Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Analytical expressions for the homotropic binding of ligand to protein dimers and trimers.

ABSTRACT: Cooperative binding of a ligand to multiple subsites on a protein is a common theme among enzymes and receptors. The analysis of cooperative binding data (either positive or negative) often relies on the assumption that free ligand concentration, L, can be approximated by the total ligand concentration, L(T). When this approximation does not hold, such analyses result in inaccurate estimates of dissociation constants. Presented here are exact analytical expressions for equilibrium concentrations of all enzyme and ligand species (in terms of K(d) values and total concentrations of protein and ligand) for homotropic dimeric and trimeric protein-ligand systems. These equations circumvent the need to approximate L and are provided in Excel worksheets suitable for simulation and least-squares fitting. The equations and worksheets are expanded to treat cases where binding signals vary with distinct site occupancy.

SUBMITTER: Lefurgy ST

PROVIDER: S-EPMC3417323 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Analytical expressions for the homotropic binding of ligand to protein dimers and trimers.

Lefurgy Scott T ST Leyh Thomas S TS

Analytical biochemistry 20111216 2

Cooperative binding of a ligand to multiple subsites on a protein is a common theme among enzymes and receptors. The analysis of cooperative binding data (either positive or negative) often relies on the assumption that free ligand concentration, L, can be approximated by the total ligand concentration, L(T). When this approximation does not hold, such analyses result in inaccurate estimates of dissociation constants. Presented here are exact analytical expressions for equilibrium concentrations ...[more]

PMID: 22230282

Similar Datasets

Metalloporphines: Dimers and Trimers.

Project description:Procedures for the purification and subsequent crystallization of the slightly soluble four-coordinate metallporphines, the simplest possible porphyrin derivatives, are described. Crystals of the porphine derivatives of cobalt(II), copper(II), platinum(II), and two polymorphs of zinc(II) were obtained. Analysis of the crystal and molecular structures shows that all except the platinum(II) derivative form an unusual trimeric species in the solid state. The isomorphous cobalt(II), copper(II), and one zinc(II) polymorph pack in the unit cell to form dimers as well as the trimers. Interplanar spacings between porphine rings are similar in both the dimers and trimers and range between 3.24 and 3.37 Å. Porphine rings are strongly overlapped with lateral shifts between ring centers in both the dimers and trimers with values between 1.52 and 1.70 Å or in Category S as originally defined by Scheidt and Lee. Periodic trends in the M-Np bond distances parallel those observed previously for tetraphenyl- and octaethylporphyrin derivatives.

| S-EPMC4988798 | biostudies-other

Homotropic cooperativity from the activation pathway of the allosteric ligand-responsive regulatory trp RNA-binding attenuation protein.

Project description:The trp RNA-binding attenuation protein (TRAP) assembles into an 11-fold symmetric ring that regulates transcription and translation of trp-mRNA in bacilli via heterotropic allosteric activation by the amino acid tryptophan (Trp). Whereas nuclear magnetic resonance studies have revealed that Trp-induced activation coincides with both microsecond to millisecond rigidification and local structural changes in TRAP, the pathway of binding of the 11 Trp ligands to the TRAP ring remains unclear. Moreover, because each of 11 bound Trp molecules is completely surrounded by protein, its release requires flexibility of Trp-bound (holo) TRAP. Here, we used stopped-flow fluorescence to study the kinetics of Trp binding by Bacillus stearothermophilus TRAP over a range of temperatures and observed well-separated kinetic steps. These data were analyzed using nonlinear least-squares fitting of several two- and three-step models. We found that a model with two binding steps best describes the data, although the structural equivalence of the binding sites in TRAP implies a fundamental change in the time-dependent structure of the TRAP rings upon Trp binding. Application of the two-binding step model reveals that Trp binding is much slower than the diffusion limit, suggesting a gating mechanism that depends on the dynamics of apo TRAP. These data also reveal that dissociation of Trp from the second binding mode is much slower than after the first Trp binding mode, revealing insight into the mechanism for positive homotropic allostery, or cooperativity. Temperature-dependent analyses reveal that both binding modes imbue increases in bondedness and order toward a more compressed active state. These results provide insight into mechanisms of cooperative TRAP activation and underscore the importance of protein dynamics for ligand binding, ligand release, protein activation, and allostery.

| S-EPMC3946311 | biostudies-literature

Crystal structures of ligand complexes of P450eryF exhibiting homotropic cooperativity.

