Positive Cooperativity Research Articles

A Molecular Replication Process Drives Supramolecular Polymerization.

Supramolecular polymers are materials in which the connections between monomers in the polymer main chain are non-covalent bonds. This area has seen rapid expansion in the last two decades and has been exploited in several applications. However, suitable contiguous hydrogen-bond arrays can be difficult to synthesize, placing some limitations on the deployment of supramolecular polymers. We have designed a hydrogen-bonded polymer assembled from a bifunctional monomer composed of two replicating templates separated by a rigid spacer. This design allows the autocatalytic formation of the polymer main chain through the self-templating properties of the replicators and drives the synthesis of the bifunctional monomer from its constituent components in solution. The template-directed 1,3-dipolar cycloaddition reaction between nitrone and maleimide proceeds with high diastereoselectivity, affording the bifunctional monomer. The high binding affinity between the self-complementary replicating templates that allows the bifunctional monomer to polymerize in solution is derived from the positive cooperativity associated with this binding process. The assembly of the polymer in solution has been investigated by diffusion-ordered NMR spectroscopy. Both microcrystalline and thin films of the polymeric material can be prepared readily and have been characterized by powder X-ray diffraction and scanning electron microscopy. These results demonstrate that the approach described here is a valid one for the construction of supramolecular polymers and can be extended to systems where the rigid spacer between the replicating templates is replaced by one carrying additional function.

Signal Transmission in Escherichia coli Cyclic AMP Receptor Protein for Survival in Extreme Acidic Conditions.

During the life cycle of enteric bacterium Escherichia coli, it encounters a wide spectrum of pH changes. The asymmetric dimer of the cAMP receptor protein, CRP, plays a key role in regulating the expressions of genes and the survival of E. coli. To elucidate the pH effects on the mechanism of signal transmission, we present a combination of results derived from ITC, crystallography, and computation. CRP responds to a pH change by inducing a differential effect on the affinity for the binding events to the two cAMP molecules, ensuing in a reversible conversion between positive and negative cooperativity at high and low pH, respectively. The structures of four crystals at pH ranging from 7.8 to 6.5 show that CRP responds by inducing a differential effect on the structures of the two subunits, particularly in the DNA binding domain. Employing the COREX/BEST algorithm, computational analysis shows the change in the stability of residues at each pH. The change in residue stability alters the connectivity between residues including those in cAMP and DNA binding sites. Consequently, the differential impact on the topology of the connectivity surface among residues in adjacent subunits is the main reason for differential change in affinity; that is, the pH-induced differential change in residue stability is the biothermodynamic basis for the change in allosteric behavior. Furthermore, the structural asymmetry of this homodimer amplifies the differential impact of any perturbations. Hence, these results demonstrate that the combination of these approaches can provide insights into the underlying mechanism of an apparent complex allostery signal and transmission in CRP.

Structural Characterization of Degrader-Induced Ternary Complexes Using Hydrogen-Deuterium Exchange Mass Spectrometry and Computational Modeling: Implications for Structure-Based Design.

