reporter gene analysis indicated that MKP-7 blocks ERK-mediated transcription. Overall, our data indicate that MKP-7 down-regulates ERK-dependent gene expression by blocking nuclear accumulation of phospho-ERK.« less

Direct Correlation of DNA Binding and Single Protein Domain Motion via Dual Illumination Fluorescence Microscopy

PubMed Central

2015-01-01

We report a dual illumination, single-molecule imaging strategy to dissect directly and in real-time the correlation between nanometer-scale domain motion of a DNA repair protein and its interaction with individual DNA substrates. The strategy was applied to XPD, an FeS cluster-containing DNA repair helicase. Conformational dynamics was assessed via FeS-mediated quenching of a fluorophore site-specifically incorporated into XPD. Simultaneously, binding of DNA molecules labeled with a spectrally distinct fluorophore was detected by colocalization of the DNA- and protein-derived signals. We show that XPD undergoes thermally driven conformational transitions that manifest in spatial separation of its two auxiliary domains. DNA binding does not strictly enforce a specific conformation. Interaction with a cognate DNA damage, however, stabilizes the compact conformation of XPD by increasing the weighted average lifetime of this state by 140% relative to an undamaged DNA. Our imaging strategy will be a valuable tool to study other FeS-containing nucleic acid processing enzymes. PMID:25204359

RACER a Coarse-Grained RNA Model for Capturing Folding Free Energy in Molecular Dynamics Simulations

NASA Astrophysics Data System (ADS)

Cheng, Sara; Bell, David; Ren, Pengyu

RACER is a coarse-grained RNA model that can be used in molecular dynamics simulations to predict native structures and sequence-specific variation of free energy of various RNA structures. RACER is capable of accurate prediction of native structures of duplexes and hairpins (average RMSD of 4.15 angstroms), and RACER can capture sequence-specific variation of free energy in excellent agreement with experimentally measured stabilities (r-squared =0.98). The RACER model implements a new effective non-bonded potential and re-parameterization of hydrogen bond and Debye-Huckel potentials. Insights from the RACER model include the importance of treating pairing and stacking interactions separately in order to distinguish folded an unfolded states and identification of hydrogen-bonding, base stacking, and electrostatic interactions as essential driving forces for RNA folding. Future applications of the RACER model include predicting free energy landscapes of more complex RNA structures and use of RACER for multiscale simulations.

Distinct modes of gene regulation by a cell-specific transcriptional activator.

PubMed

Sengupta, Tanushri; Cohet, Nathalie; Morlé, François; Bieker, James J

2009-03-17

The architectural layout of a eukaryotic RNA polymerase II core promoter plays a role in general transcriptional activation. However, its role in tissue-specific expression is not known. For example, differing modes of its recognition by general transcription machinery can provide an additional layer of control within which a single tissue-restricted transcription factor may operate. Erythroid Kruppel-like factor (EKLF) is a hematopoietic-specific transcription factor that is critical for the activation of subset of erythroid genes. We find that EKLF interacts with TATA binding protein-associated factor 9 (TAF9), which leads to important consequences for expression of adult beta-globin. First, TAF9 functionally supports EKLF activity by enhancing its ability to activate the beta-globin gene. Second, TAF9 interacts with a conserved beta-globin downstream promoter element, and ablation of this interaction by beta-thalassemia-causing mutations decreases its promoter activity and disables superactivation. Third, depletion of EKLF prevents recruitment of TAF9 to the beta-globin promoter, whereas depletion of TAF9 drastically impairs beta-promoter activity. However, a TAF9-independent mode of EKLF transcriptional activation is exhibited by the alpha-hemoglobin-stabilizing protein (AHSP) gene, which does not contain a discernable downstream promoter element. In this case, TAF9 does not enhance EKLF activity and depletion of TAF9 has no effect on AHSP promoter activation. These studies demonstrate that EKLF directs different modes of tissue-specific transcriptional activation depending on the architecture of its target core promoter.

Tumor protein D52 expression is post-transcriptionally regulated by T-cell intercellular antigen (TIA) 1 and TIA-related protein via mRNA stability.

PubMed

Motohashi, Hiromi; Mukudai, Yoshiki; Ito, Chihiro; Kato, Kosuke; Shimane, Toshikazu; Kondo, Seiji; Shirota, Tatsuo

2017-05-04

Although tumor protein D52 (TPD52) family proteins were first identified nearly 20 years ago, their molecular regulatory mechanisms remain unclear. Therefore, we investigated the post-transcriptional regulation of TPD52 family genes. An RNA immunoprecipitation (RIP) assay showed the potential binding ability of TPD52 family mRNAs to several RNA-binding proteins, and an RNA degradation assay revealed that TPD52 is subject to more prominent post-transcriptional regulation than are TPD53 and TPD54. We subsequently focused on the 3'-untranslated region (3'-UTR) of TPD52 as a cis -acting element in post-transcriptional gene regulation. Several deletion mutants of the 3'-UTR of TPD52 mRNA were constructed and ligated to the 3'-end of a reporter green fluorescence protein gene. An RNA degradation assay revealed that a minimal cis -acting region, located in the 78-280 region of the 5'-proximal region of the 3'-UTR, stabilized the reporter mRNA. Biotin pull-down and RIP assays revealed specific binding of the region to T-cell intracellular antigen 1 (TIA-1) and TIA-1-related protein (TIAR). Knockdown of TIA-1/TIAR decreased not only the expression, but also the stability of TPD52 mRNA; it also decreased the expression and stability of the reporter gene ligated to the 3'-end of the 78-280 fragment. Stimulation of transforming growth factor-β and epidermal growth factor decreased the binding ability of these factors, resulting in decreased mRNA stability. These results indicate that the 78-280 fragment and TIA-1/TIAR concordantly contribute to mRNA stability as a cis -acting element and trans -acting factor(s), respectively. Thus, we here report the specific interactions between these elements in the post-transcriptional regulation of the TPD52 gene. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

NaStEP: a proteinase inhibitor essential to self-incompatibility and a positive regulator of HT-B stability in Nicotiana alata pollen tubes.

PubMed

Jiménez-Durán, Karina; McClure, Bruce; García-Campusano, Florencia; Rodríguez-Sotres, Rogelio; Cisneros, Jesús; Busot, Grethel; Cruz-García, Felipe

2013-01-01

In Solanaceae, the self-incompatibility S-RNase and S-locus F-box interactions define self-pollen recognition and rejection in an S-specific manner. This interaction triggers a cascade of events involving other gene products unlinked to the S-locus that are crucial to the self-incompatibility response. To date, two essential pistil-modifier genes, 120K and High Top-Band (HT-B), have been identified in Nicotiana species. However, biochemistry and genetics indicate that additional modifier genes are required. We recently reported a Kunitz-type proteinase inhibitor, named NaStEP (for Nicotiana alata Stigma-Expressed Protein), that is highly expressed in the stigmas of self-incompatible Nicotiana species. Here, we report the proteinase inhibitor activity of NaStEP. NaStEP is taken up by both compatible and incompatible pollen tubes, but its suppression in Nicotiana spp. transgenic plants disrupts S-specific pollen rejection; therefore, NaStEP is a novel pistil-modifier gene. Furthermore, HT-B levels within the pollen tubes are reduced when NaStEP-suppressed pistils are pollinated with either compatible or incompatible pollen. In wild-type self-incompatible N. alata, in contrast, HT-B degradation occurs preferentially in compatible pollinations. Taken together, these data show that the presence of NaStEP is required for the stability of HT-B inside pollen tubes during the rejection response, but the underlying mechanism is currently unknown.

NaStEP: A Proteinase Inhibitor Essential to Self-Incompatibility and a Positive Regulator of HT-B Stability in Nicotiana alata Pollen Tubes1[W][OA

PubMed Central

Jiménez-Durán, Karina; McClure, Bruce; García-Campusano, Florencia; Rodríguez-Sotres, Rogelio; Cisneros, Jesús; Busot, Grethel; Cruz-García, Felipe

2013-01-01

In Solanaceae, the self-incompatibility S-RNase and S-locus F-box interactions define self-pollen recognition and rejection in an S-specific manner. This interaction triggers a cascade of events involving other gene products unlinked to the S-locus that are crucial to the self-incompatibility response. To date, two essential pistil-modifier genes, 120K and High Top-Band (HT-B), have been identified in Nicotiana species. However, biochemistry and genetics indicate that additional modifier genes are required. We recently reported a Kunitz-type proteinase inhibitor, named NaStEP (for Nicotiana alata Stigma-Expressed Protein), that is highly expressed in the stigmas of self-incompatible Nicotiana species. Here, we report the proteinase inhibitor activity of NaStEP. NaStEP is taken up by both compatible and incompatible pollen tubes, but its suppression in Nicotiana spp. transgenic plants disrupts S-specific pollen rejection; therefore, NaStEP is a novel pistil-modifier gene. Furthermore, HT-B levels within the pollen tubes are reduced when NaStEP-suppressed pistils are pollinated with either compatible or incompatible pollen. In wild-type self-incompatible N. alata, in contrast, HT-B degradation occurs preferentially in compatible pollinations. Taken together, these data show that the presence of NaStEP is required for the stability of HT-B inside pollen tubes during the rejection response, but the underlying mechanism is currently unknown. PMID:23150644

Gi- and Gs-coupled GPCRs show different modes of G-protein binding.

PubMed

Van Eps, Ned; Altenbach, Christian; Caro, Lydia N; Latorraca, Naomi R; Hollingsworth, Scott A; Dror, Ron O; Ernst, Oliver P; Hubbell, Wayne L

2018-03-06

More than two decades ago, the activation mechanism for the membrane-bound photoreceptor and prototypical G protein-coupled receptor (GPCR) rhodopsin was uncovered. Upon light-induced changes in ligand-receptor interaction, movement of specific transmembrane helices within the receptor opens a crevice at the cytoplasmic surface, allowing for coupling of heterotrimeric guanine nucleotide-binding proteins (G proteins). The general features of this activation mechanism are conserved across the GPCR superfamily. Nevertheless, GPCRs have selectivity for distinct G-protein family members, but the mechanism of selectivity remains elusive. Structures of GPCRs in complex with the stimulatory G protein, G s , and an accessory nanobody to stabilize the complex have been reported, providing information on the intermolecular interactions. However, to reveal the structural selectivity filters, it will be necessary to determine GPCR-G protein structures involving other G-protein subtypes. In addition, it is important to obtain structures in the absence of a nanobody that may influence the structure. Here, we present a model for a rhodopsin-G protein complex derived from intermolecular distance constraints between the activated receptor and the inhibitory G protein, G i , using electron paramagnetic resonance spectroscopy and spin-labeling methodologies. Molecular dynamics simulations demonstrated the overall stability of the modeled complex. In the rhodopsin-G i complex, G i engages rhodopsin in a manner distinct from previous GPCR-G s structures, providing insight into specificity determinants. Copyright © 2018 the Author(s). Published by PNAS.