Project description:Several mammalian cytochrome P450 (P450) isoforms demonstrate homotropic cooperativity with a number of substrates, including steroids and polycyclic aromatic hydrocarbons. To identify structural factors contributing to steroid and polycyclic aromatic hydrocarbon binding to P450 enzymes and to determine the location of the allosteric site, we investigated interactions of the macrolide hydroxylase P450eryF from Saccharopolyspora erythraea with androstenedione and 9-aminophenanthrene. Spectroscopic binding assays indicate that P450eryF binds androstenedione with an affinity of 365 microM and a Hill coefficient of 1.31 +/- 0.6 and coordinates with 9-aminophenanthrene with an affinity of 91 microM and a Hill coefficient of 1.38 +/- 0.2. Crystals of complexes of androstenedione and 9-aminophenanthrene with P450eryF were grown and diffracted to 2.1 A and 2.35 A, respectively. Electron density maps indicate that for both complexes two ligand molecules are simultaneously present in the active site. The P450eryF/androstenedione model was refined to an r = 18.9%, and the two androstenedione molecules have similar conformations. The proximal androstenedione is positioned such that the alpha-face of carbon-6 is closest to the heme iron, and the second steroid molecule is positioned 5.5 A distal in the active site. The P450eryF/9-aminophenanthrene model was refined to an r = 19.7% with the proximal 9-aminophenanthrene coordinated with the heme iron through the 9-amino group and the second ligand positioned approximately 6 A distal in the active site. These results establish that homotropic cooperativity in ligand binding can result from binding of two substrate molecules within the active site pocket without major conformational changes in the protein.

| S-EPMC16190 | biostudies-literature

Configurational Preference of the Glutamate Receptor Ligand Binding Domain Dimers.

Project description:Ionotropic glutamate receptors are a family of tetrameric ion channels with functional states consisting of nonconducting, conducting, and desensitized states that are starting to become well characterized by electrophysiological and biophysical studies. However, the structure and relative energetics of these states beyond the general structure of the receptor are still not well understood. It is known that the interface between monomeric subunits of the tetramer plays a major role in distinguishing these functional states. We have used umbrella sampling and multimicrosecond molecular dynamics simulations of the GluA2 AMPA subtype glutamate receptor ligand-binding domain (LBD) dimers to characterize a natural propensity of the LBD dimers for various configurational states. Our results show a proposed desensitized conformation of the LBD dimer is a highly preferable conformation of the LBD dimer without the influence of other receptor domains or crystallographic conditions. This has been demonstrated by both free protein simulations of 5 ?s duration, as well as by computed free energy difference between the active and desensitized states. At the same time, the simulations performed using the same protocols revealed that for the LBD mutant L483Y, known to lack desensitization, the postulated active state of the LBD dimer is indeed the preferred configurational state, which remained stable in the simulations. Our findings pave the path for developing more detailed hypotheses of the full receptor activation mechanism. Combined with the energetics of glutamate binding to the LBD and the energy required to open the transmembrane pore helices, our results strongly support a hypothesis that the low absolute free-energy state is the desensitized state of the intact AMPA receptor.

| S-EPMC5474840 | biostudies-literature

Enzyme-free ligation of dimers and trimers to RNA primers.