The field of targeted protein degradation (TPD) has grown exponentially over the past decade with the goal of developing therapies that mark proteins for destruction leveraging the ubiquitin-proteasome system. One common approach to achieve TPD is to employ a heterobifunctional molecule, termed as a degrader, to recruit the protein target of interest to the E3 ligase machinery. The resultant generation of an intermediary ternary complex (target-degrader-ligase) is pivotal in the degradation process. Understanding the ternary complex geometry offers valuable insight into selectivity, catalytic efficiency, linker chemistry, and rational degrader design. In this study, we utilize hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify degrader-induced protein-protein interfaces. We then use these data in conjunction with constrained protein docking to build three-dimensional models of the ternary complex. The approach was used to characterize complex formation between the E3 ligase CRBN and the first bromodomain of BRD4, a prominent oncology target. We show marked differences in the ternary complexes formed in solution based on distinct patterns of deuterium uptake for two degraders, CFT-1297 and dBET6. CFT-1297, which exhibited positive cooperativity, altered the deuterium uptake profile revealing the degrader-induced protein-protein interface of the ternary complex. For CFT-1297, the ternary complexes generated by the highest scoring HDX-constrained docking models differ markedly from those observed in the published crystal structures. These results highlight the potential utility of HDX-MS to provide rapidly accessible structural insights into degrader-induced protein-protein interfaces in solution. They further suggest that degrader ternary complexes exhibit significant conformation flexibility and that biologically relevant complexes may well not exhibit the largest interaction surfaces between proteins. Taken together, the results indicate that methods capable of incorporating linker conformation uncertainty may prove an important component in degrader design moving forward. In addition, the development of scoring functions modified to handle interfaces with no evolved complementarity, for example, through consideration of high levels of water infiltration, may prove valuable. Furthermore, the use of crystal structures as validation tools for novel degrader methods needs to be considered with caution.

Unique Positive Cooperativity Between the β-Arrestin-Biased β-Blocker Carvedilol and a Small Molecule Positive Allosteric Modulator of the β2-Adrenergic Receptor.

Among β-blockers that are clinically prescribed for heart failure, carvedilol is a first-choice agent with unique pharmacological properties. Carvedilol is distinct from other β-blockers in its ability to elicit β-arrestin–biased agonism, which has been suggested to underlie its cardioprotective effects. Augmenting the pharmacologic properties of carvedilol thus holds the promise of developing more efficacious and/or biased β-blockers. We recently identified compound-6 (cmpd-6), the first small molecule positive allosteric modulator of the β2-adrenergic receptor (β2AR). Cmpd-6 is positively cooperative with orthosteric agonists at the β2AR and enhances agonist-mediated transducer (G-protein and β-arrestin) signaling in an unbiased manner. Here, we report that cmpd-6, quite unexpectedly, displays strong positive cooperativity only with carvedilol among a panel of structurally diverse β-blockers. Cmpd-6 enhances the binding affinity of carvedilol for the β2AR and augments its ability to competitively antagonize agonist-induced cAMP generation. Cmpd-6 potentiates β-arrestin1– but not Gs-protein–mediated high-affinity binding of carvedilol at the β2AR and β-arrestin–mediated cellular functions in response to carvedilol including extracellular signal-regulated kinase phosphorylation, receptor endocytosis, and trafficking into lysosomes. Importantly, an analog of cmpd-6 that selectively retains positive cooperativity with carvedilol acts as a negative modulator of agonist-stimulated β2AR signaling. These unprecedented cooperative properties of carvedilol and cmpd-6 have implications for fundamental understanding of G-protein–coupled receptor (GPCR) allosteric modulation, as well as for the development of more effective biased beta blockers and other GPCR therapeutics.SIGNIFICANCE STATEMENTThis study reports on the small molecule–mediated allosteric modulation of the β-arrestin–biased β-blocker, carvedilol. The small molecule, compound-6 (cmpd-6), displays an exclusive positive cooperativity with carvedilol among other β-blockers and enhances the binding affinity of carvedilol for the β2-adrenergic receptor. Cooperative effects of cmpd-6 augment the β-blockade property of carvedilol while potentiating its β-arrestin–mediated signaling functions. These findings have potential implications in advancing G-protein–coupled receptor allostery, developing biased therapeutics and remedying cardiovascular ailments.

β-Arrestin-Biased Allosteric Modulator Potentiates Carvedilol-Stimulated β Adrenergic Receptor Cardioprotection.

