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Showing papers on "Cooperativity published in 2012"


Journal ArticleDOI
TL;DR: Specific types of heteroditopic receptor designs for ion pairs and the complexity of ion-pair binding are discussed to illustrate key concepts such as cooperativity.
Abstract: Ion-pair recognition is a new field of research emerging from cation and anion coordination chemistry. Specific types of heteroditopic receptor designs for ion pairs and the complexity of ion-pair binding are discussed to illustrate key concepts such as cooperativity. The importance of this area of research is reflected by the wide variety of potential applications of ion-pair receptors, including applications as membrane transport and salt solubilization agents and sensors.

208 citations


Journal ArticleDOI
TL;DR: 2-(quinolin-3-yl) acetic acid derivatives impairs both integrase-LEDGF binding and LEDGF-independent integrase catalytic activities with similar IC50 values, defining them as bona fide allosteric inhibitors of integrase function.

169 citations


Journal ArticleDOI
TL;DR: Short linkers limit the beneficial amine-silanol cooperativity in aldol condensations, resulting in lower catalytic rates than with the C3+ linkers, and the adsorptive cooperativity exhibits similar trends for CO(2) capture efficiency.
Abstract: Cooperative interactions between aminoalkylsilanes and silanols on a silica surface can be controlled by varying the length of the alkyl linker attaching the amine to the silica surface from C1 (methyl) to C5 (pentyl). The linker length strongly affects the catalytic cooperativity of amines and silanols in aldol condensations as well as the adsorptive cooperativity for CO2 capture. The catalytic cooperativity increases with the linker length up to propyl (C3), with longer, more flexible linkers (up to C5) providing no additional benefit or hindrance. Short linkers (C1 and C2) limit the beneficial amine–silanol cooperativity in aldol condensations, resulting in lower catalytic rates than with the C3+ linkers. For the same materials, the adsorptive cooperativity exhibits similar trends for CO2 capture efficiency.

158 citations


Journal ArticleDOI
TL;DR: A model that varies the size of the allosteric site and its interactions to construct a series of energy landscapes with pronounced minima corresponding to both the effector bound and unbound crystal structures is applied to three proteins with experimentally characterized transitions.
Abstract: Allostery is a phenomenon that couples effector ligand binding at an allosteric site to a structural and/or dynamic change at a distant regulated site. To study an allosteric transition, we vary the size of the allosteric site and its interactions to construct a series of energy landscapes with pronounced minima corresponding to both the effector bound and unbound crystal structures. We use molecular dynamics to sample these landscapes. The degree of perturbation by the effector, modeled by the size of the allosteric site, provides an order parameter for allostery that allows us to determine how microscopic motions give rise to commonly discussed macroscopic mechanisms: (i) induced fit, (ii) population shift, and (iii) entropy driven. These mechanisms involve decreasing structural differences between the effector bound and unbound populations. A metric (ligand-induced cooperativity) can measure how cooperatively a given regulated site responds to effector binding and therefore what kind of allosteric mechanism is involved. We apply the model to three proteins with experimentally characterized transitions: (i) calmodulin-GFP Ca2+ sensor protein, (ii) maltose binding protein, and (iii) CSL transcription factor. Remarkably, the model is able to reproduce allosteric motion and predict coupling in a manner consistent with experiment.

155 citations


Journal ArticleDOI
TL;DR: Pepcans are the first endogenous allosteric modulators identified for CB1 receptors and could play an important physiological role in modulating endocannabinoid signaling.