Project description:The template-directed formation of phosphodiester bonds between two nucleic acid components is a pivotal process in biology. To induce such a reaction in the absence of enzymes is a challenge. This challenge has been met for the extension of a primer with mononucleotides, but the ligation of short oligonucleotides (dimers or trimers) has proven difficult. Here we report a method for ligating dimers and trimers of ribonucleotides using in situ activation in aqueous buffer. All 16 different dimers and two trimers were tested. Binding studies by NMR showed low millimolar dissociation constants for complexes between representative dimers and hairpins mimicking primer-template duplexes, confirming that a weak template effect is not the cause of the poor ligating properties of these short oligomers. Rather, cyclization was found to compete with ligation, with up to 90% of dimer being converted to the cyclic form during the course of an assay. This side reaction is strongly sequence dependent and more pronounced for dimers than for trimers. Under optimized reaction conditions, high yields were observed with strongly pairing purines at the 3'-terminus. These results show that short oligomers of ribonucleotides are competent reactants in enzyme-free copying.

| S-EPMC6486630 | biostudies-literature

Endothelin receptor dimers evaluated by FRET, ligand binding, and calcium mobilization.

Project description:Endothelin-1 (ET-1) mediates physiological responses via endothelin A (ET(A)) and B (ET(B)) receptors, which may form homo- and heterodimers with unknown function. Here, we investigated ET-receptor dimerization using fluorescence resonance energy transfer (FRET) between receptors tagged with CFP (donor) and receptors tagged with tetracysteine-FlAsH (fluorescein arsenical hairpin) (acceptor) expressed in HEK293 cells. FRET efficiencies were 15%, 22%, and 27% for ET(A)/ET(A), ET(B)/ET(B), and ET(A)/ET(B), respectively, and dimerization was further supported by coimmunoprecipitation. For all dimer pairs, the natural but nonselective ligand ET-1 rapidly (<or=30 s) reduced FRET by >50%, but did not detectably reduce coimmunoprecipitation. ET-1 stimulated a transient increase in intracellular Ca(2+) ([Ca(2+)](i)) lasting 1-2 min for both homodimer pairs, and these ET-1 actions on FRET and [Ca(2+)](i) elevation were blocked by the appropriate subtype-selective antagonist. In contrast, ET(A)/ET(B) heterodimers mediated a sustained [Ca(2+)](i) increase lasting >10 min, and required a combination of ET(A) and ET(B) antagonists to block the observed FRET and [Ca(2+)](i) responses. The sensitive CFP/FlAsH FRET assay used here provides new insights into endothelin-receptor dimer function, and represents a unique approach to characterize G-protein-coupled receptor oligomers, including their pharmacology.

| S-EPMC2426627 | biostudies-literature

Combinatorial Synthesis of Structurally Diverse Triazole-Bridged Flavonoid Dimers and Trimers.

Project description:Flavonoids are a large family of compounds associated with a broad range of biologically useful properties. In recent years, synthetic compounds that contain two flavonoid units linked together have attracted attention in drug discovery and development projects. Numerous flavonoid dimer systems, incorporating a range of monomers attached via different linkers, have been reported to exhibit interesting bioactivities. From a medicinal chemistry perspective, the 1,2,3-triazole ring system has been identified as a particularly attractive linker moiety in dimeric derivatives (owing to several favourable attributes including proven biological relevance and metabolic stability) and triazole-bridged flavonoid dimers possessing anticancer and antimalarial activities have recently been reported. However, there are relatively few examples of libraries of triazole-bridged flavonoid dimers and the diversity of flavonoid subunits present within these is typically limited. Thus, this compound type arguably remains underexplored within drug discovery. Herein, we report a modular strategy for the synthesis of novel and biologically interesting triazole-bridged flavonoid heterodimers and also very rare heterotrimers from readily available starting materials. Application of this strategy has enabled step-efficient and systematic access to a library of structurally diverse compounds of this sort, with a variety of monomer units belonging to six different structural subclasses of flavonoid successfully incorporated.

| S-EPMC6273872 | biostudies-other

Refolding of Escherichia coli outer membrane protein F in detergent creates LPS-free trimers and asymmetric dimers.

