Non-cooperative Manner Research Articles

A stability analysis with time-delay of primal–dual power control in optical links

Primal-dual power control algorithms in optical networks must be designed properly to remain stable in the presence of time-delays. Control algorithms at the signal transmitters continuously update their channel powers in response to dynamic pricing information from the network links. The control algorithms optimize the optical signal-to-noise ratios (OSNRs) of the channels in a distributive and non-cooperative manner. We consider the case of a single link with multiple channels and a constant time-delay. These results also apply to single sink general network configurations if the time-delay represents the longest round-trip time in the network. We derive sufficient conditions for the stability of the closed loop system based on a tunable parameter in the control algorithm. The work combines singular perturbation theory with Lyapunov–Razumikhin time-delay techniques. We verify our results through simulations based on realistic network parameters.

Noncooperative Folding of Subdomains in Adenylate Kinase

Conformational change is regulating the biological activity of a large number of proteins and enzymes. Efforts in structural biology have provided molecular descriptions of the interactions that stabilize the stable ground states on the reaction trajectories during conformational change. Less is known about equilibrium thermodynamic stabilities of the polypeptide segments that participate in structural changes and whether the stabilities are relevant for the reaction pathway. Adenylate kinase (Adk) is composed of three subdomains: CORE, ATPlid, and AMPbd. ATPlid and AMPbd are flexible nucleotide binding subdomains where large-scale conformational changes are directly coupled to catalytic activity. In this report, the equilibrium thermodynamic stabilities of Adk from both mesophilic and hyperthermophilic bacteria were investigated using solution state NMR spectroscopy together with protein engineering experiments. Equilibrium hydrogen to deuterium exchange experiments indicate that the flexible subdomains are of significantly lower thermodynamic stability compared to the CORE subdomain. Using site-directed mutagenesis, parts of ATPlid and AMPbd could be selectively unfolded as a result of perturbation of hydrophobic clusters located in these respective subdomains. Analysis of the perturbed Adk variants using NMR spin relaxation and C(alpha) chemical shifts shows that the CORE subdomain can fold independently of ATPlid and AMPbd; consequently, folding of the two flexible subdomains occurs independently of each other. Based on the experimental results it is apparent that the flexible subdomains fold into their native structure in a noncooperative manner with respect to the CORE subdomain. These results are discussed in light of the catalytically relevant conformational change of ATPlid and AMPbd.

Multivalent Recognition of Peptides by Modular Self-Assembled Receptors

Developing nontraditional approaches to the synthesis and characterization of multivalent compounds is critical to our efforts to study and interface with biological systems and to build new noncovalent materials. This paper demonstrates a biomimetic approach to the construction of discrete, modular, multivalent receptors via molecular self-assembly in aqueous solution. Scaffolds presenting 1-3 viologen groups recruit a respective 1-3 copies of the synthetic host, cucurbit[8]uril, in a noncooperative manner and with a consistent equilibrium association constant (K(a)) value of 2 x 10(6) M(-1) per binding site. The assembled mono-, di-, and trivalent receptors bind to their cognate target peptides containing 1-3 Trp residues with K(a) values in the range 1.7 x 10(4)-4.7 x 10(6) M(-1) and in predetermined mono- or multivalent binding modes with 31-280-fold enhancements in affinity and additive enthalpies due to multivalency. The extent of valency was determined directly by measuring the visible charge-transfer absorptivity due to the viologen-indole pair. The predictable behavior of this system and its ease of synthesis and analysis make it well suited to serve as a model for multivalent binding and for the multivalent recognition of peptides by design.

Noncooperative Formation of the Off-Pathway Molten Globule during Folding of the α−β Parallel Protein Apoflavodoxin