147 citations


Journal ArticleDOI
TL;DR: The cooperative interactions between CO2 molecules are found to augment overall binding by over 7 kJmol, a significant result for CO2 capture in any porous material, and the present study applies these methods to understanding CO2 uptake in another MOF, that intuitively should give betterCO2 capture properties.
Abstract: Alkylamines, such as monoethanolamine, are used to scrub CO2 molecules from flue gas streams, however, as they form strong chemical bonds (85–105 kJmol ), the post-capture recovery of the amine is energy-intensive (130–150 8C including heating the entire aqueous solution). Alternatively, the use of less-basic amines, such as aryl amines, could favor strong physisorption (30–50 kJmol ) with CO2, rather than chemisorption. This would mean a porous compound with such amine groups could give easy-on/easy-off reversible CO2 capture balanced with selectivity. To obtain high efficiency at lower partial pressures, the material, along with having strong CO2 binding sites, needs to have reasonable surface area for capacity. Metal–organic frameworks (MOFs) are widely studied for gas sorption owing to the ability to modify pore sizes, shapes, and surfaces. Functionalizing with specific interaction sites is being actively studied as a route to selective gas capture. Computational modeling can give tremendous insight to the sorption properties of a MOF. We recently reported a zinc aminotriazolato oxalate MOF, {Zn2(Atz)2(ox)} (2), exhibiting amine-lined pores and a high heat of adsorption for CO2 (ca. 40 kJmol ). Further studies showed that the CO2 binding sites could be located crystallographically. These data offered an exceptional opportunity to validate a suite of computational methods to model not only the CO2 isotherm, but also the locations of binding sites and role of specific interactions to the overall CO2 binding enthalpy. The present study applies these methods to understanding CO2 uptake in another MOF, {Zn3(Atz)3(PO4)} (1), that intuitively should give better CO2 capture properties. In comparison to {Zn2(Atz)2(ox)}, only two-thirds of the number of trianionic phosphate groups are required to charge compensate [Zn(Atz)] layers, so larger, amine-lined pores were anticipated and observed. Despite this, the CO2 uptake (at 273 K) and heat of adsorption do not exceed those of 2. The computational methods provide crucial insight to understanding these phenomena and demonstrate the wide spread applicability of such techniques to ascertain binding details in MOFs not directly accessible by experiment. Although the role of the amine functionalities in 1 is surprisingly diminished, the cooperative interactions between CO2 molecules are found to augment overall binding by over 7 kJmol , a significant result for CO2 capture in any porous material. Solvothermal reaction of basic ZnCO3 with 3-amino-1,2,4triazole, H3PO4, and NH4OH gave {Zn3Atz3(PO4)(H2O)3.5}, 1·(H2O)3.5, in both single-crystal and bulk phases (Supporting Information, Figure S1). The aminotriazole ligand has been employed to construct otherMOFs, including with Zn ions, but has not been extensively studied for CO2 capture excepting 2. 1·(H2O)3.5 is made up of cationic Zn–Atz layers pillared by PO4 anions to form a 3D porous network (Figure 1). The Zn(Atz) layers lie in the ac plane and contain three independent Zn ions and Atz ligands. No amine groups coordinate to Zn ions; ligation is exclusively through triazole nitrogen atoms. Pillaring of these layers by the phosphate ions results in a 3D network of pores (accounting for van der

125 citations


Journal ArticleDOI
TL;DR: A recently revised version of the additive CHARMM protein force field, which includes optimization of the backbone CMAP potential to achieve good balance between different types of secondary structure, is shown to result in improved cooperativity for the helix-coil transition, and the cooperativity of β-hairpin formation is much improved relative to other force fields studied.

124 citations


Journal ArticleDOI
27 Feb 2012-PLOS ONE
TL;DR: Interestingly, no natural substrates display the obtained consensus sequence but represent sequences that show only 1–30% of the optimal cleavage rate for thrombin, which clearly indicates that maximal cleavage, excluding the help of exosite interactions, is not always desired, which may instead cause problems with dysregulated coagulation.
Abstract: Thrombin is one of the most extensively studied of all proteases. Its central role in the coagulation cascade as well as several other areas has been thoroughly documented. Despite this, its consensus cleavage site has never been determined in detail. Here we have determined its extended substrate recognition profile using phage-display technology. The consensus recognition sequence was identified as, P2-Pro, P1-Arg, P1'-Ser/Ala/Gly/Thr, P2'-not acidic and P3'-Arg. Our analysis also identifies an important role for a P3'-arginine in thrombin substrates lacking a P2-proline. In order to study kinetics of this cooperative or additive effect we developed a system for insertion of various pre-selected cleavable sequences in a linker region between two thioredoxin molecules. Using this system we show that mutations of P2-Pro and P3'-Arg lead to an approximate 20-fold and 14-fold reduction, respectively in the rate of cleavage. Mutating both Pro and Arg results in a drop in cleavage of 200-400 times, which highlights the importance of these two positions for maximal substrate cleavage. Interestingly, no natural substrates display the obtained consensus sequence but represent sequences that show only 1-30% of the optimal cleavage rate for thrombin. This clearly indicates that maximal cleavage, excluding the help of exosite interactions, is not always desired, which may instead cause problems with dysregulated coagulation. It is likely exosite cooperativity has a central role in determining the specificity and rate of cleavage of many of these in vivo substrates. Major effects on cleavage efficiency were also observed for residues as far away as 4 amino acids from the cleavage site. Insertion of an aspartic acid in position P4 resulted in a drop in cleavage by a factor of almost 20 times.

