Showing papers on "Cooperativity published in 2011"

Structure:function relationships in molecular spin-crossover complexes

Abstract: Spin-crossover compounds are becoming increasingly popular for device and sensor applications, and in soft materials, that make use of their switchable colour, paramagnetism and conductivity. The de novo design of new solid spin-crossover compounds with pre-defined switching properties is desirable for application purposes. This challenging problem of crystal engineering requires an understanding of how the temperature and cooperativity of a spin-transition are influenced by the structure of the bulk material. Towards that end, this critical review presents a survey of molecular spin-crossover compounds with good availability of crystallographic data. A picture is emerging that changes in molecular shape between the high- and low-spin states, and the ability of a lattice to accommodate such changes, can play an important role in determining the existence and the cooperativity of a thermal spin-transition in the solid state (198 references).

Flexibility, Diversity, and Cooperativity: Pillars of Enzyme Catalysis

Abstract: This brief review discusses our current understanding of the molecular basis of enzyme catalysis. A historical development is presented, beginning with steady state kinetics and progressing through modern fast reaction methods, nuclear magnetic resonance, and single-molecule fluorescence techniques. Experimental results are summarized for ribonuclease, aspartate aminotransferase, and especially dihydrofolate reductase (DHFR). Multiple intermediates, multiple conformations, and cooperative conformational changes are shown to be an essential part of virtually all enzyme mechanisms. In the case of DHFR, theoretical investigations have provided detailed information about the movement of atoms within the enzyme–substrate complex as the reaction proceeds along the collective reaction coordinate for hydride transfer. A general mechanism is presented for enzyme catalysis that includes multiple intermediates and a complex, multidimensional standard free energy surface. Protein flexibility, diverse protein conforma...

Allosteric, chelate, and interannular cooperativity: a mise au point.

Abstract: The distinction between different types of cooperativity is essential for understanding the fundamentals involved. The three title cooperative effects arise from the interplay of intermolecular binding interactions, the presence of one or more intramolecular binding interactions, and, in the latter case, their possible interplay. A master equation for the stability of an assembly is outlined that takes into account all of the three possible types of cooperativity

Quantitative models of the mechanisms that control genome-wide patterns of transcription factor binding during early Drosophila development.

Abstract: Transcription factors that drive complex patterns of gene expression during animal development bind to thousands of genomic regions, with quantitative differences in binding across bound regions mediating their activity. While we now have tools to characterize the DNA affinities of these proteins and to precisely measure their genome-wide distribution in vivo, our understanding of the forces that determine where, when, and to what extent they bind remains primitive. Here we use a thermodynamic model of transcription factor binding to evaluate the contribution of different biophysical forces to the binding of five regulators of early embryonic anterior-posterior patterning in Drosophila melanogaster. Predictions based on DNA sequence and in vitro protein-DNA affinities alone achieve a correlation of ∼0.4 with experimental measurements of in vivo binding. Incorporating cooperativity and competition among the five factors, and accounting for spatial patterning by modeling binding in every nucleus independently, had little effect on prediction accuracy. A major source of error was the prediction of binding events that do not occur in vivo, which we hypothesized reflected reduced accessibility of chromatin. To test this, we incorporated experimental measurements of genome-wide DNA accessibility into our model, effectively restricting predicted binding to regions of open chromatin. This dramatically improved our predictions to a correlation of 0.6–0.9 for various factors across known target genes. Finally, we used our model to quantify the roles of DNA sequence, accessibility, and binding competition and cooperativity. Our results show that, in regions of open chromatin, binding can be predicted almost exclusively by the sequence specificity of individual factors, with a minimal role for protein interactions. We suggest that a combination of experimentally determined chromatin accessibility data and simple computational models of transcription factor binding may be used to predict the binding landscape of any animal transcription factor with significant precision.

Ammonia formation by metal–ligand cooperative hydrogenolysis of a nitrido ligand

Abstract: One of the hurdles facing the development of effective catalysts to produce ammonia from nitrogen is the stability of the metal nitrides that form during the reaction. Now, the hydrogenolysis of nitride ligands with hydrogen is reported and attributed to PNP pincer ligand cooperativity.