β 1 adrenergic receptors (β 1ARs) are central regulators of cardiac function and a drug target for cardiac disease. As a member of the G protein-coupled receptor family, β 1ARs activate cellular signaling by primarily coupling to Gs proteins to activate adenylyl cyclase, cAMP-dependent pathways, and the multifunctional adaptor-transducer protein β-arrestin. Carvedilol, a traditional β-blocker widely used in treating high blood pressure and heart failure by blocking β adrenergic receptor-mediated G protein activation, can selectively stimulate Gs-independent β-arrestin signaling of β adrenergic receptors, a process known as β-arrestin-biased agonism. Recently, a DNA-encoded small-molecule library screen against agonist-occupied β 2 adrenergic receptors (β 2ARs) identified Compound-6 (Cmpd-6) to be a positive allosteric modulator for agonists on β 2ARs. Intriguingly, it was further discovered that Cmpd-6 is positively cooperative with the β-arrestin-biased ligand carvedilol at β 2ARs. Here we describe the surprising finding that at β 1ARs unlike β 2ARs, Cmpd-6 is cooperative only with carvedilol and not agonists. Cmpd-6 increases the binding affinity of carvedilol for β 1ARs and potentiates carvedilol-stimulated, β-arrestin-dependent β 1AR signaling, such as epidermal growth factor receptor transactivation and extracellular signal-regulated kinase activation, whereas it does not have an effect on Gs-mediated cAMP generation. In vivo, Cmpd-6 enhances the antiapoptotic, cardioprotective effect of carvedilol in response to myocardial ischemia/reperfusion injury. This antiapoptotic role of carvedilol is dependent on β-arrestins since it is lost in mice with myocyte-specific deletion of β-arrestins. Our findings demonstrate that Cmpd-6 is a selective β-arrestin-biased allosteric modulator of β 1ARs and highlight its potential clinical utility in enhancing carvedilol-mediated cardioprotection against ischemic injury. SIGNIFICANCE STATEMENT: This study demonstrates the positive cooperativity of Cmpd-6 on β1ARs as a β-arrestin-biased positive allosteric modulator. Cmpd-6 selectively enhances the affinity and cellular signaling of carvedilol, a known β-arrestin-biased β-blocker for β1ARs, whereas it has minimal effect on other ligands tested. Importantly, Cmpd-6 enhances the β-arrestin-dependent in vivo cardioprotective effect of carvedilol during ischemia/reperfusion injury-induced apoptosis. The data support the potential therapeutic application of Cmpd-6 to enhance the clinical benefits of carvedilol in the treatment of cardiac disease.

Turn-on, photostable, nontoxic and specific, iron(II) sensor

The pressing need to develop a specific analytical sensor that can identify and quantify Fe(II) without a cytotoxic response was the major motivation drive in this work. The turn-on fluorescent sensor here described can successfully detect Fe(II) and discriminate this ion from other analytes that commonly act as interferents in biological media. Moreover, this reduced fluoresceinamine-based sensor has a high photostability and high dissociation constant, which is an indication that the complex obtained between reduced fluoresceinamine (RFL) and Fe(II) is highly stable. This fluorescence-based sensor has a binding mechanism of 1:1 and a positive cooperativity was found between analyte and sensor. The detection, quantification and sensitivity parameters of the sensor were determined: 21.6 ± 0.1 μM; 65.6 ± 0.1 μM and 48 ± 3 (×107) μM, respectively. To evaluate a possible cytotoxicity effect an erythrocyte assay was performed and the obtained data were evaluated considering CdTe Quantum Dots (QDs) passivated with mercaptoacetic acid has experimental control. According to the resulting data RFL is not cytotoxic even when used in high concentrations, 660 mM. On the other hand QDs are quite different. Indeed it was proven that these heavy metal-based nanoparticles are responsible for 40% erytrocytes hemolysis in concentrations of 600 mM.

Antioxidant Potential of the Bio-Based Fucose-Rich Polysaccharide FucoPol Supports Its Use in Oxidative Stress-Inducing Systems.