During folding of many proteins, molten globules are formed. These partially folded forms of proteins have a substantial amount of secondary structure but lack virtually all tertiary side-chain packing characteristic of native structures. Molten globules are ensembles of interconverting conformers and are prone to aggregation, which can have detrimental effects on organisms. Consequently, molten globules attract considerable attention. The molten globule that is observed during folding of flavodoxin from Azotobacter vinelandii is a kinetically off-pathway species, as it has to unfold before the native state of the protein can be formed. This intermediate contains helices and can be populated at equilibrium using guanidinium hydrochloride as denaturant, allowing the use of NMR spectroscopy to follow molten globule formation at the residue level. Here, we track changes in chemical shifts of backbone amides, as well as disappearance of resonances of unfolded apoflavodoxin, upon decreasing denaturant concentration. Analysis of the data shows that structure formation within virtually all parts of the unfolded protein precedes folding to the molten globule state. This folding transition is noncooperative and involves a series of distinct transitions. Four structured elements in unfolded apoflavodoxin transiently interact and subsequently form the ordered core of the molten globule. Although hydrophobic, tryptophan side chains are not involved in the latter process. This ordered core is gradually extended upon decreasing denaturant concentration, but part of apoflavodoxin's molten globule remains random coil in the denaturant range investigated. The results presented here, together with those reported on the molten globule of alpha-lactalbumin, show that helical molten globules apparently fold in a noncooperative manner.

Thermally induced early events of ribonuclease A under reducing conditions: Evidenced by principal component analysis and two-dimensional correlation infrared spectroscopy

The thermally induced unfolding of ribonuclease A in the presence of 2-mercaptoethanol has been followed by fluorescence spectroscopy and Fourier transform infrared (FTIR) spectroscopy. Principal component analysis (PCA) in combination with two-dimensional (2D) infrared correlation spectroscopy has been employed as an attractive method for the investigation of subtle pretransitional conformational changes in the protein. Separate analyses of different stages indicated that, in stage I the change of β1(43–49) and α2(24–34) might be the main process; in stage II, β1 was further unfolded, changes of α1(3–13) and another species of β2(61–63) and β4(79–87) involved. Furthermore, quantitative analysis of the power spectra extracted from the synchronous 2D contour maps revealed that the intensity variations from pretransitional stages (below 47 °C) were 5% less than that for the main unfolding. Meanwhile, under reducing conditions the early subtle structural changes occurred in a non-cooperative manner, in contrast to what was found under non-reducing conditions.

α-Helix folding in the presence of structural constraints

We have investigated the site-specific folding kinetics of a photoswitchable cross-linked alpha-helical peptide by using single (13)C = (18)O isotope labeling together with time-resolved IR spectroscopy. We observe that the folding times differ from site to site by a factor of eight at low temperatures (6 degrees C), whereas at high temperatures (45 degrees C), the spread is considerably smaller. The trivial sum of the site signals coincides with the overall folding signal of the unlabeled peptide, and different sites fold in a noncooperative manner. Moreover, one of the sites exhibits a decrease of hydrogen bonding upon folding, implying that the unfolded state at low temperature is not unstructured. Molecular dynamics simulations at low temperature reveal a stretched-exponential behavior which originates from parallel folding routes that start from a kinetically partitioned unfolded ensemble. Different metastable structures (i.e., traps) in the unfolded ensemble have a different ratio of loop and helical content. Control simulations of the peptide at high temperature, as well as without the cross-linker at low temperature, show faster and simpler (i.e., single-exponential) folding kinetics. The experimental and simulation results together provide strong evidence that the rate-limiting step in formation of a structurally constrained alpha-helix is the escape from heterogeneous traps rather than the nucleation rate. This conclusion has important implications for an alpha-helical segment within a protein, rather than an isolated alpha-helix, because the cross-linker is a structural constraint similar to those present during the folding of a globular protein.

Noncooperative cadmium(II) binding to human metallothionein 1a

The two-domain (βα) mammalian metallothionein binds seven divalent metals, however, the binding mechanism is not well characterized and recent reports require the presence of the partially metallated protein. In this paper, step-wise metallation of the metal-free, two-domain βα-rhMT and the isolated β-rhMT using Cd(II) is shown to proceed in a noncooperative manner by analysis of electrospray ionization mass spectrometric data. Under limiting amounts of Cd(II), all intermediate metallation states up to the fully metallated Cd 3-β-rhMT and Cd 7-βα-rhMT were observed. Addition of excess Cd(II), resulted in formation of the supermetallated (metallation in excess of normal levels) Cd 4-β- and Cd 8-βα-metallothionein species. These data establish that noncooperative cadmium metallation is a property of each isolated domain and the complete two-domain protein. Our data now also establish that supermetallation is a property that may provide information about the mechanism of metal transfer to other proteins.

