Showing papers on "Cooperative binding published in 2002"
TL;DR: The models for BSA and HSA with bound SDS suggest that the surfactant could be bound at the same sites as those reported in the crystal structure for the fatty acid, suggesting that the mechanism of the interaction is the same.
549 citations
TL;DR: NMR studies of the binding of the p53-derived peptides revealed global conformational changes of the overall structure ofMDM2, stretching far beyond the binding cleft, indicating significant changes in the domain dynamics of MDM2 upon ligand binding.
368 citations
TL;DR: Analysis of wild-type and mutant Zif268 (Egr1) zinc fingers bound to microarrays containing all possible central 3 bp triplet binding sites indicates that the nucleotides of transcription factor binding sites cannot be treated independently, suggesting the importance of complete reference tables of all possible binding sites for comparing protein binding preferences for various DNA sequences.
Abstract: We can determine the effects of many possible sequence variations in transcription factor binding sites using microarray binding experiments. Analysis of wild-type and mutant Zif268 (Egr1) zinc fingers bound to microarrays containing all possible central 3 bp triplet binding sites indicates that the nucleotides of transcription factor binding sites cannot be treated independently. This indicates that the current practice of characterizing transcription factor binding sites by mutating individual positions of binding sites one base pair at a time does not provide a true picture of the sequence specificity. Similarly, current bioinformatic practices using either just a consensus sequence, or even mononucleotide frequency weight matrices to provide more complete descriptions of transcription factor binding sites, are not accurate in depicting the true binding site specificities, since these methods rely upon the assumption that the nucleotides of binding sites exert independent effects on binding affinity. Our results stress the importance of complete reference tables of all possible binding sites for comparing protein binding preferences for various DNA sequences. We also show results suggesting that microarray binding data using particular subsets of all possible binding sites can be used to extrapolate the relative binding affinities of all possible full-length binding sites, given a known binding site for use as a starting sequence for site preference refinement.
349 citations
TL;DR: Positive homotropic allosteric anion binding was observed and is ascribed to the structurally coupled nature of the two binding cavities present in the macrocycles, support for this latter contention came from energy minimization studies.
Abstract: Quinoxaline-bridged porphyrinoids (3), the first macrocycles containing dipyrrolylquinoxaline (DPQ, 1) subunits, were synthesized from the condensation of the diformyl-substituted DPQ derivatives (2) and 1,8-diaminoanthracene. The resulting structures were confirmed by X-ray analyses, which showed encapsulation of CHCl3 molecules within the columnar channels established by the stacked arrangement of the individual macrocycles. The solution phase interactions with fluoride and dihydrogenphosphate anions were studied in the case of the unsubstituted system 3a in CH2Cl2. The binding affinities for these anions, studied at the tetrabutylammonium salts, were found to be enhanced relative to those of the simple, unsubstituted monomeric DPQ “parent” system (1a), presumably as the result of the combined effects of preorganization and cooperative binding permitted by the pyrrole NH donor groups. Positive homotropic allosteric anion binding was observed and is ascribed to the structurally coupled nature of the two ...
189 citations
TL;DR: The GABAB receptor is further refined and the putative-binding site in the VFTM of GB2 is characterized, indicating that ligand binding in the GB2 V FTM is not required for activation and most residues of the binding pocket are conserved from Caenorhabditis elegans to human, no such conservation is observed in GB2.
Abstract: The GABAB receptor plays important roles in the tuning of many synapses. Although pharmacological differences have been observed between various GABAB-mediated effects, a single GABAB receptor composed of two subunits (GB1 and GB2) has been identified. Although GB1 binds GABA, GB2 plays a critical role in G-protein activation. Moreover, GB2 is required for the high agonist affinity of GB1. Like any other family 3 G-protein-coupled receptors, GB1 and GB2 are composed of a Venus Flytrap module (VFTM) that usually contains the agonist-binding site and a heptahelical domain. So far, there has been no direct demonstration that GB2 binds GABA or another endogenous ligand. Here, we have further refined the GABA-binding site of GB1 and characterized the putative-binding site in the VFTM of GB2. None of the residues important for GABA binding in GB1 appeared to be conserved in GB2. Moreover, mutation of 10 different residues, alone or in combination, within the possible binding pocket of GB2 affects neither GABA activation of the receptor nor the ability of GB2 to increase agonist affinity on GB1. These data indicate that ligand binding in the GB2 VFTM is not required for activation. Finally, although in either GB1 or the related metabotropic glutamate receptors most residues of the binding pocket are conserved from Caenorhabditis elegans to human, no such conservation is observed in GB2. This suggests that the GB2 VFTM does not constitute a binding site for a natural ligand.