118 citations


Journal ArticleDOI
TL;DR: A method for global unbiased analysis of RNA-protein interactions that uses in vitro selection, high-throughput sequencing, and sequence-specificity landscapes is described that yields affinities for a vast array of RNAs in a single experiment, including both low- and high-affinity sites.

116 citations


Journal ArticleDOI
TL;DR: There appear to be three allosterically linked binding sites on FFA1 with agonists specific for each of these sites, and these ligands with their high potencies and strong positive functional cooperativity with endogenous fatty acids have the potential to deliver therapeutic benefits.
Abstract: Activation of FFA1 (GPR40), a member of G protein-coupling receptor family A, is mediated by medium- and long-chain fatty acids and leads to amplification of glucose-stimulated insulin secretion, suggesting a potential role for free fatty acid 1 (FFA1) as a target for type 2 diabetes. It was assumed previously that there is a single binding site for fatty acids and synthetic FFA1 agonists. However, using members of two chemical series of partial and full agonists that have been identified, radioligand binding interaction studies revealed that the full agonists do not bind to the same site as the partial agonists but exhibit positive heterotropic cooperativity. Analysis of functional data reveals positive functional cooperativity between the full agonists and partial agonists in various functional assays (in vitro and ex vivo) and also in vivo. Furthermore, the endogenous fatty acid docosahexaenoic acid (DHA) shows negative or neutral cooperativity with members of both series of agonists in binding assays but displays positive cooperativity in functional assays. Another synthetic agonist is allosteric with members of both agonist series, but apparently competitive with DHA. Therefore, there appear to be three allosterically linked binding sites on FFA1 with agonists specific for each of these sites. Activation of free fatty acid 1 receptor (FFAR1) by each of these agonists is differentially affected by mutations of two arginine residues, previously found to be important for FFAR1 binding and activation. These ligands with their high potencies and strong positive functional cooperativity with endogenous fatty acids, demonstrated in vitro and in vivo, have the potential to deliver therapeutic benefits.

115 citations


Journal ArticleDOI
TL;DR: It is shown that LY2033298 can also bind to the M2 mAChR and mediate robust positive or negative allosteric effects, depending on the orthosteric ligand used as a probe of receptor activity, and this finding of striking “probe dependence” indicates that the previously described selectivity of the modulator does not arise as a consequence of selective affinity for a poorly conservedallosteric site.
Abstract: We recently described 3-amino-5-chloro-6-methoxy-4-methylthieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298) as a novel allosteric modulator of M(4) muscarinic acetylcholine (ACh) receptors (mAChRs) on the basis of its ability to preferentially potentiate the actions of ACh at the M(4) mAChR subtype. In the current study, we show that LY2033298 can also bind to the M(2) mAChR and mediate robust positive or negative allosteric effects, depending on the orthosteric ligand used as a probe of receptor activity. This finding of striking "probe dependence" indicates that the previously described selectivity of the modulator does not arise as a consequence of selective affinity for a poorly conserved allosteric site but rather is due to subtype-selective cooperativity with ACh upon interaction with a common allosteric binding site. Moreover, a comparison of the effects of the modulator on orthosteric ligand affinity relative to signaling through a [(35)S]guanosine 5'-O-(3-thio)triphosphate or extracellular signal-regulated kinase 1/2 phosphorylation assay at the M(2) mAChR revealed that, although the effects on binding were positive in all instances, the effects on signaling were either positive or strongly negative, depending on the agonist and the pathway. Mutational analysis identified residues Tyr177 and Trp99(3.28) (Ballesteros and Weinstein numbers are provided in superscript to indicate relative position of residues within the transmembrane domain) as contributing to the binding of LY2033298, whereas the orthosteric site residues, Tyr104(3.33) and Tyr403(6.51), contributed to the ability of the ligand to impose pathway-biased modulation. Taken together, these findings have important implications for the detection and validation of allosteric modulators of G protein-coupled receptors (GPCRs), because they highlight the potential for ligand misclassification or lack of appreciation of off-target allosteric activities.