Subunits act independently in a cyclic nucleotide‐activated K + channel

Ion channels gated by cyclic nucleotides have crucial roles in neuronal excitability and signal transduction of sensory neurons. Here, we studied ligand binding of a cyclic nucleotide-activated K(+) channel from Mesorhizobium loti and its isolated cyclic nucleotide-binding domain. The channel and the binding domain alone bind cyclic AMP with similar affinity in a non-cooperative manner. The cAMP sensitivities of binding and activation coincide. Thus, each subunit in the tetrameric channel acts independently of the others. The binding and gating properties of the bacterial channel are distinctively different from those of eukaryotic cyclic nucleotide-gated channels.

Decentralized inventory control in a two-stage capacitated supply chain

This paper investigates a two-stage supply chain consisting of a capacitated supplier and a retailer that faces a stationary random demand. Both the supplier and the retailer employ base stock policies for inventory replenishment. All unsatisfied demand is backlogged and the customer backorder cost is shared between the supplier and the retailer. We investigate the determination of decentralized inventory decisions when the two parties optimize their individual inventory-related costs in a noncooperative manner. We explicitly characterize the Nash equilibrium inventory strategies and identify the causes of inefficiency in the decentralized operation. We then study a set of simple linear contracts to see whether these inefficiencies can be overcome. Finally, we investigate Stackelberg games where one of the parties is assumed to be dominating.

Quantitative analysis of HP1α binding to nucleosomal arrays

Elucidating how the metazoan genome is organised into distinct functional domains is fundamental to understanding all aspects of normal cellular growth and development. The “histone code” hypothesis predicts that post-translational modifications of specific histone residues regulate genomic function by selectively recruiting nuclear factors that modify chromatin structure. A paradigm supporting this hypothesis is the preferential binding of the silencing protein heterochromatin protein 1 (HP1) to histone H3 trimethylated at K9. However, a caveat to several in vitro studies is that they employed histone N-terminal tail peptides to determine dissociation constants, thus ignoring any potential role of DNA and/or the underlying chromatin structure in the recruitment of HP1. Using a well-defined in vitro chromatin assembly system (employing a 12-208 DNA template), we describe here, the use of a fluorescence spectroscopic method that enabled us to measure and quantify the relative binding affinities of HP1α to unmodified and variant nucleosomal arrays. Using this approach, we previously demonstrated that mouse HP1α (i) binds with high affinity to naked DNA, (ii) has an intrinsic affinity for highly folded chromatin, (iii) has a 2-fold higher affinity for nucleosomal arrays when H2A is replaced with H2A.Z, and (iv) binds to DNA or chromatin in a non-cooperative manner.

CAMP Is a Ligand for the Tandem GAF Domain of Human Phosphodiesterase 10 and cGMP for the Tandem GAF Domain of Phosphodiesterase 11

N-terminal tandem GAF domains are present in 5 out of 11 mammalian phosphodiesterase (PDE) families. The ligand for the GAF domains of PDEs 2, 5, and 6 is cGMP, whereas those for PDEs 10 and 11 remained enigmatic for years. Here we used the cyanobacterial cyaB1 adenylyl cyclase, which has an N-terminal tandem GAF domain closely related to those of the mammalian PDEs, as an assay system to identify the ligands for the human PDEs 10 and 11 GAF domains. We report that a chimera between the PDE10 GAF domain and the cyanobacterial cyclase was 9-fold stimulated by cAMP (EC50= 19.8 microm), whereas cGMP had only low activity. cAMP increased Vmax in a non-cooperative manner and did not affect the Km for ATP of 27 microm. In an analogous chimeric construct with the tandem GAF domain of human PDE11A4, cGMP was identified as an allosteric activator (EC50 = 72.5 microm) that increased Vmax of the cyclase non-cooperatively 4-fold. GAF-B of PDE10 and GAF-A of PDE11A4 contain an invariant NKFDE motif present in all mammalian PDE GAF ensembles. We mutated the aspartates within this motif in both regions and found that intramolecular signaling was considerably reduced or abolished. This was in line with all data concerning GAF domains with an NKFDE motif as far as they have been tested. The data appeared to define those GAF domains as a distinct subclass within the >3100 annotated GAF domains for which we propose a tentative classification scheme.