164 citations
TL;DR: The type of cooperativity that will be the focus of this review is “allosteric cooperativity”, which has been used to describe a ligand-enzyme interaction, which results in a measurable conformational change in proximal and distal regions of that protein.
145 citations
TL;DR: The model is consistent with the results of solution, gel, and single crystal oxygen binding studies, but underestimates the population of doubly-liganded molecules determined in low-temperature electrophoresis experiments.
144 citations
TL;DR: It is shown that purified phosphatidylinositol phospholipids compete for 2,4,6,6,-trinitrophenyl (TNP)-ATP binding to the COOH termini of KATP channels with EC50 values for PIP2 between 6–8 μM, and neutral and charged lipids had weak, but significant, effects on TNP-ATPbinding.
Abstract: Inwardly rectifying, ATP-sensitive K+ channels (KATP) couple metabolism to either cell excitability (Kir6.x) or potassium secretion (Kir1.1). Phosphatidylinositol phospholipids, like PI(4,5)P2, antagonize nucleotide inhibition of KATP channels enhancing the coupling of metabolic events to cell electrical or transport activity. The mechanism by which phospholipids relieve ATP block is unclear. We have shown that maltose-binding fusion proteins (MBP) containing the COOH termini of KATP channels (Kir1.1, Kir6.1, and Kir6.2) form functional tetramers that directly bind at least two ATP molecules with negative cooperativity. Here we show that purified phosphatidylinositol phospholipids compete for 2,4,6,-trinitrophenyl (TNP)-ATP binding to the COOH termini of KATP channels with EC50 values for PIP2 between 6–8 μM. The phospholipid potency profile was PIP3 > PIP2 = PIP > PI, suggesting that net phospholipid charge was important. A role for head group charge was supported by polycations (neomycin, spermine, and polylysine) reversing the effect of PIP2 on TNP-ATP binding to the Kir1.1 channel COOH terminal fusion protein. In contrast, the water-soluble charged hydrolytic product of PIP2, inositol(1,4,5)P3 (IP3), had no effect on TNP-ATP binding, suggesting that the acyl chain of PIP2 was also necessary for its effect on TNP-ATP binding. Indeed, neutral and charged lipids had weak, but significant, effects on TNP-ATP binding. Whereas μM concentrations of PIP2 could compete with TNP-ATP, we found that mM concentrations of MgATP were required to compete with PIP2 for binding to these KATP channel COOH termini. Thus the COOH termini of KATP channels form a nucleotide- and phospholipid-modulated channel gate on which ATP and phospholipids compete for binding.
131 citations
TL;DR: Kinetic analyses of the binding properties of the heteromeric complexes suggested a sequential mechanism for the binding of IL-13 to its signaling receptor, in whichIL-13 first binds to IL- 13Rα1 and this then recruits IL-4Rα to stabilize a high affinity interaction.
116 citations
TL;DR: Results support the previous proposal that purines regulate GDH activity by altering the dynamics of the NAD binding domain, and a possible structural mechanism for negative cooperativity is presented.
114 citations
TL;DR: It is concluded that the presence of multiple binding sites that function cooperatively allow peptides such as SMAP-29 and CAP-18 to bind LPS with high affinity.
