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A simple model-free method for direct assessment of fluorescent ligand binding by linear spectral summation.

ABSTRACT: Fluorescent tagged ligands are commonly used to determine binding to proteins. However, bound and free ligand concentrations are not directly determined. Instead the response in a fluorescent ligand titration experiment is considered to be proportional to the extent of binding and, therefore, the maximum value of binding is scaled to the total protein concentration. Here, a simple model-free method is presented to be performed in two steps. In the first step, normalized bound and free spectra of the ligand are determined. In the second step, these spectra are used to fit composite spectra as the sum of individual components or linear spectral summation. Using linear spectral summation, free and bound 1-Anilinonaphthalene-8-Sulfonic Acid (ANS) fluorescent ligand concentrations are directly calculated to determine ANS binding to tear lipocalin (TL), an archetypical ligand binding protein. Error analysis shows that the parameters that determine bound and free ligand concentrations were recovered with high certainty. The linear spectral summation method is feasible when fluorescence intensity is accompanied by a spectral shift upon protein binding. Computer simulations of the experiments of ANS binding to TL indicate that the method is feasible when the fluorescence spectral shift between bound and free forms of the ligand is just 8 nm. Ligands tagged with environmentally sensitive fluorescent dyes, e.g., dansyl chromophore, are particularly suitable for this method.

SUBMITTER: Gasymov OK

PROVIDER: S-EPMC3945049 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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A simple model-free method for direct assessment of fluorescent ligand binding by linear spectral summation.

Gasymov Oktay K OK Abduragimov Adil R AR Glasgow Ben J BJ

Journal of fluorescence 20130918 1

Fluorescent tagged ligands are commonly used to determine binding to proteins. However, bound and free ligand concentrations are not directly determined. Instead the response in a fluorescent ligand titration experiment is considered to be proportional to the extent of binding and, therefore, the maximum value of binding is scaled to the total protein concentration. Here, a simple model-free method is presented to be performed in two steps. In the first step, normalized bound and free spectra of ...[more]

PMID: 24043458

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Project description:Design of selective ligands for closely related targets is becoming one of the most important tasks in the drug development. New tools, more precise than fast scoring functions and less demanding than sophisticated Free Energy Perturbation methods, are necessary to help accomplish this goal. The methods of intermediate complexity, characterizing individual contributions to the binding energy, have been an area of intense research in the past few years. Our recently developed quantum mechanical/molecular mechanical (QM/MM) modification of the Linear Response (LR) method describes the binding free energies as the sum of empirically weighted contributions of the QM/MM interaction energies and solvent-accessible surface areas for the time-averaged structures of hydrated complexes, obtained by molecular dynamics (MD) simulations. The method was applied to published data on 27 inhibitors of matrix metalloproteinase-3 (MMP-3). The two descriptors explained 90% of variance in the inhibition constants with RMSE of 0.245 log units. The QM/MM treatment is indispensable for characterization of the systems lacking suitable force-field expressions. In this case, it provided characteristics of H-bonds of the inhibitors to Glu202, charges of binding site atoms, and accurate coordination geometries of the ligands to catalytic zinc. The geometries were constrained during the MD simulations, which characterized conformational flexibility of the complexes and helped in the elucidation of the binding differences for related compounds. A comparison of the presented QM/MM LR results with those previously published for inhibition of MMP-9 by the same set of ligands showed that the QM/MM LR approach was able to distinguish subtle differences in binding affinities for MMP-3 and MMP-9, which did not exceed one order of magnitude. This precision level makes the approach a useful tool for design of selective ligands to similar targets, because the results can be safely extrapolated to maximize selectivity.

Dataset Information

A simple model-free method for direct assessment of fluorescent ligand binding by linear spectral summation.

Publications

A simple model-free method for direct assessment of fluorescent ligand binding by linear spectral summation.

Similar Datasets

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