The Predicted Coiled-coil Domain of Myosin 10 Forms a Novel Elongated Domain That Lengthens the Head

Myosin 10 contains a region of predicted coiled coil 120 residues long. However, the highly charged nature and pattern of charges in the proximal 36 residues appear incompatible with coiled-coil formation. Circular dichroism, NMR, and analytical ultracentrifugation show that a synthesized peptide containing this region forms a stable single alpha-helix (SAH) domain in solution and does not dimerize to form a coiled coil even at millimolar concentrations. Additionally, electron microscopy of a recombinant myosin 10 containing the motor, the three calmodulin binding domains, and the full-length predicted coiled coil showed that it was mostly monomeric at physiological protein concentration. In dimers the molecules were joined only at their extreme distal ends, and no coiled-coil tail was visible. Furthermore, the neck lengths of both monomers and dimers were much longer than expected from the number of calmodulin binding domains. In contrast, micrographs of myosin 5 heavy meromyosin obtained under the same conditions clearly showed a coiled-coil tail, and the necks were the predicted length. Thus the predicted coiled coil of myosin 10 forms a novel elongated structure in which the proximal region is a SAH domain and the distal region is a SAH domain (or has an unknown extended structure) that dimerizes only at its end. Sequence comparisons show that similar structures may exist in the predicted coiled-coil domains of myosins 6 and 7a and MyoM and could function to increase the size of the working stroke.

Berberine, a Strong Polyriboadenylic Acid Binding Plant Alkaloid: Spectroscopic, Viscometric, and Thermodynamic Study.

The interaction of berberine with single stranded poly(rA) structure was investigated using a combination of spectrophotometric, spectrofluorimetric, circular dichroic, viscometric, and thermodynamic studies. The interaction process was characterized by typical hypochromic and bathochromic effects in the absorption spectrum of berberine, enhancement of fluorescence intensity of berberine, increase of viscosity, and perturbation of circular dichroic spectrum of single stranded poly(rA). Scatchard plot obtained from spectrophotometric analysis showed that berberine bound strongly to single stranded poly(rA) in a non-cooperative manner. In contrast, berberine does not show any significant effect (i) in its absorbance and fluorescence spectra on binding to double stranded poly(rA), (ii) alter the circular dichroic spectrum of double stranded poly(rA), or (iii) increase of viscosity of double stranded poly(rA) indicating that it does not bind at all to double stranded poly(rA) structure. Thermodynamic parameters indicated that the binding of the alkaloid to single stranded poly(rA) is an endothermic process and entropy driven. All these findings, taken together clearly support that berberine binds strongly to single stranded poly(rA) structure by a mechanism of partial intercalation leading to its use in gene regulation in eukaryotic cells.

The interaction of the A and A* proteins of bacteriophage phi X174 with single-stranded and double-stranded phi X DNA in vitro.

The binding of the bacteriophage phi X 174-coded A and A* proteins to single-stranded (ssDNA) and double-stranded (dsDNA ) phi X DNA was studied by electron microscopy. The interaction of the A* protein with ssDNA and dsDNA was also studied by sedimentation velocity centrifugation. It was shown that the binding of the A and A* proteins to ssDNA occurs in a non-cooperative manner and requires no or very little sequence specificity under the conditions used here. Both protein-ssDNA complexes have the same compact structure caused by intrastrand cross-linking through the interaction of protein molecules with separate parts of the ssDNA molecule. The A protein does not bind to phi X dsDNA in the absence of divalent cations. The A* protein does bind to dsDNA, although it has a strong preference for binding to ssDNA. The structure of the A* protein-dsDNA complexes is different from that of the A* protein-ssDNA complexes, as the former have a rosette-like structure caused by protein-protein interactions. High ionic strengths favour the formation of large condensed aggregates.

FF domains of CA150 bind transcription and splicing factors through multiple weak interactions.

