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.

Fig. 1

Decompositions of ANS fluorescence spectra of ANS-TL complexes into bound and free components in the ligand titration experiment. Concentration of the protein was constant, 2.8 μM. Total ANS concentrations: (a) 0.59 μM; (b) 11.64 μM. Black, red, olive and blue lines represent experimental, best fit to two component spectra, fraction of bound and free ANS spectra, respectively. Decompositions of the ANS fluorescence spectra for all ligand concentrations are provided in Fig. S2 in the ESM

Fig. 2

Error analysis for the best fit performed for the ANS (11.64 μM)-W130 (2.8 μM) complex. The bound and free concentrations of ANS are estimated to be 1.72 μM and 9.92 μM, respectively

Fig. 3

Area normalized fluorescence spectra of various concentrations of ANS with W130 (constant, 2.8 μM). An isosbestic point is apparent at about 495 nm. Noise reduction of the spectra was performed for clarity. The procedure did not induce any changes in the shapes of the spectra

Fig. 4

NS binding to TL (W130) derived from linear spectral summation method (Fig. S2). Solid lines are the best fits for a rectangular hyperbola (formula 1) in (a) and linear regression for Scatchard plot in (b)

Fig. 5

Fluorescence intensity (λ= 460 nm) as a function of varying concentrations of ANS with the protein, W130 (2.8 μM). Solid circles- experimental data corrected for the contribution of free ANS; open triangles- experimental data, open circles- free ANS intensity, solid line– best fit to the one binding site (formula 2)

Fig. 6

Computer simulation of ANS binding to TL with various spectral shifts. (a)–(c) linear regressions for Scatchard plots for spectral shifts of 39 nm, 44 nm, 49 nm, respectively. Data points were recovered from linear spectral summation method shown in Fig. S4–S6, respectively; Insets: black, red, olive and blue lines represent simulated (total ANS= 9.9 μM), best fit to two component spectra, fraction of bound and free ANS spectra, respectively. The positions of the fluorescence λ_max of simulated free ANS spectra are shown.

Fig. 7

The dependence of the R² values on the spectral shift of free ANS spectrum. Dashed line indicates the R² value of 0.68.