Integrins protect cardiomyocytes from ischemia/reperfusion injury

PubMed Central

Okada, Hideshi; Lai, N. Chin; Kawaraguchi, Yoshitaka; Liao, Peter; Copps, Jeffrey; Sugano, Yasuo; Okada-Maeda, Sunaho; Banerjee, Indroneal; Schilling, Jan M.; Gingras, Alexandre R.; Asfaw, Elizabeth K.; Suarez, Jorge; Kang, Seok-Min; Perkins, Guy A.; Au, Carol G.; Israeli-Rosenberg, Sharon; Manso, Ana Maria; Liu, Zheng; Milner, Derek J.; Kaufman, Stephen J.; Patel, Hemal H.; Roth, David M.; Hammond, H. Kirk; Taylor, Susan S.; Dillmann, Wolfgang H.; Goldhaber, Joshua I.; Ross, Robert S.

2013-01-01

Ischemic damage is recognized to cause cardiomyocyte (CM) death and myocardial dysfunction, but the role of cell-matrix interactions and integrins in this process has not been extensively studied. Expression of α7β1D integrin, the dominant integrin in normal adult CMs, increases during ischemia/reperfusion (I/R), while deficiency of β1 integrins increases ischemic damage. We hypothesized that the forced overexpression of integrins on the CM would offer protection from I/R injury. Tg mice with CM-specific overexpression of integrin α7β1D exposed to I/R had a substantial reduction in infarct size compared with that of α5β1D-overexpressing mice and WT littermate controls. Using isolated CMs, we found that α7β1D preserved mitochondrial membrane potential during hypoxia/reoxygenation (H/R) injury via inhibition of mitochondrial Ca2+ overload but did not alter H/R effects on oxidative stress. Therefore, we assessed Ca2+ handling proteins in the CM and found that β1D integrin colocalized with ryanodine receptor 2 (RyR2) in CM T-tubules, complexed with RyR2 in human and rat heart, and specifically bound to RyR2 amino acids 165–175. Integrins stabilized the RyR2 interdomain interaction, and this stabilization required integrin receptor binding to its ECM ligand. These data suggest that α7β1D integrin modifies Ca2+ regulatory pathways and offers a means to protect the myocardium from ischemic injury. PMID:24091324

Plant plasma membrane aquaporins in natural vesicles as potential stabilizers and carriers of glucosinolates.

PubMed

Martínez-Ballesta, Maria Del Carmen; Pérez-Sánchez, Horacio; Moreno, Diego A; Carvajal, Micaela

2016-07-01

Their biodegradable nature and ability to target cells make biological vesicles potential nanocarriers for bioactives delivery. In this work, the interaction between proteoliposomes enriched in aquaporins derived from broccoli plants and the glucosinolates was evaluated. The vesicles were stored at different temperatures and their integrity was studied. Determination of glucosinolates, showed that indolic glucosinolates were more sensitive to degradation in aqueous solution than aliphatic glucosinolates. Glucoraphanin was stabilized by leaf and root proteoliposomes at 25°C through their interaction with aquaporins. An extensive hydrogen bond network, including different aquaporin residues, and hydrophobic interactions, as a consequence of the interaction between the linear alkane chain of glucoraphanin and Glu31 and Leu34 protein residues, were established as the main stabilizing elements. Combined our results showed that plasma membrane vesicles from leaf and root tissues of broccoli plants may be considered as suitable carriers for glucosinolate which stabilization can be potentially attributed to aquaporins. Copyright © 2016 Elsevier B.V. All rights reserved.

RNA Tertiary Interactions in a Riboswitch Stabilize the Structure of a Kink Turn

PubMed Central

Schroeder, Kersten T.; Daldrop, Peter; Lilley, David M.J.

2011-01-01

Summary The kink turn is a widespread RNA motif that introduces an acute kink into the axis of duplex RNA, typically comprising a bulge followed by a G⋅A and A⋅G pairs. The kinked conformation is stabilized by metal ions, or the binding of proteins including L7Ae. We now demonstrate a third mechanism for the stabilization of k-turn structure, involving tertiary interactions within a larger RNA structure. The SAM-I riboswitch contains an essential standard k-turn sequence that kinks a helix so that its terminal loop can make a long-range interaction. We find that some sequence variations in the k-turn within the riboswitch do not prevent SAM binding, despite preventing the folding of the k-turn in isolation. Furthermore, two crystal structures show that the sequence-variant k-turns are conventionally folded within the riboswitch. This study shows that the folded structure of the k-turn can be stabilized by tertiary interactions within a larger RNA structure. PMID:21893284

Physicochemical interactions in solid dosage forms.

PubMed

Narang, Ajit S; Desai, Divyakant; Badawy, Sherif

2012-10-01

Complete characterization and mechanistic understanding of physicochemical interactions in solid dosage forms are not only important for consistent manufacturability, stability, and bioavailability of the drug product, but are also expected under the quality-by-design paradigm of drug development. Lack of this understanding can impact successful and timely development, scale-up, and commercial manufacture of dosage forms. This article highlights the stability and bioavailability implications of physicochemical interactions in dosage forms citing a couple of examples where such interactions necessitated the recall of commercial drug products.

Modulators of 14-3-3 Protein–Protein Interactions

PubMed Central

2017-01-01

Direct interactions between proteins are essential for the regulation of their functions in biological pathways. Targeting the complex network of protein–protein interactions (PPIs) has now been widely recognized as an attractive means to therapeutically intervene in disease states. Even though this is a challenging endeavor and PPIs have long been regarded as “undruggable” targets, the last two decades have seen an increasing number of successful examples of PPI modulators, resulting in growing interest in this field. PPI modulation requires novel approaches and the integrated efforts of multiple disciplines to be a fruitful strategy. This perspective focuses on the hub-protein 14-3-3, which has several hundred identified protein interaction partners, and is therefore involved in a wide range of cellular processes and diseases. Here, we aim to provide an integrated overview of the approaches explored for the modulation of 14-3-3 PPIs and review the examples resulting from these efforts in both inhibiting and stabilizing specific 14-3-3 protein complexes by small molecules, peptide mimetics, and natural products. PMID:28968506

Chemical inhibition of prometastatic lysyl-tRNA synthetase–laminin receptor interaction

PubMed Central

Kim, Dae Gyu; Lee, Jin Young; Kwon, Nam Hoon; Fang, Pengfei; Zhang, Qian; Wang, Jing; Young, Nicolas L.; Guo, Min; Cho, Hye Young; Mushtaq, AmeeqUl; Jeon, Young Ho; Choi, Jin Woo; Han, Jung Min; Kang, Ho Woong; Joo, Jae Eun; Hur, Youn; Kang, Wonyoung; Yang, Heekyoung; Nam, Do-Hyun; Lee, Mi-Sook; Lee, Jung Weon; Kim, Eun-Sook; Moon, Aree; Kim, Kibom; Kim, Doyeun; Kang, Eun Joo; Moon, Youngji; Rhee, Kyung Hee; Han, Byung Woo; Yang, Jee Sun; Han, Gyoonhee; Yang, Won Suk; Lee, Cheolju; Wang, Ming-Wei; Kim, Sunghoon

2014-01-01

Lysyl-tRNA synthetase (KRS), a protein synthesis enzyme in the cytosol, relocates to the plasma membrane after a laminin signal and stabilizes a 67-kDa laminin receptor (67LR) that is implicated in cancer metastasis; however, its potential as an antimetastatic therapeutic target has not been explored. We found that the small compound BC-K-YH16899, which binds to KRS, impinged on interaction of KRS with 67LR and suppressed metastasis in 3 different mouse models. The compound inhibited KRS–67LR interaction in two ways. First, it directly blocked the association between KRS and 67LR. Second, it suppressed the dynamic movement of the N-terminal extension of KRS and reduced membrane localization of KRS. However, it did not affect the catalytic activity of KRS. Our results suggest that specific modulation of a cancer-related KRS–67LR interaction may offer a way to control metastasis while avoiding the toxicities associated with inhibition of the normal functions of KRS. PMID:24212136

A coarse grain model for protein-surface interactions

NASA Astrophysics Data System (ADS)

Wei, Shuai; Knotts, Thomas A.

2013-09-01

The interaction of proteins with surfaces is important in numerous applications in many fields—such as biotechnology, proteomics, sensors, and medicine—but fundamental understanding of how protein stability and structure are affected by surfaces remains incomplete. Over the last several years, molecular simulation using coarse grain models has yielded significant insights, but the formalisms used to represent the surface interactions have been rudimentary. We present a new model for protein surface interactions that incorporates the chemical specificity of both the surface and the residues comprising the protein in the context of a one-bead-per-residue, coarse grain approach that maintains computational efficiency. The model is parameterized against experimental adsorption energies for multiple model peptides on different types of surfaces. The validity of the model is established by its ability to quantitatively and qualitatively predict the free energy of adsorption and structural changes for multiple biologically-relevant proteins on different surfaces. The validation, done with proteins not used in parameterization, shows that the model produces remarkable agreement between simulation and experiment.

Statistical mechanics of neocortical interactions: Path-integral evolution of short-term memory

NASA Astrophysics Data System (ADS)

Ingber, Lester

1994-05-01

Previous papers in this series of statistical mechanics of neocortical interactions (SMNI) have detailed a development from the relatively microscopic scales of neurons up to the macroscopic scales as recorded by electroencephalography (EEG), requiring an intermediate mesocolumnar scale to be developed at the scale of minicolumns (~=102 neurons) and macrocolumns (~=105 neurons). Opportunity was taken to view SMNI as sets of statistical constraints, not necessarily describing specific synaptic or neuronal mechanisms, on neuronal interactions, on some aspects of short-term memory (STM), e.g., its capacity, stability, and duration. A recently developed c-language code, pathint, provides a non-Monte Carlo technique for calculating the dynamic evolution of arbitrary-dimension (subject to computer resources) nonlinear Lagrangians, such as derived for the two-variable SMNI problem. Here, pathint is used to explicitly detail the evolution of the SMNI constraints on STM.

Intersegmental Eye-Head-Body Interactions during Complex Whole Body Movements

PubMed Central

von Laßberg, Christoph; Beykirch, Karl A.; Mohler, Betty J.; Bülthoff, Heinrich H.

2014-01-01

Using state-of-the-art technology, interactions of eye, head and intersegmental body movements were analyzed for the first time during multiple twisting somersaults of high-level gymnasts. With this aim, we used a unique combination of a 16-channel infrared kinemetric system; a three-dimensional video kinemetric system; wireless electromyography; and a specialized wireless sport-video-oculography system, which was able to capture and calculate precise oculomotor data under conditions of rapid multiaxial acceleration. All data were synchronized and integrated in a multimodal software tool for three-dimensional analysis. During specific phases of the recorded movements, a previously unknown eye-head-body interaction was observed. The phenomenon was marked by a prolonged and complete suppression of gaze-stabilizing eye movements, in favor of a tight coupling with the head, spine and joint movements of the gymnasts. Potential reasons for these observations are discussed with regard to earlier findings and integrated within a functional model. PMID:24763143

Communication: Many-body stabilization of non-covalent interactions: Structure, stability, and mechanics of Ag3Co(CN)6 framework.