Reactive oxygen species (ROS) are dangerous sources of macromolecular damage. While most derive from mitochondrial oxidative phosphorylation, their production can be triggered by exogenous stresses, surpassing the extinction capacity of intrinsic antioxidant defense systems of cells. Here, we report the antioxidant activity of FucoPol, a fucose-rich polyanionic polysaccharide produced by Enterobacter A47, containing ca. 17 wt% of negatively charged residues in its structure. Ferric reducing antioxidant power (FRAP) assays coupled to Hill binding kinetics fitting have shown FucoPol can neutralize ferricyanide and Fe3+-TPTZ species at an EC50 of 896 and 602 µg/mL, respectively, with positive binding cooperativity (2.52 ≤ H ≤ 4.85). This reducing power is greater than most polysaccharides reported. Moreover, an optimal 0.25% w/v FucoPol concentration shown previously to be cryo- and photoprotective was also demonstrated to protect Vero cells against H2O2-induced acute exposure not only by attenuating metabolic viability decay, but also by accentuating post-stress proliferation capacity, whilst preserving cell morphology. These results on antioxidant activity provide evidence for the biopolymer’s ability to prevent positive feedback cascades of the radical-producing Fenton reaction. Ultimately, FucoPol provides a biotechnological alternative for implementation in cryopreservation, food supplementation, and photoprotective sunscreen formula design, as all fields benefit from an antioxidant functionality.

Molecular Mechanism Exploration of Potent Fluorinated PI3K Inhibitors with a Triazine Scaffold: Unveiling the Unusual Synergistic Effect of Pyridine-to-Pyrimidine Ring Interconversion and CF3 Defluorination.

The phosphatidylinostitol-3-kinase (PI3K)/AKT/mammalian target of rapamycin signaling pathway is a vital regulator of cell proliferation, growth, and survival, which is frequently overactivated in many human cancers. To this effect, PI3K, which is an important mediator of this pathway, has been pinpointed as a crucial target in cancer therapy and hence the importance of PI3K inhibitors. It was recently reported that defluorination and pyridine-to-pyrimidine ring interconversion increase the potency of specific small-molecule inhibitors of PI3K. Compound 4, an inhibitor with the difluorinated pyrimidine motif, was found to be eight times more potent against PI3K than compound 1, an inhibitor with the trifluorinated pyridine motif. This observation presents the need to rationally resolve the differential inhibitory mechanisms exhibited by both compounds. In this present work, we employed multiple computational approaches to investigate and distinguish the binding modes of 1 and 4 in addition to the effects they mediate on the secondary structure of PI3K. Likewise, we evaluated two other derivatives, compounds 2 with the difluorinated pyridine motif and 3 with the trifluorinated pyrimidine motif, to investigate the cooperativity effect between the defluorination of CF3 and pyridine-to-pyrimidine ring interconversion. Findings revealed that PI3K, upon interaction with 4, exhibited a series of structural changes that favored the binding of the inhibitor at the active-site region. Furthermore, a positive (synergistic) cooperativity effect was observed between CF3 defluorination and pyridine-to-pyrimidine ring interconversion. Moreover, there was a good correlation between the binding free energy estimated and the biological activity reported experimentally. Energy decomposition analysis revealed that the major contributing force to binding affinity variations between 1 and 4 is the electrostatic energy. Per-residue energy-based hierarchical clustering analysis further identified four hot-spot residues ASP841, TYR867, ASP964, and LYS833 and four warm-spot residues ASP836, SER806, ASP837, and LYS808, which essentially mediate the optimal and higher-affinity binding of compound 4 to PI3K relative to 1. This study therefore provides rational insights into the mechanisms by which 4 exhibited superior PI3K-inhibitory activities over 1, which is vital for future structure-based drug discovery efforts in PI3K targeting.