The human transcription factor CA150 modulates human immunodeficiency virus type 1 gene transcription and contains numerous signaling elements, including six FF domains. Repeated FF domains are present in several transcription and splicing factors and can recognize phosphoserine motifs in the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Using mass spectrometry, we identify a number of nuclear binding partners for the CA150 FF domains and demonstrate a direct interaction between CA150 and Tat-SF1, a protein involved in the coupling of splicing and transcription. CA150 FF domains recognize multiple sites within the Tat-SF1 protein conforming to the consensus motif (D/E)(2/5)-F/W/Y-(D/E)(2/5). Individual FF domains are capable of interacting with Tat-SF1 peptide ligands in an equivalent and noncooperative manner, with affinities ranging from 150 to 500 microM. Repeated FF domains therefore appear to bind their targets through multiple weak interactions with motifs comprised of negatively charged residues flanking aromatic amino acids. The RNAPII CTD represents a consensus FF domain-binding site, contingent on generation of the requisite negative charges by phosphorylation of serines 2 and 5. We propose that CA150, through the dual recognition of acidic motifs in proteins such as Tat-SF1 and the phosphorylated CTD, could mediate the recruitment of transcription and splicing factors to actively transcribing RNAPII.

Berberine, a strong polyriboadenylic acid binding plant alkaloid: spectroscopic, viscometric, and thermodynamic study

The interaction of berberine with single stranded poly(rA) structure was investigated using a combination of spectrophotometric, spectrofluorimetric, circular dichroic, viscometric, and thermodynamic studies. The interaction process was characterized by typical hypochromic and bathochromic effects in the absorption spectrum of berberine, enhancement of fluorescence intensity of berberine, increase of viscosity, and perturbation of circular dichroic spectrum of single stranded poly(rA). Scatchard plot obtained from spectrophotometric analysis showed that berberine bound strongly to single stranded poly(rA) in a non-cooperative manner. In contrast, berberine does not show any significant effect (i) in its absorbance and fluorescence spectra on binding to double stranded poly(rA), (ii) alter the circular dichroic spectrum of double stranded poly(rA), or (iii) increase of viscosity of double stranded poly(rA) indicating that it does not bind at all to double stranded poly(rA) structure. Thermodynamic parameters indicated that the binding of the alkaloid to single stranded poly(rA) is an endothermic process and entropy driven. All these findings, taken together clearly support that berberine binds strongly to single stranded poly(rA) structure by a mechanism of partial intercalation leading to its use in gene regulation in eukaryotic cells.

Molecular Aspects on the Interaction of Aristololactam-β-D-Glucoside with HL-Form Deoxyribonucleic Acid Structures

Synthetic alternating GC-rich DNA polymers can adopt Hoogsteen base-paired structures (HL-form) under the influence of low pH and temperature. The interaction of aristololactam-β-D-glucoside (ADG), a natural glucoside derivative of aristolochia group of alkaloids, with protonation-induced structures (HL-form) of poly(dG-dC)·poly(dG-dC) and poly(dG- m5dC)·poly(dG-m5dC) has been studied using different biophysical techniques. The binding of ADG to protonated DNA is characterized by typical hypochromism and bathochromism of the absorption spectrum of the alkaloid, quenching of steady state fluorescence intensity, decrease in quantum yield, increase in fluorescence polarization anisotropy values, increase in thermal transition temperature of polynucleotides following alkaloid binding and perturbation of circular dichroic spectrum of polynucleotides as a result of its interaction with the alkaloid. Scatchard analysis of the data indicates that ADG binds to protonated structures in a nonlinear noncooperative manner. The binding parameters determined from spectrophotometric titration data employing excluded site model indicate that protonated poly(dG-m5dC)·poly(dG-m5dC) is more favorable for ADG binding than the corresponding nonmethylated analog. The binding of ADG to protonated structures renders a higher degree of stabilization against thermal denaturation compared to respective B-form-ADG interactions and induces a conformational switch to a bound altered form which is different from its interaction with B- and Z-form DNA structures. Thermodynamic parameters (δG°, δH° and δS°) obtained by van't Hoff analysis of the data indicate that the binding of alkaloid to protonated structures is an exothermic process and the binding free energy arises primarily from a negative enthalpy change. Moreover, the binding leads to an increase in the contour length of protonated DNAs. These results suggest that ADG possibly binds to protonated DNAs by the mechanism of intercalation.

