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.

Conformational modulation of the farnesoid X receptor by prenylflavonoids: Insights from hydrogen deuterium exchange mass spectrometry (HDX-MS), fluorescence titration and molecular docking studies

“…Whereas, the additions of IX and 8-PN caused red shifting, suggesting that one or more tryptophan residues in FXR-LBD were exposed to solvent to a higher degree. In addition, the isosbestic point in the spectra from the IX titration at 405 nm suggests that the presence of both bound and free IX in equilibrium [51, 52]. …”

Ligand binding studies by high speed centrifugal precipitation and linear spectral summation using ultraviolet–visible absorption spectroscopy

“…The fitting algorithm uses iterative reconvolution to minimize error. The goodness of the fit is judged by the normalized root mean square deviation [ 1 ]. The appropriate scaled factors for the bound and free spectra, respectively are calculated that result in the best fit.…”

“…The appropriate scaled factors for the bound and free spectra, respectively are calculated that result in the best fit. The bound and unbound lipid concentrations can then be determined from the product of the respective scaling factors and the known concentration of the pure bound and free input spectra [ 1 ]. The experiment was repeated 3 times and the means were calculated.…”

“…Ligands often show shifts in peaks in UV–Vis absorbance or fluorescence spectra with binding. Linear spectral summation is suitable when the shift of the spectral peak is greater than about 8 nm between bound and free ligand [ 1 ]. In our case, C6 ceramide complexed to tear lipocalin showed such a shift and was amenable to the technique but not C12 ceramide ( Fig.…”

“…The concentration of the each fraction can be precisely measured by absorbance spectrophotometry. A second technique, linear spectral summation has been published for protein-ligand associations using fluorescence of labeled ligands [ 1 ]. Here, the method is altered for use with ultraviolet-visible (UV–Vis) absorption spectroscopy.…”