Journal ArticleDOI
TL;DR: Microscale thermophoresis shows that PIP2 binding to the polybasic patch of synaptotagmin-1 increases the Ca2+ affinity by >40-fold, which shows that this interplay between Ca2+, synaptolipid phosphatidylinositol 4,5-bisphosphate, and PIP1 is crucial for neurotransmitter release.

Journal ArticleDOI
TL;DR: It is demonstrated that PC190723 negatively modulates Staphylococcus aureus FtsZ polymerization cooperativity as reflected in polymerization at lower concentrations without a defined critical concentration.
Abstract: The cooperative assembly of FtsZ, the prokaryotic homologue of tubulin, plays an essential role in cell division. FtsZ is a potential drug target, as illustrated by the small-molecule cell-cycle inhibitor and antibacterial agent PC190723 that targets FtsZ. We demonstrate that PC190723 negatively modulates Staphylococcus aureus FtsZ polymerization cooperativity as reflected in polymerization at lower concentrations without a defined critical concentration. The crystal structure of the S. aureus FtsZ-PC190723 complex shows a domain movement that would stabilize the FtsZ protofilament over the monomeric state, with the conformational change mediated from the GTP-binding site to the C-terminal domain via helix 7. Together, the results reveal the molecular mechanism of FtsZ modulation by PC190723 and a conformational switch to the high-affinity state that enables polymer assembly.

Journal ArticleDOI
TL;DR: Stochastic reaction kinetic theory is used to discuss cooperative transitions in cellular biochemical processes at both the macromolecular and the cellular levels and shows that fluctuation-enhanced sensitivity (stochastic focusing) shares an essential feature with the transition in a bistable system.
Abstract: Cooperativity in classical biophysics originates from molecular interactions; nonlinear feedbacks in biochemical networks regulate dynamics inside cells. Using stochastic reaction kinetic theory, we discuss cooperative transitions in cellular biochemical processes at both the macromolecular and the cellular levels. We show that fluctuation-enhanced sensitivity (stochastic focusing) shares an essential feature with the transition in a bistable system. The same theory explains zeroth-order ultrasensitivity with temporal cooperativity. Dynamic cooperativity in fluctuating enzyme (i.e., dynamic disorder), stochastic focusing, and the recently proposed stochastic binary decision all have a shared mechanism: They are generalizations of the hyperbolic response of Michaelis-Menten kinetics x/(K+x), with fluctuating K or stochastic x. Sigmoidal dependence on substrate concentration necessarily yields affinity amplification for competing ligands; both sigmoidal response and affinity amplification exhibit a square l...

Journal ArticleDOI
TL;DR: It is shown that self-association to a catalytically viable state requires structural cooperativity of the flexible β-hairpin “flap” regions of the enzyme and that the major transition pathway is first via self-Association in the semiopen/open enzyme states, followed by enzyme conformational transition into a catalyTically viable closed state.
Abstract: HIV maturation requires multiple cleavage of long polyprotein chains into functional proteins that include the viral protease itself. Initial cleavage by the protease dimer occurs from within these precursors, and yet only a single protease monomer is embedded in each polyprotein chain. Self-activation has been proposed to start from a partially dimerized protease formed from monomers of different chains binding its own N termini by self-association to the active site, but a complete structural understanding of this critical step in HIV maturation is missing. Here, we captured the critical self-association of immature HIV-1 protease to its extended amino-terminal recognition motif using large-scale molecular dynamics simulations, thus confirming the postulated intramolecular mechanism in atomic detail. We show that self-association to a catalytically viable state requires structural cooperativity of the flexible β-hairpin “flap” regions of the enzyme and that the major transition pathway is first via self-association in the semiopen/open enzyme states, followed by enzyme conformational transition into a catalytically viable closed state. Furthermore, partial N-terminal threading can play a role in self-association, whereas wide opening of the flaps in concert with self-association is not observed. We estimate the association rate constant (kon) to be on the order of ∼1 × 104 s−1, suggesting that N-terminal self-association is not the rate-limiting step in the process. The shown mechanism also provides an interesting example of molecular conformational transitions along the association pathway.