PubMed

Liu, Xiaofei; Hermann, Jan; Tkatchenko, Alexandre

2016-12-28

Stimuli-responsive metal-organic frameworks (MOFs) and other framework materials exhibit a broad variety of useful properties, which mainly stem from an interplay of strong covalent bonds within the organic linkers with presumably weak van der Waals (vdW) interactions which determine the overall packing of the framework constituents. Using Ag 3 Co(CN) 6 as a fundamental test case-a system with a colossal positive and negative thermal expansion [A. L. Goodwin et al., Science 319, 794 (2008)]-we demonstrate that its structure, stability, dielectric, vibrational, and mechanical properties are critically influenced by many-body electronic correlation contributions to non-covalent vdW interactions. The Ag 3 Co(CN) 6 framework is a remarkable molecular crystal, being visibly stabilized, rather than destabilized, by many-body vdW correlations. A detailed comparison with H 3 Co(CN) 6 highlights the crucial role of strongly polarized metallophilic interactions in dictating the exceptional properties of denser MOFs. Beyond MOFs, our findings indicate that many-body electronic correlations can substantially stabilize polarizable materials, providing a novel mechanism for tuning the properties of nanomaterials with intricate structural motifs.

Stability and Interaction of Coherent Structure in Supersonic Reactive Wakes

NASA Technical Reports Server (NTRS)

Menon, Suresh

1983-01-01

A theoretical formulation and analysis is presented for a study of the stability and interaction of coherent structure in reacting free shear layers. The physical problem under investigation is a premixed hydrogen-oxygen reacting shear layer in the wake of a thin flat plate. The coherent structure is modeled as a periodic disturbance and its stability is determined by the application of linearized hydrodynamic stability theory which results in a generalized eigenvalue problem for reactive flows. Detailed stability analysis of the reactive wake for neutral, symmetrical and antisymmetrical disturbance is presented. Reactive stability criteria is shown to be quite different from classical non-reactive stability. The interaction between the mean flow, coherent structure and fine-scale turbulence is theoretically formulated using the von-Kaman integral technique. Both time-averaging and conditional phase averaging are necessary to separate the three types of motion. The resulting integro-differential equations can then be solved subject to initial conditions with appropriate shape functions. In the laminar flow transition region of interest, the spatial interaction between the mean motion and coherent structure is calculated for both non-reactive and reactive conditions and compared with experimental data wherever available. The fine-scale turbulent motion determined by the application of integral analysis to the fluctuation equations. Since at present this turbulence model is still untested, turbulence is modeled in the interaction problem by a simple algebraic eddy viscosity model. The applicability of the integral turbulence model formulated here is studied parametrically by integrating these equations for the simple case of self-similar mean motion with assumed shape functions. The effect of the motion of the coherent structure is studied and very good agreement is obtained with previous experimental and theoretical works for non-reactive flow. For the reactive case, lack of experimental data made direct comparison difficult. It was determined that the growth rate of the disturbance amplitude is lower for reactive case. The results indicate that the reactive flow stability is in qualitative agreement with experimental observation.

Two dynamin-like proteins stabilize FtsZ rings during Streptomyces sporulation.

PubMed

Schlimpert, Susan; Wasserstrom, Sebastian; Chandra, Govind; Bibb, Maureen J; Findlay, Kim C; Flärdh, Klas; Buttner, Mark J

2017-07-25

During sporulation, the filamentous bacteria Streptomyces undergo a massive cell division event in which the synthesis of ladders of sporulation septa convert multigenomic hyphae into chains of unigenomic spores. This process requires cytokinetic Z-rings formed by the bacterial tubulin homolog FtsZ, and the stabilization of the newly formed Z-rings is crucial for completion of septum synthesis. Here we show that two dynamin-like proteins, DynA and DynB, play critical roles in this process. Dynamins are a family of large, multidomain GTPases involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. Using a cell biological approach, we show that the two Streptomyces dynamins specifically localize to sporulation septa in an FtsZ-dependent manner. Moreover, dynamin mutants have a cell division defect due to the decreased stability of sporulation-specific Z-rings, as demonstrated by kymographs derived from time-lapse images of FtsZ ladder formation. This defect causes the premature disassembly of individual Z-rings, leading to the frequent abortion of septum synthesis, which in turn results in the production of long spore-like compartments with multiple chromosomes. Two-hybrid analysis revealed that the dynamins are part of the cell division machinery and that they mediate their effects on Z-ring stability during developmentally controlled cell division via a network of protein-protein interactions involving DynA, DynB, FtsZ, SepF, SepF2, and the FtsZ-positioning protein SsgB.

The coupling between stability and ion pair formation in magnesium electrolytes from first-principles quantum mechanics and classical molecular dynamics

DOE PAGES

Rajput, Nav Nidhi; Qu, Xiaohuui; Sa, Niya; ...

2015-02-10

Here in this work we uncover a novel effect between concentration dependent ion pair formation and anion stability at reducing potentials, e.g., at the metal anode. Through comprehensive calculations using both first-principles as well as well-benchmarked classical molecular dynamics over a matrix of electrolytes, covering solvents and salt anions with a broad range in chemistry, we elucidate systematic correlations between molecular level interactions and composite electrolyte properties, such as electrochemical stability, solvation structure, and dynamics. We find that Mg electrolytes are highly prone to ion pair formation, even at modest concentrations, for a wide range of solvents with different dielectricmore » constants, which have implications for dynamics as well as charge transfer. Specifically, we observe that, at Mg metal potentials, the ion pair undergoes partial reduction at the Mg cation center (Mg 2+ -> Mg +), which competes with the charge transfer mechanism and can activate the anion to render it susceptible to decomposition. Specifically, TFSI exhibits a significant bond weakening while paired with the transient, partially reduced Mg +. In contrast, BH 4 $-$ and BF 4 $-$ are shown to be chemically stable in a reduced ion pair configuration. Furthermore, we observe that higher order glymes as well as DMSO improve the solubility of Mg salts, but only the longer glyme chains reduce the dynamics of the ions in solution. This information provides critical design metrics for future electrolytes as it elucidates a close connection between bulk solvation and cathodic stability as well as the dynamics of the salt.« less

Salt bridge as a gatekeeper against partial unfolding.

PubMed

Hinzman, Mark W; Essex, Morgan E; Park, Chiwook

2016-05-01

Salt bridges are frequently observed in protein structures. Because the energetic contribution of salt bridges is strongly dependent on the environmental context, salt bridges are believed to contribute to the structural specificity rather than the stability. To test the role of salt bridges in enhancing structural specificity, we investigated the contribution of a salt bridge to the energetics of native-state partial unfolding in a cysteine-free version of Escherichia coli ribonuclease H (RNase H*). Thermolysin cleaves a protruding loop of RNase H(*) through transient partial unfolding under native conditions. Lys86 and Asp108 in RNase H(*) form a partially buried salt bridge that tethers the protruding loop. Investigation of the global stability of K86Q/D108N RNase H(*) showed that the salt bridge does not significantly contribute to the global stability. However, K86Q/D108N RNase H(*) is greatly more susceptible to proteolysis by thermolysin than wild-type RNase H(*) is. The free energy for partial unfolding determined by native-state proteolysis indicates that the salt bridge significantly increases the energy for partial unfolding by destabilizing the partially unfolded form. Double mutant cycles with single and double mutations of the salt bridge suggest that the partially unfolded form is destabilized due to a significant decrease in the interaction energy between Lys86 and Asp108 upon partial unfolding. This study demonstrates that, even in the case that a salt bridge does not contribute to the global stability, the salt bridge may function as a gatekeeper against partial unfolding that disturbs the optimal geometry of the salt bridge. © 2016 The Protein Society.

Surface-supported Ag islands stabilized by a quantum size effect: Their interaction with small molecules relevant to ethylene epoxidation

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

Shao, Dahai

2013-05-15

This dissertation focuses on how QSE-stabilized, surface-supported Ag nanoclusters will interact with ethylene or oxygen. Experiments are performed to determine whether the QSE-mediated Ag islands react differently toward adsorption of ethylene or oxygen, or whether the adsorption of these small molecules will affect the QSE-mediated stability of Ag islands. Studies of the interaction of oxygen with Ag/Si(111)-7×7 were previously reported, but these studies were performed at a low Ag coverage where 3D Ag islands were not formed. So the study of such a system at a higher Ag coverage will be a subject of this work. The interaction of ethylenemore » with Ag/Si(111)-7×7, as well as the interaction of oxygen with Ag/NiAl(110) are also important parts of this study.« less

Molecular Dynamics Simulation of Rap1 Myb-type domain in Saccharomyces cerevisiae

PubMed Central

Mukherjee, Koel; Pandey, Dev Mani; Vidyarthi, Ambarish Saran

2012-01-01

Telomere is a nucleoprotein complex that plays important role in stability and their maintenance and consists of random repeats of species specific motifs. In budding Saccharomyces cerevisiae, Repressor Activator Protein 1 (Rap1) is a sequence specific protein that involved in transcriptional regulation. Rap1 consist of three active domains like N-terminal BRCT-domain, DNA-binding domain and C-terminal RCT-domain. In this study the unknown 3D structure of Myb-type domain (having 61 residues) within DNAbinding domain was modeled by Modeller7, and verified using different online bioinformatics tools (ProCheck, WhatIf, Verify3D). Dynamics of Myb-type domain of Rap1was carried out through simulation studies using GROMACS software. Time dependent interactions among the molecules were analyzed by Root Mean Square Deviation (RMSD), Radius of Gyration (Rg) and Root Mean Square Fluctuation (RMSF) plots. Motional properties in reduced dimension were also performed by Principal Component Analysis (PCA). Result indicated that Rap1 interacts with DNA major groove through its Helix Turn Helix motifs. Helix 3 was rigid, less amount of fluctuation was found as it interacts with DNA major groove. Helix2 and N-terminal having considerable fluctuation in the time scale. PMID:23144544

Networks of energetic and metabolic interactions define dynamics in microbial communities.

PubMed

Embree, Mallory; Liu, Joanne K; Al-Bassam, Mahmoud M; Zengler, Karsten

2015-12-15

Microorganisms form diverse communities that have a profound impact on the environment and human health. Recent technological advances have enabled elucidation of community diversity at high resolution. Investigation of microbial communities has revealed that they often contain multiple members with complementing and seemingly redundant metabolic capabilities. An understanding of the communal impacts of redundant metabolic capabilities is currently lacking; specifically, it is not known whether metabolic redundancy will foster competition or motivate cooperation. By investigating methanogenic populations, we identified the multidimensional interspecies interactions that define composition and dynamics within syntrophic communities that play a key role in the global carbon cycle. Species-specific genomes were extracted from metagenomic data using differential coverage binning. We used metabolic modeling leveraging metatranscriptomic information to reveal and quantify a complex intertwined system of syntrophic relationships. Our results show that amino acid auxotrophies create additional interdependencies that define community composition and control carbon and energy flux through the system while simultaneously contributing to overall community robustness. Strategic use of antimicrobials further reinforces this intricate interspecies network. Collectively, our study reveals the multidimensional interactions in syntrophic communities that promote high species richness and bolster community stability during environmental perturbations.