Molecular dynamics simulations of human L-asparaginase1: Insights into structural determinants of enzymatic activity

The l-asparaginase enzyme is used in cancer therapy, mainly acute lymphoid leukemia (ALL). Commercial enzymes (EcASNase2) cause adverse reactions during treatment, such as immunogenicity. A human enzyme could be a non-immunogenic substitute. However, no candidate was found showing efficient kinetic properties. HASNase1 is an l-asparaginase that comes from the N-terminal domain of a protein called 60 kDa-lysophospholipase and its 3D structure has not been resolved. HASNase1 is homologous to EcASNase1 and gpASNase1, and this last one has shown efficient kinetic properties. Homology modeling was used to find the 3D structure of hASNase1, so one could submit it to Molecular Dynamics (MD), in order to understand structural differences that lead to different catalytic efficiency compared to EcASNase2 and gpASNase1. The interaction potential between L-Asn and active site residues showed that the substrate can rotate in the site when Region1 is open. Region1 residues sequence favors deformations and movements as shown in MD. Region2-A is linear in gpASNase1, and it features a helix portion in hASNase1, which leaves the Tyr308 position projected to the active site ratifying its role in catalytic efficiency. Analysis of Lys188 orientation and movement showed the effect of positive cooperativity in hASNase1. It was found that the presence of Asn at the allosteric site helps, not only in Region1 stabilization, but also in Lys188 stabilization for the maintenance of the triad. Despite structural similarities in hASNase1, gpASNase1, and EcASNase2, there are differences in structural determinants that, in addition to allosterism, may explain the different kinetic properties.

Community resilience for urban flood-prone areas: a methods paper on criteria selection using the Fuzzy Delphi method

PurposeCommunity resilience decides a lot on a city's ability to withstand an external shock. It has evolved naturally from a bounce-back approach to a more robust and meaningful bounce-forward process. The study explores gaps found in community resilience and finds that criteria specific to different disasters are absent.Design/methodology/approachThe study uses a multi-criteria decision analysis technique, fuzzy Delphi, to select criteria. Derivation of the initial list of criteria was from a pilot study, a focus group discussion and other literature studies which was followed by the fuzzy Delphi survey.FindingsAfter two rounds of fuzzy Delphi analysis, the consensus among 65 experts resulted in selecting 125 sub-sub-criteria within seven criteria. Findings show that many criteria previously not discussed in other pieces of literature project high fuzzy scores such as “availability of drinking water post-disaster” and “cracking down fake news spreaders by the police”. In addition, positive cooperation between political and religious institutions have proven to expedite disaster recovery.Research limitations/implicationsThe future scope also includes weighing the selected criteria using analytical hierarchy process (AHP).Practical implicationsPolicymakers in the disaster management domain can use the study findings in implementing effective disaster mitigation strategies.Originality/valueThe selection of criteria is based on the community resilience shown by the Kerala community during the floods of 2018 and 2019 (in Kerala). Measures demonstrated by the community need to be studied, which will help foster disaster mitigation better in future scenarios.

CryoEM reveals the stochastic nature of individual ATP binding events in a group II chaperonin

Chaperonins are homo- or hetero-oligomeric complexes that use ATP binding and hydrolysis to facilitate protein folding. ATP hydrolysis exhibits both positive and negative cooperativity. The mechanism by which chaperonins coordinate ATP utilization in their multiple subunits remains unclear. Here we use cryoEM to study ATP binding in the homo-oligomeric archaeal chaperonin from Methanococcus maripaludis (MmCpn), consisting of two stacked rings composed of eight identical subunits each. Using a series of image classification steps, we obtained different structural snapshots of individual chaperonins undergoing the nucleotide binding process. We identified nucleotide-bound and free states of individual subunits in each chaperonin, allowing us to determine the ATP occupancy state of each MmCpn particle. We observe distinctive tertiary and quaternary structures reflecting variations in nucleotide occupancy and subunit conformations in each chaperonin complex. Detailed analysis of the nucleotide distribution in each MmCpn complex indicates that individual ATP binding events occur in a statistically random manner for MmCpn, both within and across the rings. Our findings illustrate the power of cryoEM to characterize a biochemical property of multi-subunit ligand binding cooperativity at the individual particle level.