Journal ArticleDOI
TL;DR: The results presented here show that multivalency is expressed in both the thermodynamics and the kinetics in different ways and a spacer optimized for strong binding is suboptimal for fast pseudorotaxane formation.
Abstract: Homo- and heterodivalent crown-ammonium pseudorotaxanes with different spacers connecting the two axle ammonium binding sites have been synthesized and characterized by NMR spectroscopy and ESI mass spectrometry. The homodivalent pseudorotaxanes are investigated with respect to the thermodynamics of divalent binding and to chelate cooperativity. The shortest spacer exhibits a chelate cooperativity much stronger than that of the longer spacers. On the basis of crystal structure, this can be explained by a noninnocent spacer, which contributes to the binding strength in addition to the two binding sites. Already very subtle changes in the spacer length, i.e., the introduction of an additional methylene group, cause substantial changes in the magnitude of cooperative binding as expressed in the large differences in effective molarity. With a similar series of heterodivalent pseudorotaxanes, the spacer effects on the barrier for the intramolecular threading step has been examined with the result that the shor...

Journal ArticleDOI
TL;DR: The crystal structure of the intact Escherichia coli RelB2E2 complex at 2.8 Å resolution provides a firm basis for understanding the model of conditional cooperativity at the molecular level and supports a model in which relO is optimally bound by two adjacent RelB 2E heterotrimeric units, and is not compatible with concomitant binding of two RelB1E2 heterotetramers.

Journal ArticleDOI
13 Apr 2012-Cell
TL;DR: It is suggested that cooperativity in noncoding RNAs arose from natural selection of architectures conducive to forming a unique, stable fold.

Journal ArticleDOI
TL;DR: The primary mechanisms postulated to give rise to monomeric cooperativity are discussed and modern experimental methods that could offer new insights into the nature of this phenomenon are highlighted.

Journal ArticleDOI
TL;DR: This work reveals the biological function of conditional cooperativity by constructing a mathematical model of the well studied TA system, relBE of Escherichia coli and shows that the model with the in vivo and in vitro established parameters reproduces experimentally observed response to nutritional stress.
Abstract: Many toxin–antitoxin (TA) loci are known to strongly repress their own transcription. This auto-inhibition is often called ‘conditional cooperativity’ as it relies on cooperative binding of TA complexes to operator DNA that occurs only when toxins are in a proper stoichiometric relationship with antitoxins. There has recently been an explosion of interest in TA systems due to their role in bacterial persistence, however the role of conditional cooperativity is still unclear. We reveal the biological function of conditional cooperativity by constructing a mathematical model of the well studied TA system, relBE of Escherichia coli. We show that the model with the in vivo and in vitro established parameters reproduces experimentally observed response to nutritional stress. We further demonstrate that conditional cooperativity stabilizes the level of antitoxin in rapidly growing cells such that random induction of relBE is minimized. At the same time it enables quick removal of free toxin when the starvation is terminated.

Journal ArticleDOI
01 Oct 2012-Langmuir
TL;DR: A set of Langmuir binding models in which electrostatic cooperativity effects to protein sorption is incorporated in the spirit of Guoy-Chapman-Stern models, where the global substrate (microgel) charge state is modified by bound reactants (charged proteins).
Abstract: We present a set of Langmuir binding models in which electrostatic cooperativity effects to protein sorption is incorporated in the spirit of Guoy-Chapman-Stern models, where the global substrate (microgel) charge state is modified by bound reactants (charged proteins). Application of this approach to lysozyme sorption to oppositely charged core-shell microgels allows us to extract the intrinsic, binding affinity of the protein to the gel, which is salt concentration independent and mostly hydrophobic in nature. The total binding affinity is found to be mainly electrostatic in nature, changes many orders of magnitude during the sorption process, and is significantly influenced by osmotic deswelling effects. The intrinsic binding affinity is determined to be about 7 k(B)T for our system. We additionally show that Langmuir binding models and those based on excluded-volume interactions are formally equivalent for low to moderate protein packing, if the nature of the bound state is consistently defined. Having appreciated this, a more quantitative interpretation of binding isotherms in terms of separate physical interactions is possible in the future for a wide variety of experimental approaches.