SWI/SNF interacts with cleavage and polyadenylation factors and facilitates pre-mRNA 3' end processing.

PubMed

Yu, Simei; Jordán-Pla, Antonio; Gañez-Zapater, Antoni; Jain, Shruti; Rolicka, Anna; Östlund Farrants, Ann-Kristin; Visa, Neus

2018-05-31

SWI/SNF complexes associate with genes and regulate transcription by altering the chromatin at the promoter. It has recently been shown that these complexes play a role in pre-mRNA processing by associating at alternative splice sites. Here, we show that SWI/SNF complexes are involved also in pre-mRNA 3' end maturation by facilitating 3' end cleavage of specific pre-mRNAs. Comparative proteomics show that SWI/SNF ATPases interact physically with subunits of the cleavage and polyadenylation complexes in fly and human cells. In Drosophila melanogaster, the SWI/SNF ATPase Brahma (dBRM) interacts with the CPSF6 subunit of cleavage factor I. We have investigated the function of dBRM in 3' end formation in S2 cells by RNA interference, single-gene analysis and RNA sequencing. Our data show that dBRM facilitates pre-mRNA cleavage in two different ways: by promoting the association of CPSF6 to the cleavage region and by stabilizing positioned nucleosomes downstream of the cleavage site. These findings show that SWI/SNF complexes play a role also in the cleavage of specific pre-mRNAs in animal cells.

Size-dependent Hamaker constants for silver and gold nanoparticles

NASA Astrophysics Data System (ADS)

Pinchuk, Pavlo; Jiang, Ke

2015-08-01

Hamaker-Lifshitz constants are material specific constants that are used to calculate van der Waals interaction forces between small particles in solution. Typically, these constants are size-independent and material specific. According to the Lifshitz theory, the Hamaker-Lifshitz constants can be calculated by taking integrals that include the dielectric permittivity, as a function of frequency, of the interacting particles and the medium around particles. The dielectric permittivity of interacting metal nanoparticles can be calculated using the Drude model, which is based on the assumption of motion of free conducting electrons. For bulk metals, the Drude model does not predict any sizedependence of the dielectric permittivity. However, the conducting electrons in small noble metal nanoparticles (R ~ 10nm) exhibit surface scattering, which changes the complex permittivity function. In this work, we show theoretically that scattering of the free conducting electrons inside silver and gold nanoparticles with the size of 1 - 50 nm leads to size-dependent dielectric permittivity and Hamaker-Lifshitz constants. We calculate numerically the Hamaker-Lifshitz constants for silver and gold nanoparticles with different diameters. The results of the study might be of interests for understanding colloidal stability of metal nanoparticles.

Molecular Dynamics Simulation of Rap1 Myb-type domain in Saccharomyces cerevisiae.

PubMed

Mukherjee, Koel; Pandey, Dev Mani; Vidyarthi, Ambarish Saran

2012-01-01

Telomere is a nucleoprotein complex that plays important role in stability and their maintenance and consists of random repeats of species specific motifs. In budding Saccharomyces cerevisiae, Repressor Activator Protein 1 (Rap1) is a sequence specific protein that involved in transcriptional regulation. Rap1 consist of three active domains like N-terminal BRCT-domain, DNA-binding domain and C-terminal RCT-domain. In this study the unknown 3D structure of Myb-type domain (having 61 residues) within DNAbinding domain was modeled by Modeller7, and verified using different online bioinformatics tools (ProCheck, WhatIf, Verify3D). Dynamics of Myb-type domain of Rap1was carried out through simulation studies using GROMACS software. Time dependent interactions among the molecules were analyzed by Root Mean Square Deviation (RMSD), Radius of Gyration (Rg) and Root Mean Square Fluctuation (RMSF) plots. Motional properties in reduced dimension were also performed by Principal Component Analysis (PCA). Result indicated that Rap1 interacts with DNA major groove through its Helix Turn Helix motifs. Helix 3 was rigid, less amount of fluctuation was found as it interacts with DNA major groove. Helix2 and N-terminal having considerable fluctuation in the time scale.

A novel TPR-BEN domain interaction mediates PICH-BEND3 association.

PubMed

Pitchai, Ganesha P; Kaulich, Manuel; Bizard, Anna H; Mesa, Pablo; Yao, Qi; Sarlos, Kata; Streicher, Werner W; Nigg, Erich A; Montoya, Guillermo; Hickson, Ian D

2017-11-02

PICH is a DNA translocase required for the maintenance of chromosome stability in human cells. Recent data indicate that PICH co-operates with topoisomerase IIα to suppress pathological chromosome missegregation through promoting the resolution of ultra-fine anaphase bridges (UFBs). Here, we identify the BEN domain-containing protein 3 (BEND3) as an interaction partner of PICH in human cells in mitosis. We have purified full length PICH and BEND3 and shown that they exhibit a functional biochemical interaction in vitro. We demonstrate that the PICH-BEND3 interaction occurs via a novel interface between a TPR domain in PICH and a BEN domain in BEND3, and have determined the crystal structure of this TPR-BEN complex at 2.2 Å resolution. Based on the structure, we identified amino acids important for the TPR-BEN domain interaction, and for the functional interaction of the full-length proteins. Our data reveal a proposed new function for BEND3 in association with PICH, and the first example of a specific protein-protein interaction mediated by a BEN domain. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Molecular docking and molecular dynamics studies on the interactions of hydroxylated polybrominated diphenyl ethers to estrogen receptor alpha.

PubMed

Lu, Qun; Cai, Zhengqing; Fu, Jie; Luo, Siyi; Liu, Chunsheng; Li, Xiaolin; Zhao, Dongye

2014-03-01

Environmental estrogens have attracted great concerns. Recent studies have indicated that some hydroxylated polybrominated diphenyl ethers (HO-PBDEs) can interact with estrogen receptor (ER), and exhibit estrogenic activity. However, interactions between HO-PBDEs and ER are not well understood. In this work, molecular docking and molecular dynamics (MD) simulations were performed to characterize interactions of two HO-PBDEs (4'-HO-BDE30 and 4'-HO-BDE121) with ERα. Surflex-Dock was employed to reveal the probable binding conformations of the compounds at the active site of ERα; MD simulation was used to determine the detailed binding process. The driving forces of the binding between HO-PBDEs and ERα were van der Waals and electrostatic interactions. The decomposition of the binding free energy indicated that the hydrogen bonds between the residues Glu353, Gly521 and ligands were crucial for anchoring the ligands into the active site of ERα and stabilizing their conformations. The results showed that different interaction modes and different specific interactions with some residues were responsible for the different estrogenic activities of the two HO-PBDEs. Copyright © 2013 Elsevier Inc. All rights reserved.

Apoptosis-linked Gene-2 (ALG-2)/Sec31 Interactions Regulate Endoplasmic Reticulum (ER)-to-Golgi Transport

PubMed Central

Helm, Jared R.; Bentley, Marvin; Thorsen, Kevin D.; Wang, Ting; Foltz, Lauren; Oorschot, Viola; Klumperman, Judith; Hay, Jesse C.

2014-01-01

Luminal calcium released from secretory organelles has been suggested to play a regulatory role in vesicle transport at several steps in the secretory pathway; however, its functional roles and effector pathways have not been elucidated. Here we demonstrate for the first time that specific luminal calcium depletion leads to a significant decrease in endoplasmic reticulum (ER)-to-Golgi transport rates in intact cells. Ultrastructural analysis revealed that luminal calcium depletion is accompanied by increased accumulation of intermediate compartment proteins in COPII buds and clusters of unfused COPII vesicles at ER exit sites. Furthermore, we present several lines of evidence suggesting that luminal calcium affected transport at least in part through calcium-dependent interactions between apoptosis-linked gene-2 (ALG-2) and the Sec31A proline-rich region: 1) targeted disruption of ALG-2/Sec31A interactions caused severe defects in ER-to-Golgi transport in intact cells; 2) effects of luminal calcium and ALG-2/Sec31A interactions on transport mutually required each other; and 3) Sec31A function in transport required luminal calcium. Morphological phenotypes of disrupted ALG-2/Sec31A interactions were characterized. We found that ALG-2/Sec31A interactions were not required for the localization of Sec31A to ER exit sites per se but appeared to acutely regulate the stability and trafficking of the cargo receptor p24 and the distribution of the vesicle tether protein p115. These results represent the first outline of a mechanism that connects luminal calcium to specific protein interactions regulating vesicle trafficking machinery. PMID:25006245

Dpb11 may function with RPA and DNA to initiate DNA replication.

PubMed

Bruck, Irina; Dhingra, Nalini; Martinez, Matthew P; Kaplan, Daniel L

2017-01-01

Dpb11 is required for the initiation of DNA replication in budding yeast. We found that Dpb11 binds tightly to single-stranded DNA (ssDNA) or branched DNA structures, while its human homolog, TopBP1, binds tightly to branched-DNA structures. We also found that Dpb11 binds stably to CDK-phosphorylated RPA, the eukaryotic ssDNA binding protein, in the presence of branched DNA. A Dpb11 mutant specifically defective for DNA binding did not exhibit tight binding to RPA in the presence of DNA, suggesting that Dpb11-interaction with DNA may promote the recruitment of RPA to melted DNA. We then characterized a mutant of Dpb11 that is specifically defective in DNA binding in budding yeast cells. Expression of dpb11-m1,2,3,5,ΔC results in a substantial decrease in RPA recruitment to origins, suggesting that Dpb11 interaction with DNA may be required for RPA recruitment to origins. Expression of dpb11-m1,2,3,5,ΔC also results in diminished GINS interaction with Mcm2-7 during S phase, while Cdc45 interaction with Mcm2-7 is like wild-type. The reduced GINS interaction with Mcm2-7 may be an indirect consequence of diminished origin melting. We propose that the tight interaction between Dpb11, CDK-phosphorylated RPA, and branched-DNA may be required for the essential function of stabilizing melted origin DNA in vivo. We also propose an alternative model, wherein Dpb11-DNA interaction is required for some other function in DNA replication initiation, such as helicase activation.

Mean-field velocity difference model considering the average effect of multi-vehicle interaction

NASA Astrophysics Data System (ADS)

Guo, Yan; Xue, Yu; Shi, Yin; Wei, Fang-ping; Lü, Liang-zhong; He, Hong-di

2018-06-01

In this paper, a mean-field velocity difference model(MFVD) is proposed to describe the average effect of multi-vehicle interactions on the whole road. By stability analysis, the stability condition of traffic system is obtained. Comparison with stability of full velocity-difference (FVD) model and the completeness of MFVD model are discussed. The mKdV equation is derived from MFVD model through nonlinear analysis to reveal the traffic jams in the form of the kink-antikink density wave. Then the numerical simulation is performed and the results illustrate that the average effect of multi-vehicle interactions plays an important role in effectively suppressing traffic jam. The increase strength of the mean-field velocity difference in MFVD model can rapidly reduce traffic jam and enhance the stability of traffic system.