Tuning a p450 enzyme for methane oxidation

Abstract: Cytochrome P450 (CYP) enzymes are heme-dependent monooxygenases that catalyze the oxidation of C H bonds of endogenous and exogenous organic compounds with formation of the respective alcohols. The mechanism involves the intermediacy of a high-spin oxyferryl porphyrin radical cation which abstracts a hydrogen atom from the substrate, and the short-lived alkyl radical then undergoes C Obond formation. The binding pockets of CYPs are relatively large, therefore small compounds do not have a statistically high enough probability of being properly oriented near the oxyferryl moiety for rapid oxidation to occur; additionally there are other effects that slow down or prevent catalysis. A notorious challenge is the oxidation of methane to methanol by chemical catalysis or using enzymes of the type methane monooxygenases (MMOs). It is not only the smallest alkane, but also has the strongest C H bond (104 kcalmol ). Although CYPs represent a superfamily of monooxygenases, none have been shown to accept methane, whereas MMOs are complex enzymes (many membrane bound) that have not been expressed in heterologous hosts in any significant quantities, among other problems. Herein we show that chemical tuning of a CYP, which is based on guest/host activation using perfluoro carboxylic acids as chemically inert guests, activates the enzyme for oxidation of not only medium-sized alkanes such as n-hexane, but also of small gaseous molecules such as propane and even methane as the ultimate challenge. In the present study we chose, for practical reasons, the enzyme P450 BM3 (CYP102A1) from Bacillus megaterium, which is a self-sufficient fusion protein composed of a P450 monooxygenase and an NADPH diflavin reductase. Several crystal structures of this CYP harboring a fatty acid or fatty acid derived inhibitors, as well in the absence of such compounds have been published. To engineer mutants of P450 BM3 and of other CYPs for enhanced activity and selectivity toward a variety of different compounds, including such difficult substrates as small alkanes, rational design as well as directed evolution have proven to be successful to some extent. For example, P450 BM3 variants characterized by numerous point mutations were obtained in extensive laboratory evolution, and showed for the first time notable activity toward propane by formation of the respective alcohols (2-propanol/1-propanol= 9:1); however, the ethane to ethanol conversion remains problematic and methane oxidation has not been achieved to date. Higher activity in ethane oxidation was accomplished using mutants of P450cam, but here again methane oxidation was not reported. Our chemical approach involves a chemically inert compound that serves as a guest in the binding pocket of P450 BM3, thereby filling the space and reducing the translational freedom of small alkanes or of any other substrate. On the basis of previous reports involving CYPs harboring various substrates, such guest/host interactions can be expected to induce other modes of activation effects as well, specifically water displacement at the Fe/heme site accompanied by a change in the electronic state from the inactive low-spin state to the catalytically active high-spin states. Moreover, many studies have shown that P450 enzymes and mutants thereof can harbor two different substrates simultaneously, thus leading to cooperative effects; one example is lauric acid and palmitic acid in which cooperativity has been demonstrated by isotope labeling experiments. In yet another study regarding the metabolism of bilirubin, the addition of lauric acid or the perfluorinated analogue was reported to facilitate NADPH oxidation and substrate degradation, a finding that has implications for the treatment of jaundice, uroporphyria, and possibly cancer. It has also been shown for the case of a distantly related H2O2dependent P450 enzyme that its peroxidase activity can be influenced by the addition of fatty acids, wherein increased or decreased activity is observed depending upon their chain length. In our endeavor we were guided by the binding mode of the natural substrates, fatty acids, of P450 BM3. The binding includes H-bonds originating from their carboxy function and residues Arg 47 and Tyr 51, as well as hydrophobic interactions. The use of perfluoro carboxylic acids such as 1a–h as chemically inert, yet activating guests was therefore envisioned, because perfluoro alkyl groups are known to be resistant to oxidation while having a hydrophobic character. Moreover, it is known that a CF3 residue is sterically comparable to a CH(CH3)2 group, [11a] which means that a perfluoro fatty acid fills much more space in a P450 binding pocket than a traditional fatty acid, and can additionally induce the crucial low-spin to high-spin conversion of Fe/heme. In exploratory studies, the oxidation of n-octane and n-hexane as well as isomers thereof was studied using P450 [*] Dr. F. E. Zilly, Dr. J. P. Acevedo, Dr. W. Augustyniak, A. Deege, U. W. H usig, Prof. M. T. Reetz Max-Planck-Institut f r Kohlenforschung Kaiser-Wilhelm-Platz 1, 45470 M lheim an der Ruhr (Germany) reetz@mpiso-muelheim.mpg.de