Co-symbiosis of arbuscular mycorrhizal fungi (AMF) and diazotrophs promote biological nitrogen fixation in mangrove ecosystems

Nitrogen (N) is the most critical nutrient that affects the establishment and stability of mangrove ecosystems. Despite the importance of biological nitrogen fixation (BNF) mediated by diazotrophs in mangrove ecosystems, current studies largely overlook the influence of arbuscular mycorrhizal fungi (AMF) on diazotrophs-driven BNF process in the N-limited ecosystems. Here, we conducted a comprehensive study on root-associated AMF and their interspecific interactions with diazotrophs in mangrove ecosystems using high throughput sequencing methods. Our results showed that the richness/diversity of diazotrophs had significant positive correlations with AMF community in mangrove rhizosphere. BNF rate in the rhizosphere of all mangrove species increased with the increasing of positive co-occurrence between AMF and diazotrophs, suggesting that the positive cooperation may promote N fixation efficiency in mangrove ecosystems. We also identified the potential keystone taxa (Zi ≥ 2.5 or Pi ≥ 0.62) and determined their crucial implications for the ecological processes of N fixation in mangrove ecosystems. The random forest analysis further indicated that the AMF keystone taxa were the most important predictors of BNF in mangrove rhizosphere. In addition, path analysis indicated that the α-diversity of AMF and diazotrophs communities, N-related enzymes and sediment nutrition components (e.g., TC, TN, Fe) were the main factors driving BNF process in mangrove ecosystems. This study provides novel insights into the interactions between AMF and diazotrophic communities during BNF process and expands our knowledge of AMF ecological functions of N cycle in mangrove ecosystems.

Towards safer healthcare: qualitative insights from a process view of organisational learning from failure

ObjectivesThis study adopted a process view of organisational learning to investigate the barriers to effective organisational learning from medical errors.MethodsQualitative data were collected from 40 clinicians in high and low...

Can metal halides be electron donors in σ‐hole and π‐hole tetrel bonds? Cooperativity with an alkaline‐earth bond

AbstractThe tetrel bond in the complexes of TH3F‐MX2 (T = C‐Sn, M = Be and Mg; X = F, Cl, and Br) and F2CO‐MX2 has been explored and it is also called carbon bond for T = C. The results show that the halogen atom of metal halide can engage in a σ‐hole interaction with TH3F and a π‐hole interaction with F2CO. A linear carbon‐bonded complex is formed between CH3F and MX2, while a cyclic structure is obtained in other complexes, where a tetrel bond coexists with an alkaline‐earth bond. The carbon bond between CH3F and MX2 is weak with interaction energy less than 9 kJ/mol, depending on the nature of M and X atoms. The alkaline‐earth bond is stronger than the tetrel bond in the cyclic complexes and both interactions display positive cooperativity. The F2CO‐MX2 complex is more stable than TH3F‐MX2, mainly due to the stronger alkaline‐earth bond in the former. The π‐hole carbon bond is also stronger than the σ‐hole carbon bond when a metal halide acts as an electron donor. Such tetrel‐bonded structures involving metal halides have been found in the Cambridge Structural Database.

Pillar[4]arene[1]quinone-based pseudo[3]rotaxanes by cooperative Host-Guest binding

ABSTRACT Formation of pseudo[3]rotaxane assemblies by P4Q1 and H with bis-imidazolium and bis-pyridinium guests was established by NMR spectral analyses and high-resolution mass spectrometric analyses. Cooperativity in the formation of pseudo[3]rotaxane assemblies was thoroughly investigated from the binding constant values derived from 1H NMR titration experiments. The results indicated the preferential 1:1 host-guest binding of pillar[4]arene[1]quinone and 2:1 host-guest binding by functionalized pillar[4]arene[1]quinone via positive cooperativity. The presence of hydrogen bonding functional groups attached to the quinone motif of pillar[4]arene[1]quinone facilitated the inclusion formation, which resulted in the formation of pseudo[3]rotaxanes.