Project description:The Escherichia coli OmpF (outer-membrane protein F; matrix porin) is a homotrimeric beta-barrel and a member of the bacterial porin superfamily. It is the best characterized porin protein, but has resisted attempts to refold it efficiently in vitro. In the present paper, we report the discovery of detergent-based folding conditions, including dodecylglucoside, which can create pure samples of trimeric OmpF. Whereas outer membrane LPS (lipopolysaccharide) is clearly required for in vivo folding, the artificially refolded and LPS-free trimer has properties identical with those of the outer-membrane-derived form. Thus LPS is not required either for in vitro folding or for structural integrity. Dimeric forms of OmpF have been observed in vivo and are proposed to be folding intermediates. In vitro, dimers occur transiently in refolding of trimeric OmpF and, in the presence of dodecylmaltoside, pure dimer can be prepared. This form has less beta-structure by CD and shows lower thermal stability than the trimer. Study of these proteins at the single-molecule level is possible because each OmpF subunit forms a distinct ion channel. Whereas each trimer contains three channels of equal conductance, each dimer always contains two distinct channel sizes. This provides clear evidence that the two otherwise identical monomers adopt different structures in the dimer and indicates that the asymmetric interaction, characteristic of C3 symmetry, is formed at the dimer stage. This asymmetric dimer may be generally relevant to the folding of oligomeric proteins with odd numbers of subunits such as aspartate transcarbamoylase.

| S-EPMC1316273 | biostudies-literature

Allosteric activation of preformed EGF receptor dimers by a single ligand binding event.

Project description:Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is essential to understand the molecular mechanisms underlying its activation. Unlike previously anticipated, here, we report that purified full-length EGFR adopts a homodimeric form in vitro before and after ligand binding. Cryo-electron tomography analysis of the purified receptor also showed that the extracellular domains of the receptor dimer, which are conformationally flexible before activation, are stabilized by ligand binding. This conformational flexibility stabilization most likely accompanies rotation of the entire extracellular domain and the transmembrane domain, resulting in dissociation of the intracellular kinase dimer and, thus, rearranging it into an active form. Consistently, mutations of amino acid residues at the interface of the symmetric inactive kinase dimer spontaneously activate the receptor in vivo. Optical observation also indicated that binding of only one ligand activates the receptor dimer on the cell surface. Our results suggest how oncogenic mutations spontaneously activate the receptor and shed light on the development of novel cancer therapies.

| S-EPMC9748436 | biostudies-literature

Mechanism of bacterial signal transduction revealed by molecular dynamics of Tsr dimers and trimers of dimers in lipid vesicles.

Project description:Bacterial chemoreceptors provide an important model for understanding signalling processes. In the serine receptor Tsr from E. coli, a binding event in the periplasmic domain of the receptor dimer causes a shift in a single transmembrane helix of roughly 0.15 nm towards the cytoplasm. This small change is propagated through the ≈ 22 nm length of the receptor, causing downstream inhibition of the kinase CheA. This requires interactions within a trimer of receptor dimers. Additionally, the signal is amplified across a 53,000 nm(2) array of chemoreceptor proteins, including ≈ 5,200 receptor trimers-of-dimers, at the cell pole. Despite a wealth of experimental data on the system, including high resolution structures of individual domains and extensive mutagenesis data, it remains uncertain how information is communicated across the receptor from the binding event to the downstream effectors. We present a molecular model of the entire Tsr dimer, and examine its behaviour using coarse-grained molecular dynamics and elastic network modelling. We observe a large bending in dimer models between the linker domain HAMP and coiled-coil domains, which is supported by experimental data. Models of the trimer of dimers, built from the dimer models, are more constrained and likely represent the signalling state. Simulations of the models in a 70 nm diameter vesicle with a biologically realistic lipid mixture reveal specific lipid interactions and oligomerisation of the trimer of dimers. The results indicate a mechanism whereby small motions of a single helix can be amplified through HAMP domain packing, to initiate large changes in the whole receptor structure.

| S-EPMC3447960 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data