Telomere Structure and Stability: Covalency in Hydrogen Bonds, Not Resonance Assistance, Causes Cooperativity in Guanine Quartets

Abstract: We show that the cooperative reinforcement between hydrogen bonds in guanine quartets is not caused by resonance-assisted hydrogen bonding (RAHB). This follows from extensive computational analyses of guanine quartets (G(4)) and xanthine quartets (X(4)) based on dispersion-corrected density functional theory (DFT-D). Our investigations cover the situation of quartets in the gas phase, in aqueous solution as well as in telomere-like stacks. A new mechanism for cooperativity between hydrogen bonds in guanine quartets emerges from our quantitative Kohn-Sham molecular orbital (MO) and corresponding energy decomposition analyses (EDA). Our analyses reveal that the intriguing cooperativity originates from the charge separation that goes with donor-acceptor orbital interactions in the σ-electron system, and not from the strengthening caused by resonance in the π-electron system. The cooperativity mechanism proposed here is argued to apply, beyond the present model systems, also to other hydrogen bonds that show cooperativity effects.

Core unit of chemotaxis signaling complexes

Abstract: Bacterial chemoreceptors, histidine kinase CheA, and coupling protein CheW form clusters of chemotaxis signaling complexes. In signaling complexes kinase activity is enhanced several hundredfold and placed under receptor control. Activation is necessary to poise enzyme activity such that receptor control has physiologically relevant effects. Thus kinase activation can be considered the underlying core activity of signaling complexes. We defined the minimal physical unit that generates this activity using chemoreceptor Tar from Escherichia coli rendered water soluble by insertion into nanodiscs to (i) measure saturable binding of CheA and CheW to the smallest kinase-activating groups of receptor dimers and (ii) purify and characterize core units of signaling complexes. Purified complexes activated kinase almost as well as signaling complexes formed on arrays of receptors in isolated native membrane. Purified complexes contained two receptor trimers of dimers and two CheW for each CheA dimer, consistent with the approximately 1∶1 CheA∶CheW ratio determined by binding measurements. The 2∶2∶1 stoichiometry implied that CheA dimers, the enzymatically active form, connect two chemoreceptor trimers of dimers by interaction of one CheA protomer and a CheW with each trimer, an organization for which specific molecular interactions have previously been identified. The core unit associates six receptor dimers with a CheA dimer, providing sufficient capacity to account for much of the cooperativity and interdimer influence observed experimentally. We conclude that the 2∶2∶1 organization is the core structural and functional unit of chemotaxis signaling complexes and postulate that hexagonal arrays characteristic of signaling complexes are built from this unit.

Dynamically committed, uncommitted, and quenched states encoded in protein kinase A revealed by NMR spectroscopy

Abstract: Protein kinase A (PKA) is a ubiquitous phosphoryl transferase that mediates hundreds of cell signaling events. During turnover, its catalytic subunit (PKA-C) interconverts between three major conformational states (open, intermediate, and closed) that are dynamically and allosterically activated by nucleotide binding. We show that the structural transitions between these conformational states are minimal and allosteric dynamics encode the motions from one state to the next. NMR and molecular dynamics simulations define the energy landscape of PKA-C, with the substrate allowing the enzyme to adopt a broad distribution of conformations (dynamically committed state) and the inhibitors (high magnesium and pseudosubstrate) locking it into discrete minima (dynamically quenched state), thereby reducing the motions that allow turnover. These results unveil the role of internal dynamics in both kinase function and regulation.