Abstract: The CD spectra of SMAP-29, an antimicrobial peptide from sheep, showed disordered structure in aqueous buffers, and significant helicity in membrane-like environments, including SDS micelles, lipopolysaccharide (LPS) dispersions, and trifluoroethanol buffer systems. A structure determined by NMR in 40% perdeuterated trifluoroethanol indicated that residues 8-17 were helical, residues 18-19 formed a hinge, and residues 20-28 formed an ordered, hydrophobic segment. SMAP-29 was flexible in 40% trifluoroethanol, forming two sets of conformers that differed in the relative orientation of the N-terminal domain. We used a chromogenic Limulus assay to determine the EC50 of the peptide (the concentration that bound 50% of the added LPS). Studies with full-length and truncated SMAP-29 molecules revealed that each end of the holopeptide contained an LPS-binding domain. The higher affinity LPS-binding domain was situated in the flexible N-terminal portion. LPS binding to full-length SMAP-29 showed positive cooperativity, so the EC50 of the peptide (2.6 microm) was considerably lower than that of the individual LPS-binding domains. LPS-binding studies with a mixture of truncated peptides revealed that this cooperativity was primarily intramolecular (i.e. involving the N- and C-terminal LPS-binding sites of the same peptide molecule). CAP-18[106 -142], an antimicrobial cathelicidin peptide of rabbits, resembled SMAP-29 in that it contained N- and C-terminal LPS-binding domains, had an EC50 of 2.5 microm, and bound LPS with positive cooperativity. We conclude that the presence of multiple binding sites that function cooperatively allow peptides such as SMAP-29 and CAP-18 to bind LPS with high affinity.
TL;DR: The interaction between PG987 and other allosteric agents on [(3)H]NMS dissociation from M(3) receptors indicate that PG987 binds reversibly to a site distinct from that to which gallamine and strychnine bind: in contrast, PG987 seems to bind to the same site on M( 3) receptors as KT5720, staurosporine, and WIN 51,708.
Abstract: WIN 51,708 (17-β-hydroxy-17-α-ethynyl-5-α-androstano[3,2- b ]pyrimido[1,2- a ]benzimidazole) and WIN 62,577 (17-β-hydroxy- 17-α-ethynyl-Δ4-androstano[3,2- b ]pyrimido[1,2- a ]benzimidazole) are potent and centrally active antagonists at rat, but not human, NK1 receptors. The interactions of these compounds and some analogs with [3H] N -methyl scopolamine ([3H]NMS) and unlabeled acetylcholine (ACh) at M1-M4 muscarinic receptors have been studied using equilibrium and nonequilibrium radioligand binding methods. The results are consistent with the predictions of the allosteric ternary complex model. The WIN compounds have log affinities for the unliganded receptor in the range 5 to 6.7, and exhibit positive, negative, or neutral cooperativity with [3H]NMS and ACh, depending on the receptor subtype and nature of the interacting ligands. WIN 62,577 is an allosteric enhancer of ACh affinity at M3 receptors. Although interacting allosterically, WIN 62,577 and WIN 51,708 do not affect [3H]NMS dissociation from M3receptors. Certain analogs have higher affinities than WIN 62,577, and truncated forms of WIN 62,577, including steroids, also act allosterically. One analog, 17-β-hydroxy-17-α-Δ4-androstano[3,2- b ]pyrido[2,3- b ]indole (PG987), has the unique effect of speeding [3H]NMS dissociation; its largest effect, 2.5-fold, is at M3receptors. The interaction between PG987 and other allosteric agents on [3H]NMS dissociation from M3 receptors indicate that PG987 binds reversibly to a site distinct from that to which gallamine and strychnine bind: in contrast, PG987 seems to bind to the same site on M3 receptors as KT5720, staurosporine, and WIN 51,708. Therefore, in addition to the allosteric site that binds strychnine (and probably chloromethyl brucine, another allosteric enhancer) there is a second, nonoverlapping, pharmacologically distinct allosteric site on M3 receptors that also supports positive cooperativity with ACh.
TL;DR: The data show that the interaction of verotoxin with the Gb3 trisaccharide is highly context dependent and that a membrane environment is required for biologically relevant studies of the interaction.
TL;DR: The curvilinear Hill plots are consistent with decreasing affinity and functional valencies of the multivalent analogues upon sequential binding of lectin molecules to the carbohydrate epitopes of the analogues.
