Kinetics of binding

Ecyt Mtl followed by dissociation of mannitol at the cytoplasmic side (Ecyt Mtl Ecyt). The reverse pathway would be measured with the binding to the ISO membranes. The small rate constants for the latter two processes indicate that turnover through the cycle as depicted in Fig. 5 is very slow and has to be accelerated considerably when transport is coupled to phosphorylation. 

The interpretation of much of the binding data given so far is based upon the assumption that the high affinity binding sites represent a population of independent sites. In the unphosphorylated II these sites would open up either to the periplas-mic or cytoplasmic side of the membrane independently of each other. The assumption ignores the evidence that the enzyme is, in fact, multimeric and that the data 

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A more sensitive and rigorous method of detecting and quantifying allosteric effects is through observation of the kinetics of binding. 

In general, the kinetics of most allosteric modulators have been shown to be faster than the kinetics of binding of the tracer ligand. This is an initial assumption for this experimental approach. Under these circumstances, the rate of dissociation of the tracer ligand (pA t) n the presence of the allosteric ligand is given by [11, 12]... 

Allosteric antagonism is characterized by the fact that it attains a maximal value. A sensitive method for the detection of allosteric effects is through studying the kinetics of binding. 

Fig. 7. Two possible interpretations of the transitions of the translocator domain detected by the kinetics of binding to ISO membranes at 4°C. (A) Translocation. The states not in contact with the external medium are the states in contact with the internal medium. (B) Occlusion. The binding sites can be in a state where they are not accessible from either side of the membrane. The spots represent the substrate molecule, cyt and per represent the cytoplasmic and periplasmic side of the enzyme, respectively.

Brading With actin in isolation, can you find out anything about the kinetics of binding of kinase onto actin molecules ... 

Using a concentration jump as the perturbation, Sutherland et a/.(113) measured the kinetics of binding of fluorescein-labeled human IgG (present as an antigen in solution) to surface-immobilized sheep anti-human IgG. Two TIRF surfaces were used a planar slide and a fiber-optic cylinder. Also using a TIRF recovery after a concentration jump, Kalb et a/,(114) measured the slow ( 10 4 s 1) unbinding kinetics of anti-trinitrophenol (TNP) antibodies in solution and a TNP-derivatized lipid in a planar bilayer. 

Second, these proteins are particularly stable and because of the particularly rich spectroscopy of their heme porphyrin active sites, a wide variety of physical methods are available to monitor the thermodynamics and kinetics of binding, and subsequent electron transfer reactions, ranging from 2D NMR to laser flash techniques. 

Perhaps more important is the observation that the kinetics of binding can contribute significantly to band spreading. If the results presented in that work are general, the kinetic plate height contribution sets a lower limit on the size of particles used in RPC at about 5 m and no practical gain in column efficiency would be made by using smaller particles. 

The kinetics of binding of cyanide to the heme in micelles has been studied by stopped flow and optical spectroscopy [15, 16]. Two kinetically indistinguishable mechanisms, consistent with the above equilibrium expression, have been proposed for the binding ... 

All the direct metal ligands in Con A are retained in the lectin from favin,360 which also binds stoichiometric amounts of Mn2+ and Ca2+. In total, about 40% of the residues are identical, including the site of the proposed cis-trans isomerization underlying the locked to unlocked conformational change. It will be of interest to compare the kinetics of binding of metals to favin with the results from Con A. 

The first aptasensor reported was particularly based on optical detection [66]. The 58-mer RNA aptamer selective to L-adenosine was immobilized onto the core of multimode fiber using avidin-biotin method. The detection was based on competitive binding of FITC-labeled L-adenosine with unlabeled analyte. This sensor also allowed to study the kinetics of binding and determine equilibrium constants. 

The kinetics of binding of N02 to the four subunit methemoprotein, methemo-globin (metHb) were investigated. The rate constants for fast and slow reactions were comparable with literature values.325 The thermally derived activation parameters indicated that the reactions all proceed by a dissociative mechanism. It was predicted that hydrostatic pressure could affect the compressibility of the four subunits and quaternary structure of metHb, and therefore volume parameters of reliable value for the reactions of metMb would not be obtained. 

The kinetics of binding of ligands to a binding site provides information about the way the ligands reach the site. They may bind fast, indicating an easily accessible site, or they may bind slowly indicating an occluded site. They may bind in a single... 

Fig. 8 Kinetics of binding of Flutaxl to microtubules at 35 °C. In the stopped-flow device a 1 pM solution of Flutaxl was mixed with 25 pM pure tubulin assembled into microtubules (concentration of sites 20 pM) (final concentrations of 500 nM Flutax and 10 pM sites) in the absence (a) and presence (b) of 50 pM docetaxel. Curve a is fitted to an exponential decay. Inset residues between the experimental and theoretical curves. Taken from [10]...

Ligand- macromolecule complex Stoichiometry of complex Kinetics of binding Location of interacting sites Orientation of bound ligand Structure of complex Dynamics of complex Chemical shift titration Line width, titration analysis HSQC, isotope editing NOE docking 3D/4D NMR Relaxation time measurements... 

Palleros, D. R., Welch, W. J., and Fink, A. L. (1991). Interaction of hsp70 with unfolded proteins Effects of temperature and nucleotides on the kinetics of binding. Proc. Natl. Acad. Sci. U.S.A. 88, 5719-5723. 

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