Relating Form and Function of EF-hand Calcium Binding Proteins

Abstract: The EF hand, a helix-loop-helix structure, is one of the most common motifs found in animal genomes, and EF-hand Ca(2+)-binding proteins (EFCaBPs) are widely distributed throughout the cell. However, researchers remain confounded by a lack of understanding of how peptide sequences code for specific functions and by uncertainty about the molecular mechanisms that enable EFCaBPs to distinguish among many diverse cellular targets. Such knowledge could define the roles of EFCaBPs in health and disease and ultimately enable control or even design of Ca(2+)-dependent functions in medicine and biotechnology. In this Account, we describe our structural and biochemical research designed to understand the sequence-to-function relationship in EFCaBPs. The first structural goal was to define conformational changes induced by binding Ca(2+), and our group and others established that solution NMR spectroscopy is well suited for this task. We pinpointed residues critical to the differences in Ca(2+) response of calbindin D(9k) and calmodulin (CaM), homologous EFCaBPs from different functional classes, by using direct structure determination with site-directed mutagenesis and protein engineering. Structure combined with biochemistry provided the foundation for identifying the fundamental mechanism of cooperativity in the binding of Ca(2+) ions: this cooperativity provides EFCaBPs with the ability to detect the relatively small changes in concentration that constitute Ca(2+) signals. Using calbindin D(9k) as a model system, studies of the structure and fast time scale dynamics of each of the four ion binding states in a typical EF-hand domain provided direct evidence that site-site communication lowers the free energy cost of reorganization for binding the second ion. Our work has also extended models of how EFCaBPs interact with their cellular targets. We determined the unique dimeric architecture of S100 proteins, a specialized subfamily of EFCaBPs found exclusively in vertebrates. We described the implications for how these proteins transduce signals and went on to characterize interactions with peptide fragments of important cellular targets. Studies of the CaM homolog centrin revealed novel characteristics of its binding of Ca(2+) and its interaction with its cellular target Kar1. These results provided clear examples of how subtle differences in sequence fine-tune EFCaBPs to interact with their specific targets. The structural approach stands at a critical crossroad, shifting in emphasis from descriptive structural biochemistry to integrated biology and medicine. We present our dual-molecular-switch model for Ca(2+) regulation of gating functions of voltage-gated sodium channels in which both CaM and an intrinsic EF-hand domain serve as coupled Ca(2+) sensors. A second example involves novel EFCaBP extracellular function, that is, the role of S100A8/S100A9 heterodimer in the innate immune response to bacterial pathogens. A mechanism for the antimicrobial activity of S100A8/S100A9 was discovered. We describe interactions of S100A8/S100A9 and S100B with the cell surface receptor for advanced glycation end products. Biochemical and structural studies are now uncovering the mechanisms by which EFCaBPs work and are helping to define their biological activities, while simultaneously expanding knowledge of the roles of these proteins in normal cellular physiology and the pathology of disease.

Bifunctional Acid–Base Cooperativity in Heterogeneous Catalytic Reactions: Advances in Silica Supported Organic Functional Groups

Abstract: Emulating nature is a powerful way to realize selective catalytic reactions and to develop structurally complex organic molecules. A well understood fact in biochemistry is that enzymes are capable of accelerating reactions through cooperative interactions between accurately positioned functional groups present in their active sites. Substrate recognition and activation may proceed through electrostatic, hydrogen bonding, or covalent interactions. Following the concept of ‘site isolation’, significant progress has recently been achieved in the immobilization of mutually destructive catalysts for multistep cascade reactions. ‘Site isolation’ or ‘compartmentalization’ is thus considered as one of the paradigms in heterogeneous catalysis especially in the separation of mutually incompatible catalysts, such as acids and bases. This concept, adopted from nature and through which incompatible sites are spatially separated, aids in avoiding undesired interactions. Thereby tedious work up procedures and purification can be greatly minimized. Cascade reactions—defined as two or more reactions occurring in one reactor—are an emerging strategy in modern synthetic chemistry, as a) several bonds can be formed in a single reaction step in the same reaction vessel without isolation of intermediates, b) they allow a rapid and efficient synthesis of complex molecules from simple starting materials, and c) the overall process is ‘green’ in terms of waste and cost reduction. However, it is not trivial for material chemists to try to mimic biological catalysts by fixing antagonist groups in solid supports for various onestep cooperative catalytic reactions. In a classical concept, a support material serves to handle and recover the catalyst, whereas recent systematic investigations suggest that surfaces will often act cooperatively to stabilize specific catalyst structures. Thus during the last years researchers focused their efforts to precisely position bior multifunctional groups on solid supports for cooperative catalytic reactions. Beyond doubt, this concept of multifunctional heterogeneous or heterogenized catalysts is one way to economic and ecologically benign processes. In this context, organic–inorganic hybrid materials have shown their applicability in a broad variety of heterogeneous catalytic reactions. They show high mechanical, thermal, and chemical stability provided by the inorganic components, while the organic compartments can be independently selected and optimized for specific catalytic applications. Organic modifications of structured inorganic solids, for example mesoporous materials, can be performed by three different ways: by the grafting method, by the co-condensation method, and by the formation of an organosilica material (Scheme 1). By employing one of these methods a series of monoor bifunctional organic groups can be successfully fixed inside the channels of silica supports. Heterogeneous bifunctional catalysts can provide a continuous range of functional groups and offer advantages, such as an enhancement of reactivity and stability of antagonist func-