Journal ArticleDOI
TL;DR: Five cryo‐EM structures of TRiC in apo and nucleotide‐induced states without imposing symmetry during the 3D reconstruction reveal the intra‐ and inter‐ring subunit interaction pattern changes during the ATPase cycle.
Abstract: The eukaryotic group II chaperonin TRiC/CCT is a 16-subunit complex with eight distinct but similar subunits arranged in two stacked rings. Substrate folding inside the central chamber is triggered by ATP hydrolysis. We present five cryo-EM structures of TRiC in apo and nucleotide-induced states without imposing symmetry during the 3D reconstruction. These structures reveal the intra- and inter-ring subunit interaction pattern changes during the ATPase cycle. In the apo state, the subunit arrangement in each ring is highly asymmetric, whereas all nucleotide-containing states tend to be more symmetrical. We identify and structurally characterize an one-ring closed intermediate induced by ATP hydrolysis wherein the closed TRiC ring exhibits an observable chamber expansion. This likely represents the physiological substrate folding state. Our structural results suggest mechanisms for inter-ring-negative cooperativity, intra-ring-positive cooperativity, and protein-folding chamber closure of TRiC. Intriguingly, these mechanisms are different from other group I and II chaperonins despite their similar architecture.

Journal ArticleDOI
TL;DR: An operational model of allosterism that allows quantitative estimation of modulator affinity and cooperativity values is validated and can be applied to PAM and NAM potency curves in combination with maximal fold-shift data to derive reliable estimates of modulators affinities.
Abstract: Drug discovery programs increasingly are focusing on allosteric modulators as a means to modify the activity of G protein-coupled receptor (GPCR) targets. Allosteric binding sites are topographically distinct from the endogenous ligand (orthosteric) binding site, which allows for co-occupation of a single receptor with the endogenous ligand and an allosteric modulator that can alter receptor pharmacological characteristics. Negative allosteric modulators (NAMs) inhibit and positive allosteric modulators (PAMs) enhance the affinity and/or efficacy of orthosteric agonists. Established approaches for estimation of affinity and efficacy values for orthosteric ligands are not appropriate for allosteric modulators, and this presents challenges for fully understanding the actions of novel modulators of GPCRs. Metabotropic glutamate receptor 5 (mGlu5) is a family C GPCR for which a large array of allosteric modulators have been identified. We took advantage of the many tools for probing allosteric sites on mGlu5 to validate an operational model of allosterism that allows quantitative estimation of modulator affinity and cooperativity values. Affinity estimates derived from functional assays fit well with affinities measured in radioligand binding experiments for both PAMs and NAMs with diverse chemical scaffolds and varying degrees of cooperativity. We observed modulation bias for PAMs when we compared mGlu5-mediated Ca2+ mobilization and extracellular signal-regulated kinase 1/2 phosphorylation data. Furthermore, we used this model to quantify the effects of mutations that reduce binding or potentiation by PAMs. This model can be applied to PAM and NAM potency curves in combination with maximal fold-shift data to derive reliable estimates of modulator affinities.

Journal ArticleDOI
TL;DR: It is shown that GMP can stabilize the closed (active) state of T1R1 by binding to the outer vestibule of the so‐called Venus flytrap domain of the receptor, thus suggesting a molecular mechanism of cooperativity between GMP and glutamate.
Abstract: The fifth taste quality, umami, arises from binding of glutamate to the umami receptor T1R1/T1R3. The umami taste is enhanced several-fold upon addition of free nucleotides such as guanosine-5′-monophosphate (GMP) to glutamate-containing food. GMP may operate via binding to the ligand-binding domain of the T1R1 part of the umami receptor at an allosteric site. Using molecular dynamics simulations, we show that GMP can stabilize the closed (active) state of T1R1 by binding to the outer vestibule of the so-called Venus flytrap domain of the receptor. The transition between the closed and open conformations was accessed in the simulations. Using principal component analysis, we show that the dynamics of the Venus flytrap domain along the hinge-bending motion that activates signaling is dampened significantly upon binding of glutamate, and further slows down upon binding of GMP at an allosteric site, thus suggesting a molecular mechanism of cooperativity between GMP and glutamate.