No complexity–stability relationship in empirical ecosystems

PubMed Central

Jacquet, Claire; Moritz, Charlotte; Morissette, Lyne; Legagneux, Pierre; Massol, François; Archambault, Philippe; Gravel, Dominique

2016-01-01

Understanding the mechanisms responsible for stability and persistence of ecosystems is one of the greatest challenges in ecology. Robert May showed that, contrary to intuition, complex randomly built ecosystems are less likely to be stable than simpler ones. Few attempts have been tried to test May's prediction empirically, and we still ignore what is the actual complexity–stability relationship in natural ecosystems. Here we perform a stability analysis of 116 quantitative food webs sampled worldwide. We find that classic descriptors of complexity (species richness, connectance and interaction strength) are not associated with stability in empirical food webs. Further analysis reveals that a correlation between the effects of predators on prey and those of prey on predators, combined with a high frequency of weak interactions, stabilize food web dynamics relative to the random expectation. We conclude that empirical food webs have several non-random properties contributing to the absence of a complexity–stability relationship. PMID:27553393

Wing-wake interaction destabilizes hover equilibrium of a flapping insect-scale wing.

PubMed

Bluman, James; Kang, Chang-Kwon

2017-06-15

Wing-wake interaction is a characteristic nonlinear flow feature that can enhance unsteady lift in flapping flight. However, the effects of wing-wake interaction on the flight dynamics of hover are inadequately understood. We use a well-validated 2D Navier-Stokes equation solver and a quasi-steady model to investigate the role of wing-wake interaction on the hover stability of a fruit fly scale flapping flyer. The Navier-Stokes equations capture wing-wake interaction, whereas the quasi-steady models do not. Both aerodynamic models are tightly coupled to a flight dynamic model, which includes the effects of wing mass. The flapping amplitude, stroke plane angle, and flapping offset angle are adjusted in free flight for various wing rotations to achieve hover equilibrium. We present stability results for 152 simulations which consider different kinematics involving the pitch amplitude and pitch axis as well as the duration and timing of pitch rotation. The stability of all studied motions was qualitatively similar, with an unstable oscillatory mode present in each case. Wing-wake interaction has a destabilizing effect on the longitudinal stability, which cannot be predicted by a quasi-steady model. Wing-wake interaction increases the tendency of the flapping flyer to pitch up in the presence of a horizontal velocity perturbation, which further destabilizes the unstable oscillatory mode of hovering flight dynamics.

Involvement of a bifunctional, paired-like DNA-binding domain and a transpositional enhancer in Sleeping Beauty transposition.

PubMed

Izsvák, Zsuzsanna; Khare, Dheeraj; Behlke, Joachim; Heinemann, Udo; Plasterk, Ronald H; Ivics, Zoltán

2002-09-13

Sleeping Beauty (SB) is the most active Tc1/mariner-like transposon in vertebrate species. Each of the terminal inverted repeats (IRs) of SB contains two transposase-binding sites (DRs). This feature, termed the IR/DR structure, is conserved in a group of Tc1-like transposons. The DNA-binding region of SB transposase, similar to the paired domain of Pax proteins, consists of two helix-turn-helix subdomains (PAI + RED = PAIRED). The N-terminal PAI subdomain was found to play a dominant role in contacting the DRs. Transposase was able to bind to mutant sites retaining the 3' part of the DRs; thus, primary DNA binding is not sufficient to determine the specificity of the transposition reaction. The PAI subdomain was also found to bind to a transpositional enhancer-like sequence within the left IR of SB, and to mediate protein-protein interactions between transposase subunits. A tetrameric form of the transposase was detected in solution, consistent with an interaction between the IR/DR structure and a transposase tetramer. We propose a model in which the transpositional enhancer and the PAI subdomain stabilize complexes formed by a transposase tetramer bound at the IR/DR. These interactions may result in enhanced stability of synaptic complexes, which might explain the efficient transposition of Sleeping Beauty in vertebrate cells.

Receptor dimer stabilization by hierarchical plasma membrane microcompartments regulates cytokine signaling

PubMed Central

You, Changjiang; Marquez-Lago, Tatiana T.; Richter, Christian Paolo; Wilmes, Stephan; Moraga, Ignacio; Garcia, K. Christopher; Leier, André; Piehler, Jacob

2016-01-01

The interaction dynamics of signaling complexes is emerging as a key determinant that regulates the specificity of cellular responses. We present a combined experimental and computational study that quantifies the consequences of plasma membrane microcompartmentalization for the dynamics of type I interferon receptor complexes. By using long-term dual-color quantum dot (QD) tracking, we found that the lifetime of individual ligand-induced receptor heterodimers depends on the integrity of the membrane skeleton (MSK), which also proved important for efficient downstream signaling. By pair correlation tracking and localization microscopy as well as by fast QD tracking, we identified a secondary confinement within ~300-nm-sized zones. A quantitative spatial stochastic diffusion-reaction model, entirely parameterized on the basis of experimental data, predicts that transient receptor confinement by the MSK meshwork allows for rapid reassociation of dissociated receptor dimers. Moreover, the experimentally observed apparent stabilization of receptor dimers in the plasma membrane was reproduced by simulations of a refined, hierarchical compartment model. Our simulations further revealed that the two-dimensional association rate constant is a key parameter for controlling the extent of MSK-mediated stabilization of protein complexes, thus ensuring the specificity of this effect. Together, experimental evidence and simulations support the hypothesis that passive receptor confinement by MSK-based microcompartmentalization promotes maintenance of signaling complexes in the plasma membrane. PMID:27957535

A chemical equilibrium model for metal adsorption onto bacterial surfaces

NASA Astrophysics Data System (ADS)

Fein, Jeremy B.; Daughney, Christopher J.; Yee, Nathan; Davis, Thomas A.

1997-08-01

This study quantifies metal adsorption onto cell wall surfaces of Bacillus subtilis by applying equilibrium thermodynamics to the specific chemical reactions that occur at the water-bacteria interface. We use acid/base titrations to determine deprotonation constants for the important surface functional groups, and we perform metal-bacteria adsorption experiments, using Cd, Cu, Pb, and Al, to yield site-specific stability constants for the important metal-bacteria surface complexes. The acid/base properties of the cell wall of B. subtilis can best be characterized by invoking three distinct types of surface organic acid functional groups, with pK a values of 4.82 ± 0.14, 6.9 ± 0.5, and 9.4 ± 0.6. These functional groups likely correspond to carboxyl, phosphate, and hydroxyl sites, respectively, that are displayed on the cell wall surface. The results of the metal adsorption experiments indicate that both the carboxyl sites and the phosphate sites contribute to metal uptake. The values of the log stability constants for metal-carboxyl surface complexes range from 3.4 for Cd, 4.2 for Pb, 4.3 for Cu, to 5.0 for Al. These results suggest that the stabilities of the metal-surface complexes are high enough for metal-bacterial interactions to affect metal mobilities in many aqueous systems, and this approach enables quantitative assessment of the effects of bacteria on metal mobilities.

Development of a stable cation modified graphene oxide membrane for water treatment

NASA Astrophysics Data System (ADS)

Yu, Wenzheng; (Yet Yu, Tong; Graham, Nigel

2017-12-01

Membranes prepared from layers of graphene oxide (GO) offer substantial advantages over conventional materials for water treatment (e.g. greater flux), but the stability of GO membranes in water has not been achieved until now. In this study the behavior of GO membranes prepared with different quantities and species of cations has been investigated to establish the feasibility of their application in water treatment. A range of cation-modified GO membranes were prepared and exposed to aqueous solutions containing specific chemical constituents. In pure water, unmodified and Na-modified GO membranes were highly unstable, while GO membranes modified with multivalent cations were stable provided there were sufficient quantities of cations present; their relative capability to achieve GO stability was as follows: Al3+  >  Ca2+  >  Mg2+  >  Na+. It is believed that the mechanism of cross-linking, and membrane stability, is via metal-carboxylate chelates and cation-graphite surface interactions (cation-π interaction), and that the latter appears to increase with increasing cation valency. The instability of cation (Ca or Al)-modified GO membranes by NaCl solutions during permeation occurred as Na+ exchanged with the incorporated multivalent cations, but a high content of Al3+ in the GO membrane impeded Al3+/Na+ exchange and thus retained membrane stability. In solutions containing biopolymers representative of surface waters or seawater (protein and polysaccharide solutions), Ca-GO membranes (even with high Ca2+ content) were not stable, while Al-GO membranes were stable if the Al3+ content was sufficiently high; Al-formed membranes also had a greater flux than Ca-GO membranes.

A new approach for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G1.

PubMed

De Nardis, Camilla; Hendriks, Linda J A; Poirier, Emilie; Arvinte, Tudor; Gros, Piet; Bakker, Alexander B H; de Kruif, John

2017-09-01

Bispecific antibodies combine two different antigen-binding sites in a single molecule, enabling more specific targeting, novel mechanisms of action, and higher clinical efficacies. Although they have the potential to outperform conventional monoclonal antibodies, many bispecific antibodies have issues regarding production, stability, and pharmacokinetic properties. Here, we describe a new approach for generating bispecific antibodies using a common light chain format and exploiting the stable architecture of human immunoglobulin G 1 We used iterative experimental validation and computational modeling to identify multiple Fc variant pairs that drive efficient heterodimerization of the antibody heavy chains. Accelerated stability studies enabled selection of one Fc variant pair dubbed "DEKK" consisting of substitutions L351D and L368E in one heavy chain combined with L351K and T366K in the other. Solving the crystal structure of the DEKK Fc region at a resolution of 2.3 Å enabled detailed analysis of the interactions inducing CH3 interface heterodimerization. Local shifts in the IgG backbone accommodate the introduction of lysine side chains that form stabilizing salt-bridge interactions with substituted and native residues in the opposite chain. Overall, the CH3 domain adapted to these shifts at the interface, yielding a stable Fc conformation very similar to that in wild-type IgG. Using the DEKK format, we generated the bispecific antibody MCLA-128, targeting human EGF receptors 2 and 3. MCLA-128 could be readily produced and purified at industrial scale with a standard mammalian cell culture platform and a routine purification protocol. Long-term accelerated stability assays confirmed that MCLA-128 is highly stable and has excellent biophysical characteristics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Mammalian Sterile 20-like Kinase 1 (Mst1) Enhances the Stability of Forkhead Box P3 (Foxp3) and the Function of Regulatory T Cells by Modulating Foxp3 Acetylation.