Size and Sequence and the Volume Change of Protein Folding

Abstract: The application of hydrostatic pressure generally leads to protein unfolding, implying, in accordance to LeChatelier’s principle, that the unfolded state has smaller molar volume than the folded state. However, the origin of the volume change upon unfolding, ΔVu, has yet to be determined. We have examined systematically the effects of protein size and sequence on the value of ΔVu using as a model system a series of deletion variants of the ankyrin repeat domain of the Notch receptor. The results provide strong evidence in support of the notion that the major contributing factor to pressure effects on proteins is their imperfect internal packing in the folded state. These packing defects appear to be specifically localized in the 3D structure, in contrast to the uniformly distributed effects of temperature and denaturants which depend upon hydration of exposed surface area upon unfolding. Given its local nature, the extent to which pressure globally affects protein structure can inform on the degree of cooperativity and long range coupling intrinsic to the folded state. We also show that the energetics of the protein’s conformations can significantly modulate their volumetric properties, providing further insight into protein stability.

Abnormal N-heterocyclic Carbenes: More than Just Exceptionally Strong Donor Ligands

Abstract: Complexes comprising a so-called abnormal carbene ligand, which displays pronounced mesoionic character, have recently been shown to be competent catalyst precursors for bond activation processes and oxidative transformations, including base-free alcohol oxidation and water oxidation. In this highlight we propose that these abnormal carbene ligands are not just useful spectator ligands but also actively participate in the bond activation step. This mode of action is partially based on the exceptionally strong donor properties of the ligand and, specifically, on the mesoionic character of these abnormal carbenes. The mesoionic properties provide a reservoir for charges and holes and thus induce efficient ligand-metal cooperativity, which is beneficial in particular for oxidation catalysis that involves concerted proton and electron transfer processes.

Cross species comparison of C/EBPα and PPARγ profiles in mouse and human adipocytes reveals interdependent retention of binding sites

Abstract: The transcription factors peroxisome proliferator activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) are key transcriptional regulators of adipocyte differentiation and function. We and others have previously shown that binding sites of these two transcription factors show a high degree of overlap and are associated with the majority of genes upregulated during differentiation of murine 3T3-L1 adipocytes. Here we have mapped all binding sites of C/EBPα and PPARγ in human SGBS adipocytes and compared these with the genome-wide profiles from mouse adipocytes to systematically investigate what biological features correlate with retention of sites in orthologous regions between mouse and human. Despite a limited interspecies retention of binding sites, several biological features make sites more likely to be retained. First, co-binding of PPARγ and C/EBPα in mouse is the most powerful predictor of retention of the corresponding binding sites in human. Second, vicinity to genes highly upregulated during adipogenesis significantly increases retention. Third, the presence of C/EBPα consensus sites correlate with retention of both factors, indicating that C/EBPα facilitates recruitment of PPARγ. Fourth, retention correlates with overall sequence conservation within the binding regions independent of C/EBPα and PPARγ sequence patterns, indicating that other transcription factors work cooperatively with these two key transcription factors. This study provides a comprehensive and systematic analysis of what biological features impact on retention of binding sites between human and mouse. Specifically, we show that the binding of C/EBPα and PPARγ in adipocytes have evolved in a highly interdependent manner, indicating a significant cooperativity between these two transcription factors.

Stepwise effective molarities in porphyrin oligomer complexes: preorganization results in exceptionally strong chelate cooperativity.

Abstract: Complexes of zinc porphyrin oligomers with multivalent ligands can be denatured by adding a large excess of a monodentate ligand, such as quinuclidine. We have used denaturation titrations to determine the stabilities of the complexes of a cyclic zinc–porphyrin hexamer with multidentate ligands with two to six pyridyl coordination sites. The corresponding complexes of linear porphyrin oligomers were also investigated. The results reveal that the stepwise effective molarities (EMs) for the third through sixth intramolecular coordination events with the cyclic hexamer are extremely high (EM = 102–103 M), whereas the values for the linear porphyrin oligomers are modest (EM ≈ 0.05 M). The speciation profiles for the denaturation reactions demonstrate that intermediate species are not significantly populated and that these equilibria are well described by a highly cooperative two-state model.