Abstract: Our previous study demonstrated that isothermal titration microcalorimetry (ITC) could be used to determine the thermodynamics of binding of a series of synthetic multivalent carbohydrates to the Man/Glc-specific lectins concanavalin A (ConA) and Dioclea grandiflora lectin (DGL) [Dam, T. K., Roy, R., Das, S. K., Oscarson, S. and Brewer, C. F. (2000) J. Biol. Chem. 275, 14223−14230]. The higher affinities of the multivalent carbohydrates for the two lectins were shown to be due to their greater positive entropy of binding contributions relative to monovalent analogues. In the present study, ITC data from our previous report for binding of di-, tri-, and tetravalent carbohydrate analogues possessing terminal 3,6-di-O-(α-d-mannopyranosyl)-α-d-mannopyranoside residues to ConA and DGL were subjected to Hill plot analysis. Hill plots of the binding of monovalent methyl 3,6-di-O-(α-d-mannopyranosyl)-α-d-mannopyranoside to ConA and DGL are linear with slopes near 1.0, demonstrating a lack of binding cooperativity...
TL;DR: Together, the binding properties of Pot1pN suggest that the protein anchors itself at the very 3' end of a chromosome and then fills in very efficiently, coating the entire single-stranded overhang of the telomere.
Abstract: The fission yeast Pot1 (protection of telomeres) protein is a single-stranded telomeric DNA-binding protein and is required to protect the ends of chromosomes. Its N-terminal DNA-binding domain, Pot1pN, shows sequence similarity to the first OB fold of the telomere-binding protein alpha subunit of Oxytricha nova. The minimal-length telomeric ssDNA required to bind Pot1pN was determined to consist of six nucleotides, GGTTAC, by gel filtration chromatography and filter-binding assay (K(D) = 83 nM). Pot1pN is a monomer, and each monomer binds one hexanucleotide. Experiments with nucleotide substitutions demonstrated that the central four nucleotides are crucial for binding. The dependence of Pot1pN-ssDNA binding on salt concentration was consistent with a single ionic contact between the protein and the ssDNA phosphate backbone, such that at physiological salt condition 83% of the free energy of binding is nonelectrostatic. Subsequent binding experiments with longer ssDNAs indicated that Pot1pN binds to telomeric ssDNA with 3' end preference and in a highly cooperative manner that mainly results from DNA-induced protein-protein interactions. Together, the binding properties of Pot1pN suggest that the protein anchors itself at the very 3' end of a chromosome and then fills in very efficiently, coating the entire single-stranded overhang of the telomere.
TL;DR: The concerted use of NMR and isothermal titration calorimetry is reported to determine the intrinsic and cooperative binding free energies for a ligand–protein complex and observe that human ileal bile acid binding protein binds two molecules of glycocholic acid with low intrinsic affinity but an extraordinarily high degree of positive cooperativity.
Abstract: Proteins with multiple binding sites exhibit a complex behavior that depends on the intrinsic affinities for each site and the energetic communication between the sites. The contributions from intrinsic affinity and cooperativity are difficult to deconvolute using conventional binding experiments that lack information about the occupancies of individual sites. Here, we report the concerted use of NMR and isothermal titration calorimetry to determine the intrinsic and cooperative binding free energies for a ligand–protein complex. The NMR measurements provided the site-specific information necessary to resolve the binding parameters. Using this approach, we observed that human ileal bile acid binding protein binds two molecules of glycocholic acid with low intrinsic affinity but an extraordinarily high degree of positive cooperativity. The highly cooperative nature of the binding provides insights into the protein's biological mechanism. With ongoing improvements in sensitivity and resolution, NMR methods are becoming more amenable to dissecting the complex binding energetics of multisite systems.
TL;DR: An analysis by NMR of a 58 kDa complex of the core domain of the tumour suppressor p53 with DNA that complements and extends the crystal structure analysis, suggesting that the affected residues contribute to the stability of p53 core domain-DNA complex.
TL;DR: The data reported here provide a basis for understanding the mechanisms by which HS modulates RANTES functions and a complex binding model that involved dimerization of the chemokine through a mechanism of positive cooperativity is presented.