A non-cooperative neuro-fuzzy system for integrating ATIS and ATMS decisions

This paper addresses the problem of integrating successful existing implementations of Advanced Traveler Information Systems (ATIS) and Advanced Traffic Management Systems (ATMS). The methodologies and challenges to integrate ATIS and ATMS are addressed in details. This paper discusses in details the development of a rule-based neuro-fuzzy logic to integrate existing ATIS and ATMS when they operate in a non-cooperative manner. The individual existing logics are initially upgraded to the so-called ATIS and ATMS stand-alone systems, which may be regarded as bi-level optimization systems. The upper level (of the bi-level optimization system) represents an augmented process for guessing the counter system's decisions, and the lower level represents the existing logic (of ATIS or ATMS). The stand-alone systems are then replicated by simulation-based optimization algorithms, which are used to generate the training data necessary to calibrate the neuro-fuzzy logic. The role of the neural nets and the methodology of fuzzy-logic calibration are discussed in details. The effectiveness and robustness (of the neuro-fuzzy integrated ATIS/ATMS system) are assessed using simulation-based experiments with two different hypothetical networks.

Spectroscopic and Thermodynamic Studies on the Binding of Sanguinarine and Berberine to Triple and Double Helical DNA and RNA Structures

A comparative study on the interaction of sanguinarine and berberine with DNA and RNA triplexes and their parent duplexes was performed, by using a combination of spectrophotometric, UV thermal melting, circular dichroic and thermodynamic techniques. Formation of the DNA and RNA triplexes was confirmed from UV-melting and circular dichroic measurements. The interaction process was characterized by increase of thermal melting temperature, perturbation in circular dichroic spectrum and the typical hypochromic and bathochromic effects in the absorption spectrum. Scatchard analysis indicated that both the alkaloids bound to the triplex and duplex structures in a non-cooperative manner and the binding was stronger to triplexes than to parent duplexes. Thermal melting studies further indicated that sanguinarine stabilized the Hoogsteen base paired third strand of both DNA and RNA triplexes more tightly compared to their Watson-Crick strands, while berberine stabilized the third strand only without affecting the Watson-Crick strand. However, sanguinarine stabilized the parent duplexes while no stabilization was observed with berberine under identical conditions. Circular dichroic studies were also consistent with the observation that perturbations of DNA and RNA triplexes were more compared to their parent duplexes in presence of the alkaloids. Thermodynamic data revealed that binding of sanguinarine and berberine to triplexes (T·AxT and U·AxU) and duplexes (A·T and A·U) showed negative enthalpy changes and positive entropy changes but that of sanguinarine to C·GxC+ triplex and G·C duplex exhibited negative enthalpy and negative entropy changes. Taken together, these results suggest that both sanguinarine and berberine can bind and stabilize the DNA and RNA triplexes more strongly than their respective parent duplexes.

Brain-specific metallothionein-3 has higher metal-binding capacity than ubiquitous metallothioneins and binds metals noncooperatively.

Zinc metabolism in the cells is largely regulated by ubiquitous small proteins, metallothioneins (MT). Metallothionein-3 is specifically expressed in the brain and is down regulated in Alzheimer's disease. We demonstrate by mass spectrometry that MT-3, in contrast to common MTs, binds Zn(2+) and Cd(2+) in a noncooperative manner and can also bind higher stoichiometries of metals than seven. MT-3 reconstituted with seven metals exists in a dynamic equilibrium of different metalloforms, where the prevalent metalloform is Me(7)MT-3, but metalloforms with 6, 8, and even 9 metals are also present. The results from pH and stability studies demonstrate that the heterogeneity of metalloforms originates from the N-terminal beta-cluster, whereas the C-terminal alpha-cluster of MT-3 binds four metal ions such as that of common MTs. Experiments with EDTA demonstrate that the beta-cluster of ZnMT-3 has a higher metal transfer potential than the beta-cluster of Zn(7)MT-2. Moreover, ZnMT-3 loses metals during ultrafiltration. MT-3, reconstituted with an excess of Zn(2+) or Cd(2+), exists as a dynamic mixture of metalloforms with higher than 7 metal stoichiometries (8-11). Such forms of ZnMT-3 are unstable and decompose partly already during a rapid gel filtration, whereas CdMT-3 forms are more stable. Extra metal ions may bind to the beta-cluster region as well as to the carboxylates of MT-3. The specific metal-binding properties of MT-3 could be functionally implemented for buffering of fluctuating concentrations of zinc in zincergic neurons and for transfer of zinc to synaptic vesicles.