Rationalizing Tight Ligand Binding through Cooperative Interaction Networks

Abstract: Small modifications of the molecular structure of a ligand sometimes cause strong gains in binding affinity to a protein target, rendering a weakly active chemical series suddenly attractive for further optimization. Our goal in this study is to better rationalize and predict the occurrence of such interaction hot-spots in receptor binding sites. To this end, we introduce two new concepts into the computational description of molecular recognition. First, we take a broader view of noncovalent interactions and describe protein–ligand binding with a comprehensive set of favorable and unfavorable contact types, including for example halogen bonding and orthogonal multipolar interactions. Second, we go beyond the commonly used pairwise additive treatment of atomic interactions and use a small world network approach to describe how interactions are modulated by their environment. This approach allows us to capture local cooperativity effects and considerably improves the performance of a newly derived empirica...

The oligomeric state sets GABAB receptor signalling efficacy

Abstract: G protein-coupled receptors (GPCRs) have key roles in cell-cell communication. Recent data suggest that these receptors can form large complexes, a possibility expected to expand the complexity of this regulatory system. Among the brain GPCRs, the heterodimeric GABA(B) receptor is one of the most abundant, being distributed in most brain regions, on either pre- or post-synaptic elements. Here, using specific antibodies labelled with time-resolved FRET compatible fluorophores, we provide evidence that the heterodimeric GABA(B) receptor can form higher-ordered oligomers in the brain, as suggested by the close proximity of the GABA(B1) subunits. Destabilizing the oligomers using a competitor or a GABA(B1) mutant revealed different G protein coupling efficiencies depending on the oligomeric state of the receptor. By examining, in heterologous system, the G protein coupling properties of such GABA(B) receptor oligomers composed of a wild-type and a non-functional mutant heterodimer, we provide evidence for a negative functional cooperativity between the GABA(B) heterodimers.

Allosteric interactions across native adenosine-A3 receptor homodimers: quantification using single-cell ligand-binding kinetics

Abstract: A growing awareness indicates that many G-protein-coupled receptors (GPCRs) exist as homodimers, but the extent of the cooperativity across the dimer interface has been largely unexplored. Here, measurement of the dissociation kinetics of a fluorescent agonist (ABA-X-BY630) from the human A1 or A3 adenosine receptors expressed in CHO-K1 cells has provided evidence for highly cooperative interactions between protomers of the A3-receptor dimer in single living cells. In the absence of competitive ligands, the dissociation rate constants of ABA-X-BY630 from A1 and A3 receptors were 1.45 ± 0.05 and 0.57 ± 0.07 min−1, respectively. At the A3 receptor, this could be markedly increased by both orthosteric agonists and antagonists [15-, 9-, and 19-fold for xanthine amine congener (XAC), 5′-(N-ethyl carboxamido)adenosine (NECA), and adenosine, respectively] and reduced by coexpression of a nonbinding (N250A) A3-receptor mutant. The changes in ABA-X-BY630 dissociation were much lower at the A1 receptor (1.5-, 1.4-, and 1.5-fold). Analysis of the pEC50 values of XAC, NECA, and adenosine for the ABA-X-BY630-occupied A3-receptor dimer yielded values of 6.0 ± 0.1, 5.9 ± 0.1, and 5.2 ± 0.1, respectively. This study provides new insight into the spatial and temporal specificity of drug action that can be provided by allosteric modulation across a GPCR homodimeric interface.—May, L. T., Bridge, L. J., Stoddart, L. A., Briddon, S. J., Hill, S. J. Allosteric interactions across native adenosine-A3 receptor homodimers: quantification using single-cell ligand-binding kinetics.