Abstract: Heparan sulfate (HS) and heparin bind to virtually all chemokines and have been shown to play critical roles in the regulation of their activities. However, both binding mechanisms and structural features involved in chemokine-HS interactions remain poorly defined. In the study presented here, we analyzed the binding of heparin to RANTES(9-68), a N-terminally truncated form of the CC-chemokine RANTES. Using biochemical and surface plasmon resonance (BIAcore system) approaches, we showed that the RANTES(9-68)-heparin interaction was characterized by a complex binding model that involved dimerization of the chemokine through a mechanism of positive cooperativity. Since RANTES(9-68) remains monomeric in solution, we concluded that heparin induced chemokine dimerization. The structure of a complex involving a RANTES dimer and a heparin heptadecasaccharide was proposed by molecular modeling. This model was used to design a dimer of "head to head" coupled octasaccharides that would fit the internal symmetry of the chemokine dimer. This engineered oligosaccharide bound RANTES(9-68) much better than a natural heparin fragment of the same length, further supporting the interaction process and the proposed structural model. Altogether, the data reported here provide a basis for understanding the mechanisms by which HS modulates RANTES functions.
TL;DR: It is the first time that ETS-1 was shown to be able to counteract its own autoinhibition, and required the 245–330-residue region of the protein that is encoded by exon VII of the gene.
TL;DR: It is proposed that selective binding of p53 to various promoters may be determined by the DNA conformation within p53 cognate sites.
TL;DR: An R826M mutation causes nearly equal decreases in affinity of NBD2 for both ATP and ADP, indicating that at this site, the sensor-2 provides binding energy, but does not act to sense the difference between these nucleotides.
Abstract: Hsp104 from Saccharomyces cerevisiae is a hexameric protein with two AAA ATPase domains (N- and C-terminal nucleotide-binding domains NBD1 and NBD2, respectively) per monomer. Our previous analysis of the Hsp104 ATP hydrolysis cycle revealed that NBD1 and NBD2 have very different catalytic properties, but each shows positive cooperativity in hydrolysis. There is also communication between the two domains, in that ATP hydrolysis at NBD1 depends on the nucleotide that is bound to NBD2. Here, we extend our understanding of the Hsp104 ATP hydrolysis cycle through mutagenesis of the AAA sensor-2 motif in NBD2. To do so, we took advantage of the lack of tryptophan residues in Hsp104 to place a single tryptophan in the C-terminal domain (Y819W). The Y819W substitution has no significant effects on folding stability of the C-terminal domain or on ATP hydrolysis by NBD1 or NBD2. The fluorescence of this tryptophan changes in response to ATP and ADP binding, allowing the Kd and Hill coefficient to be determined for each nucleotide. By using this site-specific probe of binding, we analyze the effect of mutating the conserved arginine residue in the sensor-2 motif in Hsp104 NBD2. An R826M mutation causes nearly equal decreases in affinity of NBD2 for both ATP and ADP, indicating that at this site, the sensor-2 provides binding energy, but does not act to sense the difference between these nucleotides. In addition, the rate of ATP hydrolysis at NBD1 is decreased by the R826M mutation, providing further evidence for interdomain communication in the Hsp104 ATP hydrolysis cycle.
TL;DR: The interplay between calcium ions and phosphate groups or phospholipid molecules in the C2 domain of PKCalpha is supported by the specificity and spatial organisation of the binding sites in the domain and by the variable occupancies of ligands found in the different crystal structures.
TL;DR: It is shown that GRP94 contains a peptide-binding site in its N-terminal 355 amino acids, which is distinct from the radicicol-binding pocket, because both can bind to the N-Terminal fragment simultaneously.
TL;DR: The present study demonstrates "reverse" ITC experiments in which the lectin is titrated into solutions of di- and trivalent analogues, and shows an 18-fold greater microscopic affinity constant of ConA for the first epitope of the divalent analogue versus its second epitope and a 53-foldgreater microscopic affinity constants ofconA binding to the first symbol of the trivalents.