Journal ArticleDOI
01 Jun 2012-RNA
TL;DR: Dual-color fluorescence cross-correlation spectroscopy (dcFCCS) shows that the binding affinity of several proteins is significantly changed during the Prp2-mediated transition of precatalytic B(act) splICEosomes to catalytically activated B* spliceosomes from Saccharomyces cerevisiae, and suggests high cooperativity of multiple Prp 2-mediated structural rearrangements at the spliceOSome's catalytic core.
Abstract: The compositional and conformational changes during catalytic activation of the spliceosome promoted by the DEAH box ATPase Prp2 are only poorly understood. Here, we show by dual-color fluorescence cross-correlation spectroscopy (dcFCCS) that the binding affinity of several proteins is significantly changed during the Prp2-mediated transition of precatalytic B act spliceosomes to catalytically activated B* spliceosomes from Saccharomyces cerevisiae. During this step, several proteins, including the zinc-finger protein Cwc24, are quantitatively displaced from the B* complex. Consistent with this, we show that Cwc24 is required for step 1 but not for catalysis per se. The U2-associated SF3a and SF3b proteins Prp11 and Cus1 remain bound to the B* spliceosome under near-physiological conditions, but their binding is reduced at high salt. Conversely, high-affinity binding sites are created for Yju2 and Cwc25 during catalytic activation, consistent with their requirement for step 1 catalysis. Our results suggest high cooperativity of multiple Prp2-mediated structural rearrangements at the spliceosome’s catalytic core. Moreover, dcFCCS represents a powerful tool ideally suited to study quantitatively spliceosomal protein dynamics in equilibrium.

Journal ArticleDOI
TL;DR: Polypeptoids, which lack backbone hydrogen bonding and chirality, are used to probe the exclusive effect of hydrophobicity on the coil-to-globule collapse and two sequences containing the same composition of only hydrophobic “H’ N-methylglycine and polar “P” N-(2-carboxyethyl)glycines are shown to have very different globule collapse behaviors.
Abstract: Understanding the driving forces for the collapse of a polymer chain from a random coil to a globule would be invaluable in enabling scientists to predict the folding of polypeptide sequences into defined tertiary structures. The HP model considers hydrophobic collapse to be the major driving force for protein folding. However, due to the inherent presence of chirality and hydrogen bonding in polypeptides, it has been difficult to experimentally test the ability of hydrophobic forces to independently drive structural transitions. In this work, we use polypeptoids, which lack backbone hydrogen bonding and chirality, to probe the exclusive effect of hydrophobicity on the coil-to-globule collapse. Two sequences containing the same composition of only hydrophobic "H" N-methylglycine and polar "P" N-(2-carboxyethyl)glycine monomers are shown to have very different globule collapse behaviors due only to the difference in their monomer sequence. As compared to a repeating sequence with an even distribution of H and P monomers, a designed protein-like sequence collapses into a more compact globule in aqueous solution as evidenced by small-angle X-ray scattering, dynamic light scattering, and probing with environmentally sensitive fluorophores. The free energy change for the coil-to-globule transition was determined by equilibrium denaturant titration with acetonitrile. Using a two-state model, the protein-like sequence is shown to have a much greater driving force for globule formation, as well as a higher m value, indicating increased cooperativity for the collapse transition. This difference in globule collapse behavior validates the ability of the HP model to describe structural transitions based solely on hydrophobic forces.

Journal ArticleDOI
TL;DR: This analysis of the complex activation mechanism of homotetrameric HCN2 channels by confocal patch-clamp fluorometry and kinetically quantify all ligand binding steps and closed-open isomerizations of the intermediate states suggests a dimeric organization of the activation process.
Abstract: The four subunits of the tetrameric voltage-sensitive HCN channel have different cAMP binding affinities, with the second and fourth binding events being positively cooperative and the third being negatively cooperative, suggesting a double-dimeric organization of the channel. Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric membrane proteins that generate electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are primarily activated by voltage but are receptors as well, binding the intracellular ligand cyclic AMP. The molecular mechanism of channel activation is still unknown. Here we analyze the complex activation mechanism of homotetrameric HCN2 channels by confocal patch-clamp fluorometry and kinetically quantify all ligand binding steps and closed-open isomerizations of the intermediate states. For the binding affinity of the second, third and fourth ligand, our results suggest pronounced cooperativity in the sequence positive, negative and positive, respectively. This complex interaction of the subunits leads to a preferential stabilization of states with zero, two or four ligands and suggests a dimeric organization of the activation process: within the dimers the cooperativity is positive, whereas it is negative between the dimers.