Comparison of P⋯D (D = P,N) with other noncovalent bonds in molecular aggregates

Abstract: All the minima on the potential energy surfaces of homotrimers and tetramers of PH3 are identified and analyzed as to the source of their stability. The same is done with mixed trimers in which one PH3 molecule is replaced by either NH3 or PFH2. The primary noncovalent attraction in all global minima is the BP⋯D (D = N,P) bond which is characterized by the transfer of charge from a lone pair of the donor D to a σ* B–P antibond of the partner molecule which is turned away from D, the same force earlier identified in the pertinent dimers. Examination of secondary minima reveals the presence of other weaker forces, some of which do not occur within the dimers. Examples of the latter include PH⋯P, NH⋯P, and PH⋯F H-bonds, and “reverse” H-bonds in which the source of the electron density is the smaller tail lobe of the donor lone pair. The global minima are cyclic structures in all cases, and exhibit some cooperativity, albeit to a small degree. The energy spacing of the oligomers is much smaller than that in the corresponding strongly H-bonded complexes such as the water trimer.

Temperature sensitivity trends and multi-stimuli sensitive behavior in amphiphilic oligomers.

Abstract: A series of oligomers, containing oligo(ethylene glycol) (OEG) moieties, with the same composition of amphiphilic functionalities has been designed, synthesized, and characterized on the basis of their temperature-sensitive behavior. The non-covalent amphiphilic aggregates, formed from these molecules, influence their temperature sensitivity. Covalent tethering of the amphiphilic units also has a significant influence on their temperature sensitivity. The lower critical solution temperatures of these oligomers show increasingly sharp transitions with increasing numbers of OEG functional groups, indicating enhanced cooperativity in dehydration of the OEG moieties when they are covalently tethered. These molecules were also engineered to be concurrently sensitive to enzymatic reaction and pH. This possibility was investigated using porcine liver esterase as the enzyme; we show that enzymatic action on the pentamer lowers its temperature sensitivity. The product moiety from the enzymatic reaction also gives ...

Theoretical Study on Cooperativity Effects between Anion–π and Halogen‐Bonding Interactions

Abstract: This article analyzes the interplay between lone pair-π (lp-π) or anion-π interactions and halogen-bonding interactions. Interesting cooperativity effects are observed when lp/anion-π and halogen-bonding interactions coexist in the same complex, and they are found even in systems in which the distance between the anion and halogen-bond donor molecule is longer than 9 A. These effects are studied theoretically in terms of energetic and geometric features of the complexes, which are computed by ab initio methods. Bader's theory of "atoms in molecules" is used to characterize the interactions and to analyze their strengthening or weakening depending upon the variation of charge density at critical points. The physical nature of the interactions and cooperativity effects are studied by means of molecular interaction potential with polarization partition scheme. By taking advantage of all aforementioned computational methods, the present study examines how these interactions mutually influence each other. Additionally, experimental evidence for such interactions is obtained from the Cambridge Structural Database (CSD).

Sensing cooperativity in ATP hydrolysis for single multisubunit enzymes in solution

Abstract: In order to operate in a coordinated fashion, multisubunit enzymes use cooperative interactions intrinsic to their enzymatic cycle, but this process remains poorly understood. Accordingly, ATP number distributions in various hydrolyzed states have been obtained for single copies of the mammalian double-ring multisubunit chaperonin TRiC/CCT in free solution using the emission from chaperonin-bound fluorescent nucleotides and closed-loop feedback trapping provided by an Anti-Brownian ELectrokinetic trap. Observations of the 16-subunit complexes as ADP molecules are dissociating shows a peak in the bound ADP number distribution at 8 ADP, whose height falls over time with little shift in the position of the peak, indicating a highly cooperative ADP release process which would be difficult to observe by ensemble-averaged methods. When AlFx is added to produce ATP hydrolysis transition state mimics (ADP·AlFx) locked to the complex, the peak at 8 nucleotides dominates for all but the lowest incubation concentrations. Although ensemble averages of the single-molecule data show agreement with standard cooperativity models, surprisingly, the observed number distributions depart from standard models, illustrating the value of these single-molecule observations in constraining the mechanism of cooperativity. While a complete alternative microscopic model cannot be defined at present, the addition of subunit-occupancy-dependent cooperativity in hydrolysis yields distributions consistent with the data.

Competing allosteric mechanisms modulate substrate binding in a dimeric enzyme

Abstract: Allostery has been studied for many decades, yet it remains challenging to determine experimentally how it occurs at a molecular level. We have developed an approach combining isothermal titration calorimetry, circular dichroism and nuclear magnetic resonance spectroscopy to quantify allostery in terms of protein thermodynamics, structure and dynamics. This strategy was applied to study the interaction between aminoglycoside N-(6')-acetyltransferase-Ii and one of its substrates, acetyl coenzyme A. It was found that homotropic allostery between the two active sites of the homodimeric enzyme is modulated by opposing mechanisms. One follows a classical Koshland-Nemethy-Filmer (KNF) paradigm, whereas the other follows a recently proposed mechanism in which partial unfolding of the subunits is coupled to ligand binding. Competition between folding, binding and conformational changes represents a new way to govern energetic communication between binding sites.