Abstract: The preceding paper [Dam, T. K., Roy, R., Page, D., and Brewer, C. F. (2002) Biochemistry 41, 1351−1358] demonstrated that Hill plots of isothermal titration microcalorimetry (ITC) data for the binding of di-, tri-, and tetravalent carbohydrate analogues possessing terminal 3,6-di-O-(α-d-mannopyranosyl)-α-d-mannopyranoside residues to the lectin concanavalin A (ConA) show increasing negative cooperativity upon binding of the analogues to the lectin. The present study demonstrates “reverse” ITC experiments in which the lectin is titrated into solutions of di- and trivalent analogues. The results provide direct determinations of the thermodynamics of binding of ConA to the individual epitopes of the two multivalent analogues. The n values (number of binding sites per carbohydrate molecule) derived from reverse ITC demonstrate two functional binding epitopes on both the di- and trivalent analogues, confirming previous “normal” ITC results with the two carbohydrates [Dam, T. K., Roy, R., Das, S. K., Oscarson,...
TL;DR: Assays designed to characterize the interaction of DbpA with its RNA and ATP substrates reveal cooperative binding between larger RNAs and ATP with cooperative energies of approximately 1.3 kcal mol(-1).
Abstract: Unlike most DEAD/H proteins, the purified Escherichia coli protein DbpA demonstrates high specificity for its 23S rRNA substrate in vitro. Here we describe several assays designed to characterize the interaction of DbpA with its RNA and ATP substrates. Electrophoretic mobility shift assays reveal a sub-nanomolar binding affinity for a 153 nucleotide RNA substrate (R153) derived from the 23S rRNA. High affinity RNA binding requires both hairpin 92 and helix 90, as substrates lacking these structures bind DbpA with lower affinity. AMPPNP inhibition assays and ATP/ADP binding assays provide binding constants for ATP and ADP to DbpA with and without RNA substrates. These data have been used to describe a minimal thermodynamic scheme for the binding of the RNA and ATP substrates to DbpA, which reveals cooperative binding between larger RNAs and ATP with cooperative energies of ∼1.3 kcal mol-1. This cooperativity is lost upon removal of helix 89 from R153, suggesting this helix is either the preferred target fo...
TL;DR: Conformational changes within the meta two-arm host result in significantly enhanced electrochemical anion sensing compared with the more conformationally rigid three- arm host.
Abstract: A series of podands based on two or three hydrogen bonding “arms” situated in mutually ortho, meta, or para relationships about an aryl core have been prepared, and their affinities for simple inorganic anions were measured. Of the two-arm hosts the meta compound and to a lesser extent the ortho host exhibit a cooperative anion binding effect. The two arms function essentially independently in the para derivative. The mutually meta three-arm host shows dramatically enhanced cooperative binding. Conformational changes within the meta two-arm host result in significantly enhanced electrochemical anion sensing compared with the more conformationally rigid three-arm host.
TL;DR: The distinctions between the S·CEACAM interaction and other virus-receptor complexes involved in receptor-triggered entry are discussed, suggesting that S1 harbored the primary oligomerization determinants.
TL;DR: It is found that specific amino acid residues in a conserved region immediately preceding the HMG domain of Sox10 are required for cooperative binding, shedding new light on the architectural function of Sox proteins.
Abstract: The high-mobility-group (HMG) domain containing transcription factor Sox10 is an important regulator of various processes including the development of neural crest cells and glial cells. Target gene promoters contain multiple Sox10-binding sites, which either support monomeric or cooperative, dimeric binding. The latter is unusual for Sox proteins and might contribute to functional specificity of Sox10. We find that specific amino acid residues in a conserved region immediately preceding the HMG domain of Sox10 are required for cooperative binding. These residues cooperate with the HMG domain during dimeric binding in a manner dependent on specific determinants within the first two α-helices of the HMG domain. Cooperativity of DNA binding is surprisingly refractory to changes in the overall conformation of the DNA-bound dimer. Whereas maintenance of cooperativity is essential for full activation of the promoter of the myelin protein zero target gene, dimer-dependent conformational changes such as the exact bending angle introduced into the promoter appear to be less important, shedding new light on the architectural function of Sox proteins.