PubMed

Li, Jiang; Du, Xingrong; Shi, Hao; Deng, Kejing; Chi, Hongbo; Tao, Wufan

2015-12-25

Regulatory T cells (Tregs) play crucial roles in maintaining immune tolerance. The transcription factor Foxp3 is a critical regulator of Treg development and function, and its expression is regulated at both transcriptional and post-translational levels. Acetylation by lysine acetyl transferases/lysine deacetylases is one of the main post-translational modifications of Foxp3, which regulate Foxp3's stability and transcriptional activity. However, the mechanism(s) by which the activities of these lysine acetyl transferases/lysine deacetylases are regulated to preserve proper Foxp3 acetylation during Treg development and maintenance of Treg function remains to be determined. Here we report that Mst1 can enhance Foxp3 stability, its transcriptional activity, and Treg function by modulating the Foxp3 protein at the post-translational level. We discovered that Mst1 could increase the acetylation of Foxp3 by inhibiting Sirt1 activity, which requires the Mst1 kinase activity. We also found that Mst1 could attenuate Sirt1-mediated deacetylation of Foxp3 through directly interacting with Foxp3 to prevent or interfere the interaction between Sirt1 and Foxp3. Therefore, Mst1 can regulate Foxp3 stability in kinase-dependent and kinase-independent manners. Finally, we showed that treatment of Mst1(-/-) Tregs with Ex-527, a Sirt1-specific inhibitor, partially restored the suppressive function of Mst1(-/-) Tregs. Our studies reveal a novel mechanism by which Mst1 enhances Foxp3 expression and Treg function at the post-translational level. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A new approach for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G1

PubMed Central

De Nardis, Camilla; Hendriks, Linda J. A.; Poirier, Emilie; Arvinte, Tudor; Gros, Piet; Bakker, Alexander B. H.; de Kruif, John

2017-01-01

Bispecific antibodies combine two different antigen-binding sites in a single molecule, enabling more specific targeting, novel mechanisms of action, and higher clinical efficacies. Although they have the potential to outperform conventional monoclonal antibodies, many bispecific antibodies have issues regarding production, stability, and pharmacokinetic properties. Here, we describe a new approach for generating bispecific antibodies using a common light chain format and exploiting the stable architecture of human immunoglobulin G1. We used iterative experimental validation and computational modeling to identify multiple Fc variant pairs that drive efficient heterodimerization of the antibody heavy chains. Accelerated stability studies enabled selection of one Fc variant pair dubbed “DEKK” consisting of substitutions L351D and L368E in one heavy chain combined with L351K and T366K in the other. Solving the crystal structure of the DEKK Fc region at a resolution of 2.3 Å enabled detailed analysis of the interactions inducing CH3 interface heterodimerization. Local shifts in the IgG backbone accommodate the introduction of lysine side chains that form stabilizing salt-bridge interactions with substituted and native residues in the opposite chain. Overall, the CH3 domain adapted to these shifts at the interface, yielding a stable Fc conformation very similar to that in wild-type IgG. Using the DEKK format, we generated the bispecific antibody MCLA-128, targeting human EGF receptors 2 and 3. MCLA-128 could be readily produced and purified at industrial scale with a standard mammalian cell culture platform and a routine purification protocol. Long-term accelerated stability assays confirmed that MCLA-128 is highly stable and has excellent biophysical characteristics. PMID:28655766

Influence of outdoor running fatigue and medial tibial stress syndrome on accelerometer-based loading and stability.

PubMed

Schütte, Kurt H; Seerden, Stefan; Venter, Rachel; Vanwanseele, Benedicte

2018-01-01

Medial tibial stress syndrome (MTSS) is a common overuse running injury with pathomechanics likely to be exaggerated by fatigue. Wearable accelerometry provides a novel alternative to assess biomechanical parameters continuously while running in more ecologically valid settings. The purpose of this study was to determine the influence of outdoor running fatigue and MTSS on both dynamic loading and dynamic stability derived from trunk and tibial accelerometery. Runners with (n=14) and without (n=16) history of MTSS performed an outdoor fatigue run of 3200m. Accelerometer-based measures averaged per lap included dynamic loading of the trunk and tibia (i.e. axial peak positive acceleration, signal power magnitude, and shock attenuation) as well as dynamic trunk stability (i.e. tri-axial root mean square ratio, step and stride regularity, and sample entropy). Regression coefficients from generalised estimating equations were used to evaluate group by fatigue interactions. No evidence could be found for dynamic loading being higher with fatigue in runners with MTSS history (all measures p>0.05). One significant group by running fatigue interaction effect was detected for dynamic stability. Specifically, in MTSS only, decreases mediolateral sample entropy i.e. loss of complexity was associated with running fatigue (p<0.01). The current results indicate that entire acceleration waveform signals reflecting mediolateral trunk control is related to MTSS history, a compensation that went undetected in the non-fatigued running state. We suggest that a practical outdoor running fatigue protocol that concurrently captures trunk accelerometry-based movement complexity warrants further prospective investigation as an in-situ screening tool for MTSS individuals. Copyright © 2017 Elsevier B.V. All rights reserved.

Stability analysis of oil yield in oil palm (Elaeis guineensis) progenies in different environments.

PubMed

Rafii, M Y; Jalani, B S; Rajanaidu, N; Kushairi, A; Puteh, A; Latif, M A

2012-10-04

We evaluated 38 dura x pisifera (DP) oil palm progenies in four locations in Malaysia for genotype by environment interaction and genotypic stability studies. The DP progenies derived from crosses between pisifera palms of AVROS, Serdang S27B, Serdang 29/36, and Lever Cameroon were chosen to be the males' parent and Deli dura palms designated as females' parent. All the locations differed in terms of soil physical and chemical properties, and the soil types ranged from coastal clay to inland soils. The genotype by environment interaction and stability of the individual genotypes were analyzed for oil yield trait using several stability techniques. A genotype by environment interaction was detected for oil yield and it had a larger variance component than genotypic variance (σ(2)(gl)/σ(2)(g) = 139.7%). Genotype by environment interaction of oil yield was largely explained by a non-linear relationship between genotypic and environmental values. Overall assessment of individual genotypic stability showed that seven genotypes were highly stable and had consistent performance over the environments for the oil yield trait [total individual genotype stability scored more than 10 and mean oil yielded above the average of the environment (genotype means are more than 34.37 kg·palm(-1)·year(-1))]. These genotypes will be useful for oil palm breeding and tissue culture programs for developing high oil yielding planting materials with stable performance.

Mapping Interactions between Myosin Relay and Converter Domains That Power Muscle Function*

PubMed Central

Kronert, William A.; Melkani, Girish C.; Melkani, Anju; Bernstein, Sanford I.

2014-01-01

Intramolecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile508, Asn509, and Asp511) in communicating with converter domain residue Arg759. We found that the N509K relay mutation suppressed defects in myosin ATPase, in vitro motility, myofibril stability, and muscle function associated with the R759E converter mutation. Through molecular modeling, we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disabled motor function and myofibril assembly, suggesting that productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle. PMID:24627474

A first principle analysis of the structure of oligoanilines doped with alkylsulfonic acids.

PubMed

Casanovas, Jordi; Canales, Manel; Ferreira, Carlos A; Alemán, Carlos

2009-07-30

The interaction of polyaniline with alkylsulfonate dopants have been investigated at the atomic level using quantum mechanical methods. Calculations have been performed on complexes formed by dopant molecules with an alkyl group ranging from methyl to nonyl and model oligoanilines of different sizes. The stabilization provided by the formation of the alkylsulfonate...oligoaniline complexes (70-90 kcal/mol) is significantly higher than that found for conventional hydrogen bonds (5-12 kcal/mol) but lower than that obtained for methylsulfate...alkylammonium and methylsulfate...Na(+) systems (120-135 kcal/mol). On the other hand, the influence of size of the alkyl group contained in the dopant on the interaction is practically negligible, whereas, in opposition, the number of aniline units used to represent polyaniline significantly affects the energetics of the interaction. Specifically, the interaction energy of an alkyl-dopant molecule and an infinite polyaniline chain has been predicted to be around -65 kcal/mol. The overall results allow the conclusion that the interaction between alkylsulfonate dopants and polyaniline is a very local phenomenon.

Inter-subunit electrostatic interactions in ferritin molecule: comparison with inter-molecular interactions in crystals

NASA Astrophysics Data System (ADS)

Takahashi, Takuya; Hogyoku, Michiru; Nagayama, Kuniaki

1996-10-01

We evaluated the contribution of electrostatic interactions to the stability of macromolecular assembly in a horse L ferritin molecule composed of 24 subunits and the three-dimensional crystal of the ferritin molecules with numerical calculation of Poisson-Boltzmann equation based on dielectric model. The calculation showed that the electrostatic energy both favors the assembly of the 24 subunits and the crystalline assembly of the ferritin molecules (i.e., 24-mers). Short-range interactions less than 5 Å such as salt bridges and hydrogen bonds were important for both the subunit assembly and the crystalline assembly. To elucidate the strong stabilization by electrostatic interactions in both the ferritin 24-mer and its crystal, we analyzed the contribution of individual atoms. It revealed that the stabilization was arising from buried salt bridges or hydrogen bonds, which yielded more than 5 kcal/mol in some interactions. These large electrostatic stabilization and also the unexpected small ionic strength dependence was different from those of bovine pancreatic trypsin inhibitor (BPTI) orthorhombic and pig-insulin cubic crystals previously calculated. We also evaluated changes of the accessible surface area (ASA) and hydration free energy in accordance with the process of the subunit assembly. The change of hydration free energy, which was very large (i.e. ˜ + 100 kcal/mol/subunit) and unfavorable for the assembly, was proportional to the electrostatic hydration energy (i.e. Born energy change in hydration process). Hydrophobic groups were likely to appear more frequently than hydrophilic groups at the subunit interfaces. These results suggest that the molecular structure of the ferritin 24-mer and the crystal structure of the 24-mers were both stabilized by local electrostatic interactions, in particular. We view protein crystals as an extension of the protein oligomer to an infinite number of subunits association.

Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes.

PubMed

Bobone, Sara; Hilsch, Malte; Storm, Julian; Dunsing, Valentin; Herrmann, Andreas; Chiantia, Salvatore

2017-06-15

Influenza A virus matrix protein 1 (M1) is an essential component involved in the structural stability of the virus and in the budding of new virions from infected cells. A deeper understanding of the molecular basis of virion formation and the budding process is required in order to devise new therapeutic approaches. We performed a detailed investigation of the interaction between M1 and phosphatidylserine (PS) (i.e., its main binding target at the plasma membrane [PM]), as well as the distribution of PS itself, both in model membranes and in living cells. To this end, we used a combination of techniques, including Förster resonance energy transfer (FRET), confocal microscopy imaging, raster image correlation spectroscopy, and number and brightness (N&B) analysis. Our results show that PS can cluster in segregated regions in the plane of the lipid bilayer, both in model bilayers constituted of PS and phosphatidylcholine and in living cells. The viral protein M1 interacts specifically with PS-enriched domains, and such interaction in turn affects its oligomerization process. Furthermore, M1 can stabilize PS domains, as observed in model membranes. For living cells, the presence of PS clusters is suggested by N&B experiments monitoring the clustering of the PS sensor lactadherin. Also, colocalization between M1 and a fluorescent PS probe suggest that, in infected cells, the matrix protein can specifically bind to the regions of PM in which PS is clustered. Taken together, our observations provide novel evidence regarding the role of PS-rich domains in tuning M1-lipid and M1-M1 interactions at the PM of infected cells. IMPORTANCE Influenza virus particles assemble at the plasma membranes (PM) of infected cells. This process is orchestrated by the matrix protein M1, which interacts with membrane lipids while binding to the other proteins and genetic material of the virus. Despite its importance, the initial step in virus assembly (i.e., M1-lipid interaction) is still not well understood. In this work, we show that phosphatidylserine can form lipid domains in physical models of the inner leaflet of the PM. Furthermore, the spatial organization of PS in the plane of the bilayer modulates M1-M1 interactions. Finally, we show that PS domains appear to be present in the PM of living cells and that M1 seems to display a high affinity for them. Copyright © 2017 American Society for Microbiology.

Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes

PubMed Central

Bobone, Sara; Hilsch, Malte; Storm, Julian; Dunsing, Valentin; Herrmann, Andreas

2017-01-01

ABSTRACT Influenza A virus matrix protein 1 (M1) is an essential component involved in the structural stability of the virus and in the budding of new virions from infected cells. A deeper understanding of the molecular basis of virion formation and the budding process is required in order to devise new therapeutic approaches. We performed a detailed investigation of the interaction between M1 and phosphatidylserine (PS) (i.e., its main binding target at the plasma membrane [PM]), as well as the distribution of PS itself, both in model membranes and in living cells. To this end, we used a combination of techniques, including Förster resonance energy transfer (FRET), confocal microscopy imaging, raster image correlation spectroscopy, and number and brightness (N&B) analysis. Our results show that PS can cluster in segregated regions in the plane of the lipid bilayer, both in model bilayers constituted of PS and phosphatidylcholine and in living cells. The viral protein M1 interacts specifically with PS-enriched domains, and such interaction in turn affects its oligomerization process. Furthermore, M1 can stabilize PS domains, as observed in model membranes. For living cells, the presence of PS clusters is suggested by N&B experiments monitoring the clustering of the PS sensor lactadherin. Also, colocalization between M1 and a fluorescent PS probe suggest that, in infected cells, the matrix protein can specifically bind to the regions of PM in which PS is clustered. Taken together, our observations provide novel evidence regarding the role of PS-rich domains in tuning M1-lipid and M1-M1 interactions at the PM of infected cells. IMPORTANCE Influenza virus particles assemble at the plasma membranes (PM) of infected cells. This process is orchestrated by the matrix protein M1, which interacts with membrane lipids while binding to the other proteins and genetic material of the virus. Despite its importance, the initial step in virus assembly (i.e., M1-lipid interaction) is still not well understood. In this work, we show that phosphatidylserine can form lipid domains in physical models of the inner leaflet of the PM. Furthermore, the spatial organization of PS in the plane of the bilayer modulates M1-M1 interactions. Finally, we show that PS domains appear to be present in the PM of living cells and that M1 seems to display a high affinity for them. PMID:28356535

Counteraction of antibiotic production and degradation stabilizes microbial communities

PubMed Central

Kelsic, Eric D.; Zhao, Jeffrey; Vetsigian, Kalin; Kishony, Roy

2015-01-01

Summary A major challenge in theoretical ecology is understanding how natural microbial communities support species diversity1-8, and in particular how antibiotic producing, sensitive and resistant species coexist9-15. While cyclic “rock-paper-scissors” interactions can stabilize communities in spatial environments9-11, coexistence in unstructured environments remains an enigma12,16. Here, using simulations and analytical models, we show that the opposing actions of antibiotic production and degradation enable coexistence even in well-mixed environments. Coexistence depends on 3-way interactions where an antibiotic degrading species attenuates the inhibitory interactions between two other species. These 3-way interactions enable coexistence that is robust to substantial differences in inherent species growth rates and to invasion by “cheating” species that cease producing or degrading antibiotics. At least two antibiotics are required for stability, with greater numbers of antibiotics enabling more complex communities and diverse dynamical behaviors ranging from stable fixed-points to limit cycles and chaos. Together, these results show how multi-species antibiotic interactions can generate ecological stability in both spatial and mixed microbial communities, suggesting strategies for engineering synthetic ecosystems and highlighting the importance of toxin production and degradation for microbial biodiversity. PMID:25992546

Assessing Specific Oligonucleotides and Small Molecule Antibiotics for the Ability to Inhibit the CRD-BP-CD44 RNA Interaction

PubMed Central

Thomsen, Dana; Lee, Chow H.

2014-01-01

Studies on Coding Region Determinant-Binding Protein (CRD-BP) and its orthologs have confirmed their functional role in mRNA stability and localization. CRD-BP is present in extremely low levels in normal adult tissues, but it is over-expressed in many types of aggressive human cancers and in neonatal tissues. Although the exact role of CRD-BP in tumour progression is unclear, cumulative evidence suggests that its ability to physically associate with target mRNAs is an important criterion for its oncogenic role. CRD-BP has high affinity for the 3′UTR of the oncogenic CD44 mRNA and depletion of CRD-BP in cells led to destabilization of CD44 mRNA, decreased CD44 expression, reduced adhesion and disruption of invadopodia formation. Here, we further characterize the CRD-BP-CD44 RNA interaction and assess specific antisense oligonucleotides and small molecule antibiotics for their ability to inhibit the CRD-BP-CD44 RNA interaction. CRD-BP has a high affinity for binding to CD44 RNA nts 2862–3055 with a Kd of 645 nM. Out of ten antisense oligonucleotides spanning nts 2862–3055, only three antisense oligonucleotides (DD4, DD7 and DD10) were effective in competing with CRD-BP for binding to 32P-labeled CD44 RNA. The potency of DD4, DD7 and DD10 in inhibiting the CRD-BP-CD44 RNA interaction in vitro correlated with their ability to specifically reduce the steady-state level of CD44 mRNA in cells. The aminoglycoside antibiotics neomycin, paramomycin, kanamycin and streptomycin effectively inhibited the CRD-BP-CD44 RNA interaction in vitro. Assessing the potential inhibitory effect of aminoglycoside antibiotics including neomycin on the CRD-BP-CD44 mRNA interaction in cells proved difficult, likely due to their propensity to non-specifically bind nucleic acids. Our results have important implications for future studies in finding small molecules and nucleic acid-based inhibitors that interfere with protein-RNA interactions. PMID:24622399

Assessing specific oligonucleotides and small molecule antibiotics for the ability to inhibit the CRD-BP-CD44 RNA interaction.

PubMed

King, Dustin T; Barnes, Mark; Thomsen, Dana; Lee, Chow H

2014-01-01

Studies on Coding Region Determinant-Binding Protein (CRD-BP) and its orthologs have confirmed their functional role in mRNA stability and localization. CRD-BP is present in extremely low levels in normal adult tissues, but it is over-expressed in many types of aggressive human cancers and in neonatal tissues. Although the exact role of CRD-BP in tumour progression is unclear, cumulative evidence suggests that its ability to physically associate with target mRNAs is an important criterion for its oncogenic role. CRD-BP has high affinity for the 3'UTR of the oncogenic CD44 mRNA and depletion of CRD-BP in cells led to destabilization of CD44 mRNA, decreased CD44 expression, reduced adhesion and disruption of invadopodia formation. Here, we further characterize the CRD-BP-CD44 RNA interaction and assess specific antisense oligonucleotides and small molecule antibiotics for their ability to inhibit the CRD-BP-CD44 RNA interaction. CRD-BP has a high affinity for binding to CD44 RNA nts 2862-3055 with a Kd of 645 nM. Out of ten antisense oligonucleotides spanning nts 2862-3055, only three antisense oligonucleotides (DD4, DD7 and DD10) were effective in competing with CRD-BP for binding to 32P-labeled CD44 RNA. The potency of DD4, DD7 and DD10 in inhibiting the CRD-BP-CD44 RNA interaction in vitro correlated with their ability to specifically reduce the steady-state level of CD44 mRNA in cells. The aminoglycoside antibiotics neomycin, paramomycin, kanamycin and streptomycin effectively inhibited the CRD-BP-CD44 RNA interaction in vitro. Assessing the potential inhibitory effect of aminoglycoside antibiotics including neomycin on the CRD-BP-CD44 mRNA interaction in cells proved difficult, likely due to their propensity to non-specifically bind nucleic acids. Our results have important implications for future studies in finding small molecules and nucleic acid-based inhibitors that interfere with protein-RNA interactions.

Apparatus for the production of gel beads containing a biocatalyst

DOEpatents

Scott, C.D.; Scott, T.C.; Davison, B.H.

1998-03-19

An apparatus is described for the large-scale and continuous production of gel beads containing a biocatalyst. The apparatus is a columnar system based on the chemical cross-linking of hydrocolloidal gels that contain and immobilize a biocatalyst, the biocatalyst being a microorganism or an enzyme. Hydrocolloidal gels, such as alginate, carrageenan, and a mixture of bone gelatin and modified alginate, provide immobilization matrices that can be used to entrap and retain the biocatalyst while allowing effective contact with substrates and release of products. Such immobilized biocatalysts are generally formulated into small spheres or beads that have high concentrations of the biocatalyst within the gel matrix. The columnar system includes a gel dispersion nozzle submerged in a heated non-interacting liquid, typically an organic liquid, that is immiscible with water to allow efficient formation of spherical gel droplets, the non-interacting liquid having a specific gravity that is less than water so that the gel droplets will fall through the liquid by the force of gravity. The heated non-interacting liquid is in direct contact with a chilled upflowing non-interacting liquid that will provide sufficient residence time for the gel droplets as they fall through the liquid so that they will be cooled below the gelling temperature and form solid spheres. The upflowing non-interacting liquid is in direct contact with an upflowing temperature-controlled aqueous solution containing the necessary chemicals for cross-linking or fixing of the gel beads to add the necessary stability. The flow rates of the two liquid streams can be varied to control the proper residence time in each liquid section to accommodate the production of gel beads of differing settling velocities. A valve is provided for continuous removal of the stabilized gel beads from the bottom of the column. 1 fig.

Apparatus and method for the production of gel beads containing a biocatalyst

DOEpatents

Scott, Charles D.; Scott, Timothy C.; Davison, Brian H.

1998-01-01

An apparatus and method for the large-scale and continuous production of gel beads containing a biocatalyst. The apparatus is a columnar system based on the chemical cross-linking of hydrocolloidal gels that contain and immobilize a biocatalyst, the biocatalyst being a microorganism or an enzyme. Hydrocolloidal gels, such as alginate, carrageenan, and a mixture of bone gelatin and modified alginate, provide immobilization matrices that can be used to entrap and retain the biocatalyst while allowing effective contact with substrates and release of products. Such immobilized biocatalysts are generally formulated into small spheres or beads that have high concentrations of the biocatalyst within the gel matrix. The columnar system includes a gel dispersion nozzle submerged in a heated non-interacting liquid, typically an organic liquid, that is immiscible with water to allow efficient formation of spherical gel droplets, the non-interacting liquid having a specific gravity that is less than water so that the gel droplets will fall through the liquid by the force of gravity. The heated non-interacting liquid is in direct contact with a chilled upflowing non-interacting liquid that will provide sufficient residence time for the gel droplets as they fall through the liquid so that they will be cooled below the gelling temperature and form solid spheres. The upflowing non-interacting liquid is in direct contact with an upflowing temperature-controlled aqueous solution containing the necessary chemicals for cross-linking or fixing of the gel beads to add the necessary stability. The flow rates of the two liquid streams can be varied to control the proper residence time in each liquid section to accommodate the production of gel beads of differing settling velocities. A valve is provided for continuous removal of the stabilized gel beads from the bottom of the column.

