Binding proteins kinetic approach

The isolation of both specific and nonspecific binding proteins on affinity matrices bearing bioactive compounds hinders the identification of drug cellular targets. While solid-phase elution or the competition methods are conventionally used to distinguish between specific and nonspecific receptor-ligand interactions, these approaches are often severely restricted by low ligand solubility and/or slow kinetic dissociation (8). This low solubility of these compounds are not uncommon, since the hydrophobic properties of these compounds are often vital for their bioactivity and/or membrane permeability. 

The experiments described above indicate that technology is available to couple SPR with mass spectrometry. These methods should be useful for protein-protein interaction mapping. For example, immobilized proteins can be used as hooks for fishing binding partners from complex protein mixtures under native conditions. The coupling of techniques can lead not only to the rapid identification of interacting proteins but will also provide information on the kinetic parameters of the interaction. This approach should serve as an excellent complement to the use of in vivo techniques such as the yeast two-hybrid system. 

The original proposal of the approach, supported by a Monte Carlo simulation study [36], has been further validated with both pre-clinical [38, 39] and clinical studies [40]. It has been shown to be robust and accurate, and is not highly dependent on the models used to fit the data. The method can give poor estimates of absorption or bioavailability in two sets of circumstances (i) when the compound shows nonlinear pharmacokinetics, which may happen when the plasma protein binding is nonlinear, or when the compound has cardiovascular activity that changes blood flow in a concentration-dependent manner or (ii) when the rate of absorption is slow, and hence flip-flop kinetics are observed, i.e., when the apparent terminal half-life is governed by the rate of drug input. 

Aleshin and coworkers (49) have reported the X-ray crystal structure at 2.2-A resolution of a G2-type variant produced by Aspergillus awamori. Meanwhile, an attempt was made to determine the amino acid residues that participate in the substrate binding and catalysis provided by G2 of A. niger (52). The results of the chemical approach indicated that the Asp-176, Glu-179, and Glu-180 form an acidic cluster crucial to the functioning of the enzyme. This conclusion was then tested by site-specific mutagenesis of these amino acid residues, which were replaced, one at a time, with Asn, Gin, and Gin, respectively (53). The substitution at Glu-179 provided an inactive protein. The other two substitutions affected the kinetic parameters but were not of crucial importance to the maintenance of activity. The crystal structure (49) supports the conclusion that Glu-179 functions as the catalytic acid but Asp-17 6 does not appear to be a good candidate for provision of catalytic base. Thus, there still exists considerable uncertainty as to how the disaccharide is accepted into the combining site for hydrolysis. Nevertheless, the kind of scheme presented by Svensson and coworkers (52) almost surely prevails. 

Selected entries from Methods in Enzymology [vol, page(s)] Analysis of GTP-binding/GTPase cycle of G protein, 237, 411-412 applications, 240, 216-217, 247 246, 301-302 [diffusion rates, 246, 303 distance of closest approach, 246, 303 DNA (Holliday junctions, 246, 325-326 hybridization, 246, 324 structure, 246, 322-324) dye development, 246, 303, 328 reaction kinetics, 246, 18, 302-303, 322] computer programs for testing, 240, 243-247 conformational distribution determination, 240, 247-253 decay evaluation [donor fluorescence decay, 240, 230-234, 249-250, 252 exponential approximation of exact theoretical decay, 240, 222-229 linked systems, 240, 234-237, 249-253 randomly distributed fluorophores, 240, 237-243] diffusion coefficient determination, 240, 248, 250-251 diffusion-enhanced FRET, 246, 326-328 distance measurement [accuracy, 246, 330 effect of dye orientation, 246, 305, 312-313 limitations, 246,... 

The apparent permeability of 11 test compounds was measured in the presence and absence of human serum albumin in the donor compartment, and by solving the differential equations describing the kinetics of membrane permeability, membrane retention and protein binding, the authors were able to obtain the Kd. With the protein in solution rather than immobilized and without the need for mass balance or equilibrium conditions, this approach provides an attractive alternative to existing methods with the potential to be applied to an array of other soluble proteins. 

Many methods of investigation of protein-ligand binding kinetics that are based on linear processes are of a pump-probe type. In this approach an optical pulse, called a pump, starts a photoreaction (such as dissociation of MbCO into Mb and CO), and its progress is probed a time At later. The probe could be, for example, a weak laser pulse, which detects the spectral changes in the heme during the protein-ligand recombination, or an x-ray pulse, which allows determination of the protein structure at a particular instant in time. 

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