Journal ArticleDOI
TL;DR: The calculated binding free energies were found to be in good agreement with the values predicted from a simple 2-ligand binding kinetic model as well as to successfully reproduce the experimental titration curve, which confirms the general applicability of rapid free-energy methods to study challenging biomolecular systems like cytochromes P450, which are characterized by a large flexibility and malleability of their active sites.
Abstract: Cytochrome P450 3A4 (CYP3A4) metabolizes more than 50% of clinically used drugs and is often involved in adverse drug–drug interactions. It displays atypical binding and kinetic behavior toward a number of ligands characterized by a sigmoidal shape of the corresponding titration curves, which is indicative of a positive homotropic cooperativity. This requires a participation of at least two ligand molecules, whereby the binding of the first ligand molecule increases the affinity of CYP3A4 for the binding of the second ligand molecule. In the current study, a combination of molecular dynamics simulations and free-energy calculations was applied to elucidate the physicochemical origin of the observed positive homotropic cooperativity in ketoconazole binding to CYP3A4. The binding of the first ketoconazole molecule was established to increase the affinity for the binding of the second ketoconazole molecule by 5 kJ mol–1, which explains and quantifies the experimentally observed cooperative behavior of CYP3A4...

Journal ArticleDOI
Zheng Yan1, Mian Li1, Hui-Ling Gao1, Xiao-Chun Huang1, Dan Li1 
TL;DR: Three 3D metal-organic framework polymorphs with the chemical formula [Fe(2)(H(0.67)bdt)(3)]·xH(2)O (H(1,4-phenylene)bis(1H-tetrazole)), all of which are constructed from similar Fe(II)-tetazole rod secondary building units (SBUs) via covalent links, but exhibit diverse spin states regulated by inter-chain cooperativity.

Journal ArticleDOI
TL;DR: The relationship between DNA sequence recognition and catalytic specificity in a DNA-modifying enzyme was explored using paramagnetic Cu2+ ions as probes for ESR spectroscopic and biochemical studies, shedding light on the structural and electrostatic factors that affect site-specific catalysis by this class of endonucleases.
Abstract: The relationship between DNA sequence recognition and catalytic specificity in a DNA-modifying enzyme was explored using paramagnetic Cu2+ ions as probes for ESR spectroscopic and biochemical studies. Electron spin echo envelope modulation spectroscopy establishes that Cu2+ coordinates to histidine residues in the EcoRI endonuclease homodimer bound to its specific DNA recognition site. The coordinated His residues were identified by a unique use of Cu2+-ion based long-range distance constraints. Double electron-electron resonance data yield Cu2+-Cu2+ and Cu2+-nitroxide distances that are uniquely consistent with one Cu2+ bound to His114 in each subunit. Isothermal titration calorimetry confirms that two Cu2+ ions bind per complex. Unexpectedly, Mg2+-catalyzed DNA cleavage by EcoRI is profoundly inhibited by Cu2+ binding at these hitherto unknown sites, 13 A away from the Mg2+ positions in the catalytic centers. Molecular dynamics simulations suggest a model for inhibition of catalysis, whereby the Cu2+ ions alter critical protein-DNA interactions and water molecule positions in the catalytic sites. In the absence of Cu2+, the Mg2+-dependence of EcoRI catalysis shows positive cooperativity, which would enhance EcoRI inactivation of foreign DNA by irreparable double-strand cuts, in preference to readily repaired single-strand nicks. Nonlinear Poisson-Boltzmann calculations suggest that this cooperativity arises because the binding of Mg2+ in one catalytic site makes the surface electrostatic potential in the distal catalytic site more negative, thus enhancing binding of the second Mg2+. Taken together, our results shed light on the structural and electrostatic factors that affect site-specific catalysis by this class of endonucleases.