Search results for "Cooperative binding"
showing 10 items of 16 documents
Analysis of substrate binding in individual active sites of bifunctional human ATIC
2018
Aminoimidazolecarboxamide ribonucleotide formyl transferase (AICARFT): Inosine monophosphate cyclohydrolase (IMPCH, collectively called ATIC) is a bifunctional enzyme that catalyses the penultimate and final steps in the purine de novo biosynthesis pathway. The bifunctional protein is dimeric and each monomer contains two different active sites both of which are capable of binding nucleotide substrates, this means to a potential total of four distinct binding events might be observed. Within this work we used a combination of site-directed and truncation mutants of ATIC to independently investigate the binding at these two sites using calorimetry. A single S10W mutation is sufficient to blo…
Nested MWC model describes hydrolysis of GroEL without assuming negative cooperativity in binding
2002
Folding assistance and ATPase activity of GroEL are based on the existence of different conformations. In order to characterise these conformations, published data on steady state ATPase activity in the absence of GroES were reanalysed simultaneously in terms of the Nested MWC model. This model is a hierarchical extension of the symmetry-model of Monod et al. [J. Mol. Biol. 12 (1965) 88]. An unique set of GroEL specific parameters was obtained. This set was supported by comparison of predictions arising from this set of values with experimental data for hydrolysis of ATP in the presence of ADP and ATPgammaS, binding of ATPgammaS and ADP to GroEL in the absence of ATP, and binding of ATP as …
Convection, diffusion and reaction in a surface-based biosensor: Modeling of cooperativity and binding site competition on the surface and in the hyd…
2005
We study theoretically the transport and kinetic processes underlying the operation of a biosensor (particularly the surface plasmon sensor "Biacore") used to study the surface binding kinetics of biomolecules in solution to immobilized receptors. Unlike previous studies, we concentrate mainly on the modeling of system-specific phenomena rather than on the influence of mass transport limitations on the intrinsic kinetic rate constants determined from binding data. In the first problem, the case of two-site binding where each receptor unit on the surface can accommodate two analyte molecules on two different sites is considered. One analyte molecule always binds first to a specific site. Sub…
In vivo detection, RNA-binding properties and characterization of the RNA-binding domain of the p7 putative movement protein from carnation mottle ca…
1999
Biochemical and structural characterization studies on the p7 putative movement protein from a Spanish isolate of carnation mottle carmovirus (CarMV) have been conducted. The CarMV p7 gene was fused to a sequence coding for a six-histidine tag and expressed in bacteria, allowing the purification of CarMV p7 and the production of a specific antiserum. This antiserum led to the immunological identification of CarMV p7 in infected leaf tissue from the experimental host Chenopodium quinoa. Putative nucleic acid-binding properties of the CarMV p7 have been explored and demonstrated with both electrophoretic mobility shift and RNA-protein blot in vitro assays using digoxigenin-labeled riboprobes.…
Cooperatively Enhanced Ion Pair Binding with a Hybrid Receptor
2015
A simple 18-crown-6-based bis-urea receptor R(1) was synthesized in three steps from a commercial starting material. The receptor's behavior toward anions, cations, and ion pairs was studied in solution with (1)H NMR, in solid state with single-crystal X-ray diffraction, and in gas phase with mass spectrometry. In 4:1 CDCl3/dimethyl sulfoxide solution the receptor's binding preference of halide anions is I(-) < Br(-) < Cl(-) following the trend of the hydrogen-bonding acceptor ability of the anions. The receptor shows a remarkable positive cooperativity toward halide anions Cl(-), Br(-), and I(-) when complexed with Na(+), K(+), or Rb(+). The solid-state binding modes of R(1) with alkali an…
Control of kinetics by cooperative interactions.
2011
Cooperative effects in ligand binding and dissociation kinetics are much less investigated than steady state kinetics or equilibrium binding. Nevertheless, cooperativity in ligand binding leads necessarily to characteristic properties with respect to kinetic properties of the system. In case of positive cooperativity as found in oxygen binding proteins, a typical property is an autocatalytic ligand dissociation behavior leading to a time dependent, apparent ligand dissociation rate. To follow systematically the influence of the various potentially involved parameters on this characteristic property, simulations based on the simple MWC model were performed which should be relevant for all ty…
Role of two operators in regulating the plasmid-borne raf operon of Escherichia coli
1994
The plasmid-borne raf operon encodes functions required for the inducible uptake and utilization of raffinose in Escherichia coli K12. The expression of three structural genes for alpha-galactosidase (rafA), Raf permease (rafB) and sucrose hydrolase (rafD) is negatively controlled by the binding of RafR repressor (rafR) to two operator sites, O1 and O2, that flank the -35 sequence of the raf promoter, PA. In vitro, O1 and O2 are occupied on increasing the concentration of RafR, without detectable preference for one site or the other or any indication of cooperative binding. Nucleotide substitutions at positions 3, 4 or 5 in an operator half-site prevented repressor binding, supporting a mod…
Involvement of carboxyl groups in chloride transport and reversible DIDS binding to band 3 protein in human erythrocytes
2011
AbstractNoncovalent DIDS binding to Band 3 (AE1) protein in human erythrocyte membranes, modified by non-penetrating, water soluble 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide (EAC), was studied at 0°C in the presence of 165 mM KCl. Under experimental conditions applied up to (48 ± 5) % of irreversible chloride self-exchange inhibition was observed. The apparent dissociation constant, KD, for “DIDS-Band 3” complex, determined from the chloride transport experiments, was (34 ± 3) nM and (80 ± 12) nM for control and EAC-treated resealed ghosts, respectively. The inhibition constant, Ki, for DIDS was (35 ± 6) nM and (60 ± 8) nM in control and EAC-treated ghosts, respectively. T…
Negative cooperativity in Root-effect hemoglobins: role of heterogeneity.
2011
In some animals, the oxygen transport capacity of blood decreases when pH is lowered, yielding oxygen binding curves with Hill-coefficients smaller than unity. This so-called Root effect is observed in several fishes and is important for creating large oxygen partial pressures locally, for example in the swim bladder. While there is general agreement on the physiological advantages of this effect, its molecular basis remains ambiguous. Various studies show that isoforms of hemoglobins usually are present in the hemolymph, when the Root effect is observed. Here, we show that in such a case the mixture of these isoforms can exhibit apparent negative cooperativity, although each component take…
Chelate Cooperativity and Spacer Length Effects on the Assembly Thermodynamics and Kinetics of Divalent Pseudorotaxanes
2011
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…