Unsuspected pathway of the allosteric transition in hemoglobin.

Abstract: Large conformational transitions play an essential role in the function of many proteins, but experiments do not provide the atomic details of the path followed in going from one end structure to the other. For the hemoglobin tetramer, the transition path between the unliganded (T) and tetraoxygenated (R) structures is not known, which limits our understanding of the cooperative mechanism in this classic allosteric system, where both tertiary and quaternary changes are involved. The conjugate peak refinement algorithm is used to compute an unbiased minimum energy path at atomic detail between the two end states. Although the results confirm some of the proposals of Perutz [Perutz MF (1970) Stereochemistry of cooperative effects in haemoglobin. Nature 228:726–734], the subunit motions do not follow the textbook description of a simple rotation of one αβ-dimer relative to the other. Instead, the path consists of two sequential quaternary rotations, each involving different subdomains and axes. The quaternary transitions are preceded and followed by phases of tertiary structural changes. The results explain the recent photodissociation measurements, which suggest that the quaternary transition has a fast (2 μs) as well as a slow (20 μs) component and provide a testable model for single molecule FRET experiments.

Is there a connection between fragility of glass forming systems and dynamic heterogeneity/cooperativity?

Abstract: Although fragility of glass forming liquids is traditionally related to cooperativity in molecular motion, the connection between those parameters remains unclear. In this paper we present the estimates of cooperativity (heterogeneity) length scale ξ obtained from the boson peak spectra. We demonstrate that ξ agrees well with the dynamic heterogeneity length scale for the structural relaxation estimated by 4-dimensional NMR, justifying the use of ξ. Presented analysis of large number of materials reveals no clear correlation between ξ and fragility. However, there is a strong correlation between the cooperativity volume ξ 3 and the activation volume measured at T g . This observation suggests that only the volume (pressure) dependence of structural relaxation time correlates directly with the cooperativity size. However, the pure thermal (energetic) contribution to the structural relaxation, the so-called isochoric fragility, exhibits no correlation to the heterogeneity length scale ξ, or the amount of structural units in ξ 3 . The presented results call for a revision of traditional view on the role of cooperativity/heterogeneity in structural relaxation of glass forming systems.

The structural basis for homotropic and heterotropic cooperativity of midazolam metabolism by human cytochrome P450 3A4.

Abstract: Human cytochrome P450 3A4 (CYP3A4) metabolizes a significant portion of clinically relevant drugs and often exhibits complex steady-state kinetics that can involve homotropic and heterotropic cooperativity between bound ligands. In previous studies, the hydroxylation of the sedative midazolam (MDZ) exhibited homotropic cooperativity via a decrease in the ratio of 1′-OH-MDZ to 4-OH-MDZ at higher drug concentrations. In this study, MDZ exhibited heterotropic cooperativity with the antiepileptic drug carbamazepine (CBZ) with characteristic decreases in the 1′-OH-MDZ to 4-OH-MDZ ratios. To unravel the structural basis of MDZ cooperativity, we probed MDZ and CBZ bound to CYP3A4 using longitudinal T1 nuclear magnetic resonance (NMR) relaxation and molecular docking with AutoDock 4.2. The distances calculated from longitudinal T1 NMR relaxation were used during simulated annealing to constrain the molecules to the substrate-free X-ray crystal structure of CYP3A4. These simulations revealed that either two MDZ mo...

Enhancing Binding Affinity by the Cooperativity between Host Conformation and Host–Guest Interactions

Abstract: Glutamate-functionalized oligocholate foldamers bound Zn(OAc)2, guanidine, and even amine compounds with surprisingly high affinities. The conformational change of the hosts during binding was crucial to the enhanced binding affinity. The strongest cooperativity between the conformation and guest-binding occurred when the hosts were unfolded but near the folding–unfolding transition. These results suggest that high binding affinity in molecular recognition may be more easily obtained from large hosts capable of strong cooperative conformational changes instead of those with rigid, preorganized structures.