Apparatus for the production of gel beads containing a biocatalyst

DOEpatents

Scott, Charles D.; Scott, Timothy C.; Davison, Brian H.

1998-01-01

An apparatus for the large-scale and continuous production of gel beads containing a biocatalyst. The apparatus is a columnar system based on the chemical cross-linking of hydrocolloidal gels that contain and immobilize a biocatalyst, the biocatalyst being a microorganism or an enzyme. Hydrocolloidal gels, such as alginate, carrageenan, and a mixture of bone gelatin and modified alginate, provide immobilization matrices that can be used to entrap and retain the biocatalyst while allowing effective contact with substrates and release of products. Such immobilized biocatalysts are generally formulated into small spheres or beads that have high concentrations of the biocatalyst within the gel matrix. The columnar system includes a gel dispersion nozzle submerged in a heated non-interacting liquid, typically an organic liquid, that is immiscible with water to allow efficient formation of spherical gel droplets, the non-interacting liquid having a specific gravity that is less than water so that the gel droplets will fall through the liquid by the force of gravity. The heated non-interacting liquid is in direct contact with a chilled upflowing non-interacting liquid that will provide sufficient residence time for the gel droplets as they fall through the liquid so that they will be cooled below the gelling temperature and form solid spheres. The upflowing non-interacting liquid is in direct contact with an upflowing temperature-controlled aqueous solution containing the necessary chemicals for cross-linking or fixing of the gel beads to add the necessary stability. The flow rates of the two liquid streams can be varied to control the proper residence time in each liquid section to accommodate the production of gel beads of differing settling velocities. A valve is provided for continuous removal of the stabilized gel beads from the bottom of the column.

Organism-Sediment Interactions Govern Post-Hypoxia Recovery of Ecosystem Functioning

PubMed Central

Van Colen, Carl; Rossi, Francesca; Montserrat, Francesc; Andersson, Maria G. I.; Gribsholt, Britta; Herman, Peter M. J.; Degraer, Steven; Vincx, Magda; Ysebaert, Tom; Middelburg, Jack J.

2012-01-01

Hypoxia represents one of the major causes of biodiversity and ecosystem functioning loss for coastal waters. Since eutrophication-induced hypoxic events are becoming increasingly frequent and intense, understanding the response of ecosystems to hypoxia is of primary importance to understand and predict the stability of ecosystem functioning. Such ecological stability may greatly depend on the recovery patterns of communities and the return time of the system properties associated to these patterns. Here, we have examined how the reassembly of a benthic community contributed to the recovery of ecosystem functioning following experimentally-induced hypoxia in a tidal flat. We demonstrate that organism-sediment interactions that depend on organism size and relate to mobility traits and sediment reworking capacities are generally more important than recovering species richness to set the return time of the measured sediment processes and properties. Specifically, increasing macrofauna bioturbation potential during community reassembly significantly contributed to the recovery of sediment processes and properties such as denitrification, bedload sediment transport, primary production and deep pore water ammonium concentration. Such bioturbation potential was due to the replacement of the small-sized organisms that recolonised at early stages by large-sized bioturbating organisms, which had a disproportionately stronger influence on sediment. This study suggests that the complete recovery of organism-sediment interactions is a necessary condition for ecosystem functioning recovery, and that such process requires long periods after disturbance due to the slow growth of juveniles into adult stages involved in these interactions. Consequently, repeated episodes of disturbance at intervals smaller than the time needed for the system to fully recover organism-sediment interactions may greatly impair the resilience of ecosystem functioning. PMID:23185440

IraL Is an RssB Anti-adaptor That Stabilizes RpoS during Logarithmic Phase Growth in Escherichia coli and Shigella

PubMed Central

Hryckowian, Andrew J.; Battesti, Aurelia; Lemke, Justin J.; Meyer, Zachary C.

2014-01-01

ABSTRACT RpoS (σS), the general stress response sigma factor, directs the expression of genes under a variety of stressful conditions. Control of the cellular σS concentration is critical for appropriately scaled σS-dependent gene expression. One way to maintain appropriate levels of σS is to regulate its stability. Indeed, σS degradation is catalyzed by the ClpXP protease and the recognition of σS by ClpXP depends on the adaptor protein RssB. Three anti-adaptors (IraD, IraM, and IraP) exist in Escherichia coli K-12; each interacts with RssB and inhibits RssB activity under different stress conditions, thereby stabilizing σS. Unlike K-12, some E. coli isolates, including uropathogenic E. coli strain CFT073, show comparable cellular levels of σS during the logarithmic and stationary growth phases, suggesting that there are differences in the regulation of σS levels among E. coli strains. Here, we describe IraL, an RssB anti-adaptor that stabilizes σS during logarithmic phase growth in CFT073 and other E. coli and Shigella strains. By immunoblot analyses, we show that IraL affects the levels and stability of σS during logarithmic phase growth. By computational and PCR-based analyses, we reveal that iraL is found in many E. coli pathotypes but not in laboratory-adapted strains. Finally, by bacterial two-hybrid and copurification analyses, we demonstrate that IraL interacts with RssB by a mechanism distinct from that used by other characterized anti-adaptors. We introduce a fourth RssB anti-adaptor found in E. coli species and suggest that differences in the regulation of σS levels may contribute to host and niche specificity in pathogenic and nonpathogenic E. coli strains. PMID:24865554

Protein Stability in Mixed Solvents: A Balance of Contact Interaction and Excluded Volume

PubMed Central

Schellman, John A.

2003-01-01

Changes in excluded volume and contact interaction with the surface of a protein have been suggested as mechanisms for the changes in stability induced by cosolvents. The aim of the present paper is to present an analysis that combines both effects in a quantitative manner. The result is that both processes are present in both stabilizing and destabilizing interactions and neither can be ignored. Excluded volume was estimated using accessible surface area calculations of the kind introduced by Lee and Richards. The change in excluded volume on unfolding, ΔX, is quite large. For example, ΔX for ribonuclease is 6.7 L in urea and ∼16 L in sucrose. The latter number is greater than the molar volume of the protein. Direct interaction with the protein is represented as the solvent exchange mechanism, which differs from ordinary association theory because of the weakness of the interaction and the high concentrations of cosolvents. The balance between the two effects and their contribution to overall stability are most simply presented as bar diagrams as in Fig. 3. Our finding for five proteins is that excluded volume contributes to the stabilization of the native structure and that contact interaction contributes to destabilization. This is true for five proteins and four cosolvents including both denaturants and osmolytes. Whether a substance stabilizes a protein or destabilizes it depends on the relative size of these two contributions. The constant for the cosolvent contact with the protein is remarkably uniform for four of the proteins, indicating a similarity of groups exposed during unfolding. One protein, staphylococcus nuclease, is anomalous in almost all respects. In general, the strength of the interaction with guanidinium is about twice that of urea, which is about twice that of trimethylamine-N-oxide and sucrose. Arguments are presented for the use of volume fractions in equilibrium equations and the ignoring of activity coefficients of the cosolvent. It is shown in the Appendix that both the excluded volume and the direct interaction can be extracted in a unified way from the McMillan-Mayer formula for the second virial coefficient. PMID:12829469

Loss of conformational stability in calmodulin upon methionine oxidation.

PubMed Central

Gao, J; Yin, D H; Yao, Y; Sun, H; Qin, Z; Schöneich, C; Williams, T D; Squier, T C

1998-01-01

We have used electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and fluorescence spectroscopy to investigate the secondary and tertiary structural consequences that result from oxidative modification of methionine residues in wheat germ calmodulin (CaM), and prevent activation of the plasma membrane Ca-ATPase. Using ESI-MS, we have measured rates of modification and molecular mass distributions of oxidatively modified CaM species (CaMox) resulting from exposure to H2O2. From these rates, we find that oxidative modification of methionine to the corresponding methionine sulfoxide does not predispose CaM to further oxidative modification. These results indicate that methionine oxidation results in no large-scale alterations in the tertiary structure of CaMox, because the rates of oxidative modification of individual methionines are directly related to their solvent exposure. Likewise, CD measurements indicate that methionine oxidation results in little change in the apparent alpha-helical content at 28 degrees C, and only a small (0.3 +/- 0.1 kcal mol(-1)) decrease in thermal stability, suggesting the disruption of a limited number of specific noncovalent interactions. Fluorescence lifetime, anisotropy, and quenching measurements of N-(1-pyrenyl)-maleimide (PMal) covalently bound to Cys26 indicate local structural changes around PMal in the amino-terminal domain in response to oxidative modification of methionine residues in the carboxyl-terminal domain. Because the opposing globular domains remain spatially distant in both native and oxidatively modified CaM, the oxidative modification of methionines in the carboxyl-terminal domain are suggested to modify the conformation of the amino-terminal domain through alterations in the structural features involving the interdomain central helix. The structural basis for the linkage between oxidative modification and these global conformational changes is discussed in terms of possible alterations in specific noncovalent interactions that have previously been suggested to stabilize the central helix in CaM. PMID:9512014

Application of D-optimal experimental design method to optimize the formulation of O/W cosmetic emulsions.

PubMed

Djuris, J; Vasiljevic, D; Jokic, S; Ibric, S

2014-02-01

This study investigates the application of D-optimal mixture experimental design in optimization of O/W cosmetic emulsions. Cetearyl glucoside was used as a natural, biodegradable non-ionic emulsifier in the relatively low concentration (1%), and the mixture of co-emulsifiers (stearic acid, cetyl alcohol, stearyl alcohol and glyceryl stearate) was used to stabilize the formulations. To determine the optimal composition of co-emulsifiers mixture, D-optimal mixture experimental design was used. Prepared emulsions were characterized with rheological measurements, centrifugation test, specific conductivity and pH value measurements. All prepared samples appeared as white and homogenous creams, except for one homogenous and viscous lotion co-stabilized by stearic acid alone. Centrifugation testing revealed some phase separation only in the case of sample co-stabilized using glyceryl stearate alone. The obtained pH values indicated that all samples expressed mild acid value acceptable for cosmetic preparations. Specific conductivity values are attributed to the multiple phases O/W emulsions with high percentages of fixed water. Results of the rheological measurements have shown that the investigated samples exhibited non-Newtonian thixotropic behaviour. To determine the influence of each of the co-emulsifiers on emulsions properties, the obtained results were evaluated by the means of statistical analysis (ANOVA test). On the basis of comparison of statistical parameters for each of the studied responses, mixture reduced quadratic model was selected over the linear model implying that interactions between co-emulsifiers play the significant role in overall influence of co-emulsifiers on emulsions properties. Glyceryl stearate was found to be the dominant co-emulsifier affecting emulsions properties. Interactions between the glyceryl stearate and other co-emulsifiers were also found to significantly influence emulsions properties. These findings are especially important as they can be used for development of the product that meets users' requirements, as represented in the study. © 2013 Society of Cosmetic Scientists and the Société Française de Cosmétologie.