CAMP binding to closed pacemaker ion channels is non-cooperative.

Electrical activity in the brain and heart depends on rhythmic generation of action potentials by pacemaker ion channels (HCN) whose activity is regulated by cAMP binding1. Previous work has uncovered evidence for both positive and negative cooperativity in cAMP binding2,3, but such bulk measurements suffer from limited parameter resolution. Efforts to eliminate this ambiguity using single-molecule techniques have been hampered by the inability to directly monitor binding of individual ligand molecules to membrane receptors at physiological concentrations. Here we overcome these challenges using nanophotonic zero-mode waveguides4 to directly resolve binding dynamics of individual ligands to multimeric HCN1 and HCN2 ion channels. We show that cAMP binds independently to all four subunits when the pore is closed, despite a subsequent conformational isomerization to a flip state at each site. The different dynamics in binding and isomerization are likely to underlie physiologically distinct responses of each isoform to cAMP5 and provide direct validation of the ligand-induced flip-state model6-9. This approach for observing stepwise binding in multimeric proteins at physiologically relevant concentrations can directly probe binding allostery at single-molecule resolution in other intact membrane proteins and receptors.

Positive cooperativity during Azotobacter vinelandii nitrogenase-catalyzed acetylene reduction

The MoFe protein component of the nitrogenase enzyme complex is the substrate reducing site and contains two sets of symmetrically arrayed metallo centers called the P (Fe8S7) and the FeMoco (MoFe7S9-C-homocitrate) centers. The ATP-binding Fe protein is the specific reductant for the MoFe protein. Both symmetrical halves of the MoFe protein are thought to function independently during nitrogenase catalysis. Forming [AlF4]− transition-state complexes between the MoFe protein and the Fe protein of Azotobacter vinelandii ranging from 0 to 2 Fe protein/MoFe protein produced a series of complexes whose specific activity decreases with increase in bound Fe protein/MoFe protein ratio. Reduction of 2H+ to H2 was inhibited in a linear manner with an x-intercept at 2.0 with increasing Fe protein binding, whereas acetylene reduction to ethylene decreased more rapidly with an x-intercept near 1.5. H+ reduction is a distinct process occurring independently at each half of the MoFe protein but acetylene reduction decreases more rapidly than H+ reduction with increasing Fe protein/MoFe protein ratio, suggesting that a response is transmitted between the two αβ halves of the MoFe protein for acetylene reduction as Fe protein is bound. A mechanistic model is derived to investigate this behavior. The model predicts that each site functions independently for 2H+ reduction to H2. For acetylene reduction, the model predicts positive (synchronous) not negative cooperativity arising from acetylene binding to both sites before substrate reduction occurs. When this model is applied to inhibition by Cp2 and modified Av2 protein (L127∆) that form strong, non-dissociable complexes, positive cooperativity is absent and each site acts independently. The results suggest a new paradigm for the catalytic function of the MoFe protein during nitrogenase catalysis.

Thymidylate kinase (TMK) of the photosynthetic, nitrogen-fixing cyanobacterium Nostoc sp. strain PCC7120: Biophysical, biochemical and physiological characterisation