TL;DR: It is proposed that Ca2+-bound myr-E85Q may represent a stable intermediate state in the kinetic mechanism of the calcium-myristoyl switch, and Fluorescence and NMR spectra of the E85Q mutation do not alter the stability and structure of the Ca2-free protein.
Abstract: Recoverin, a member of the EF-hand superfamily, serves as a calcium sensor in retinal rod cells. A myristoyl or related fatty acyl group covalently attached to the N-terminus of recoverin facilitates the binding of recoverin to retinal disk membranes by a mechanism known as the Ca2+-myristoyl switch. Previous structural studies revealed that the myristoyl group of recoverin is sequestered inside the protein core in the absence of calcium. The cooperative binding of two calcium ions to the second and third EF-hands (EF-2 and EF-3) of recoverin leads to the extrusion of the fatty acid. Here we present nuclear magnetic resonance (NMR), fluorescence, and calcium-binding studies of a myristoylated recoverin mutant (myr-E85Q) designed to abolish high-affinity calcium binding to EF-2 and thereby trap the myristoylated protein with calcium bound solely to EF-3. Equilibrium calcium-binding studies confirm that only one Ca2+ binds to myr-E85Q under the conditions of this study with a dissociation constant of 100 microM. Fluorescence and NMR spectra of the Ca2+-free myr-E85Q are identical to those of Ca2+-free wild type, indicating that the E85Q mutation does not alter the stability and structure of the Ca2+-free protein. In contrast, the fluorescence and NMR spectra of half-saturated myr-E85Q (one bound Ca2+) look different from those of Ca2+-saturated wild type (two bound Ca2+), suggesting that half-saturated myr-E85Q may represent a structural intermediate. We report here the three-dimensional structure of Ca2+-bound myr-E85Q as determined by NMR spectroscopy. The N-terminal myristoyl group of Ca2+-bound myr-E85Q is sequestered within a hydrophobic cavity lined by many aromatic residues (F23, W31, Y53, F56, F83, and Y86) resembling that of Ca2+-free recoverin. The structure of Ca2+-bound myr-E85Q in the N-terminal region (residues 2-90) is similar to that of Ca2+-free recoverin, whereas the C-terminal region (residues 100-202) is more similar to that of Ca2+-bound wild type. Hence, the structure of Ca2+-bound myr-E85Q represents a hybrid between the structures of recoverin with zero and two Ca2+ bound. The binding of Ca2+ to EF-3 leads to local structural changes within the EF-hand that alter the domain interface and cause a 45 degrees swiveling of the N- and C-terminal domains, resulting in a partial unclamping of the myristoyl group. We propose that Ca2+-bound myr-E85Q may represent a stable intermediate state in the kinetic mechanism of the calcium-myristoyl switch.
TL;DR: A rapid, simple and nonhazardous assay method for endcrine disruptors was developed using an estrogen receptor (ER) and fluorescence polarization (FP) using the 17alpha-fluorescein-labeled estradiol derivative as the most suitable ligand for the ER binding assay.
Abstract: A rapid, simple and nonhazardous assay method for endcrine disruptors was developed using an estrogen receptor (ER) and fluorescence polarization (FP). Among the fluorescent compounds, the 17alpha-fluorescein-labeled estradiol derivative was selected as the most suitable ligand for the ER binding assay, since it showed the highest affinity to ER. In the Scatchard plot analysis, its convex curve exhibited a positive cooperative binding, indicating the induction of a conformational change of the ER with the binding of the ligand to form a dimer and to increase the affinity for the additional ligand. On the basis of the Hill plot analysis, its dissociation constant and Hill coefficient were 10.4 nM and 1.63, respectively. A competitive binding assay with an unlabeled 17beta-estradiol (E2) yielded an IC50 value of 2.82 nM and a Hill coefficient of 1.67, thus providing a Ki value of 0.65 nM. In the same manner, the Hill coefficients for estrone, estriol, diethylstilbestrol, and tamoxifen were determined to be 0.99, 1.17, 1.59, and 2.44, respectively.