Thymidylate kinase (TMK/TMPK) is an important enzyme in DNA biosynthesis and catalyses the conversion of dTMP to dTDP. Due to its therapeutic potential, the focus has been on characterizing the TMK proteins of pathogens and human origin, with very little information available on the TMK proteins of photosynthetic organisms and agriculturally important nitrogen-fixing organisms. In this work we report the characterisation of TMK in an evolutionarily ancient organism, cyanobacteria. The TMK protein of the photosynthetic, nitrogen-fixing cyanobacterium Nostoc PCC7120 (AnTMK) was found to have low conformational stability, which related to its low Tm of ~46 °C confirmed by Differential Scanning Fluorimetry (DSF) and Differential Scanning Calorimetry (DSC) techniques. The AnTMK protein exhibited substrate specificity for dTMP and ATP with Km of 20.74 ± 1.47 μM and 20.17 ± 2.96 μM respectively. The enzyme kinetics data and the positive co-operativity observed between dTMP and ATP binding correlated well with the data obtained from Isothermal Titration Calorimetry (ITC). Homology model of the enzyme suggested that the binding mode of substrate nucleotides to the enzyme is conserved. When overexpressed constitutively in Nostoc PCC7120 (Antmk+), it supported faster growth measured in terms of chlorophyll a content under normal growth conditions, but exhibited lower photosynthetic efficiency. Compared to the vector control recombinant Nostoc AnpAM, the Antmk + cells exhibited higher photoinhibition at higher light irradiance with more open reaction centres and lower dissipation of heat, indicative of damage to photosynthetic machinery. This indicated that the TMK is likely to have a significant role in photosynthetic organisms.

Positive Cooperative Protonation of a Metal-Organic Framework: pH-Responsive Fluorescence and Proton Conduction.

Positive cooperative binding, a phenomenon prevalent in biological processes, holds great appeal for the design of highly sensitive responsive molecules and materials. It has been demonstrated that metal-organic frameworks (MOFs) can show positive cooperative adsorption to the benefit of gas separation, but potential binding cooperativity is largely ignored in the study of sensory MOFs. Here, we report the first demonstration of positive cooperative protonation of a MOF and the relevant pH response in fluorescence and proton conduction. The MOF is built of Zr-O clusters and bipyridyl-based tetracarboxylate linkers and has excellent hydrolytic stability. It shows a unique pH response that features two synchronous abrupt turn-off and turn-on fluorescent transitions. The abrupt transitions, which afford high sensitivity to small pH fluctuations, are due to cooperative protonation of the pyridyl sites with a Hill coefficient of 1.6. The synchronous dual-emission response, which leads to visual color change, is ascribable to proton-triggered switching between (n, π*) and (π, π*) emissions. The latter emission can be quenched by electron donating anion-dependent through photoinduced electron transfer and ground-state charge transfer. Associated with cooperative protonation, the proton conductivity of the MOF is abruptly enhanced at low pH by two orders, but overhigh acid concentration is adverse because excessive anions can interrupt the conducting networks. Our work shows new perspectives of binding cooperativity in MOFs and should shed new light on the development of responsive fluorescent MOFs and proton conductive materials.

Cooperativity of α-Synuclein Binding to Lipid Membranes.

Cooperative binding is a key feature of metabolic pathways, signaling, and transport processes. It provides tight regulation over a narrow concentration interval of a ligand, thus enabling switching to be triggered by small concentration variations. The data presented in this work reveal strong positive cooperativity of α-synuclein binding to phospholipid membranes. Fluorescence cross-correlation spectroscopy, confocal microscopy, and cryo-TEM results show that in excess of vesicles α-synuclein does not distribute randomly but binds only to a fraction of all available vesicles. Furthermore, α-synuclein binding to a supported lipid bilayer observed with total internal reflection fluorescence microscopy displays a much steeper dependence of bound protein on total protein concentration than expected for independent binding. The same phenomenon was observed in the case of α-synuclein binding to unilamellar vesicles of sizes in the nm and μm range as well as to flat supported lipid bilayers, ruling out that nonuniform binding of the protein is governed by differences in membrane curvature. Positive cooperativity of α-synuclein binding to lipid membranes means that the affinity of the protein to a membrane is higher where there is already protein bound compared to a bare membrane. The phenomenon described in this work may have implications for α-synuclein function in synaptic transmission and other membrane remodeling events.