Characterization of fluorescence of ANS-tear lipocalin complex: evidence for multiple-binding modes

Abstract

ANS is widely used as a probe for locating binding sites of proteins and studying structural changes under various external conditions. However, the nature of ANS-binding sites in proteins and the accompanying changes in fluorescence properties are controversial. We examined the steady-state and time-resolved fluorescence of the ANS-protein complexes for tear lipocalin (TL) and its mutants in order to discern the origin of lifetime components via analysis that included the multiexponential decay and the model-free maximum entropy methods. Fluorescence lifetimes of ANS-TL complexes can be grouped into two species, 14.01-17.42 ns and 2.72-4.37 ns. The log-normal analyses of fluorescence spectral shapes reveal the heterogeneous nature of both long- and short-lifetime species. The constructed time-resolved emission, amplitude (TRES) and area normalized (TRANES), and decay-associated spectra are consistent with a model that includes heterogeneous modes of ANS binding with two separate lifetime components. The two lifetime components are not derived from solvent relaxation, but rather may represent different binding modes.

Figure 1

A single log-normal component fitting of the ANS spectra in different solvents. Symbols: square, in dioxane; circle, in ethanol; triangle, in acetonitril; star, in buffer. Solid lines represent log-normal fitting.

Figure 2

The steady state fluorescence spectra and DAS for the mixture of apo-WTL (5 μm) and ANS (15 μm) constructed from global triple-decay time analysis. Symbols: square, 0.25 ns DAS (λ_max∼536 nm); circle, 4.09 ns DAS (broad composite spectrum); triangle, 17.33 ns DAS (λ_max∼464 nm). Solid line represents the steady-state fluorescence spectrum (λ_max∼463 nm). Dashed lines are log-normal fitting of DAS components.

Figure 3

The steady-state fluorescence spectra and DAS for the mixture of apo-G59W (5 μm) and ANS (15 μm) constructed from global triple-decay time analysis. Symbols: square, 0.25 ns DAS (λ_max∼523 nm); circle, 4.46 ns DAS (λ_max∼472 nm); triangle, 15.82 ns DAS (λ_max = 469 ± 0.3 nm). Solid line represents steady-state fluorescence spectrum (λ_max∼472 nm). Dashed lines are log-normal fitting of DAS components.

Figure 4

Fluorescence intensity decays of apoWTL (5 μm) ANS (15 μm) (a) and apoG59W (5 μm) ANS (15 μm) (b) complexes at various wavelengths. Symbols: (a) square, 419 nm; triangle, 464 nm; circle, 534 nm and (b) square, 416 nm; triangle, 461 nm; circle, 521 nm. Dashed lines are instrument response function. Solid curves represent the global triple-exponential decay fit.

Figure 5

The lifetime distributions for the mixture of apo-G59W (5 μm) and ANS (15 μm) at different emission wavelengths by MEM decay analysis.

Figure 6

The steady-state fluorescence spectra and DAS for the mixture of apo-G59W (5 μm) and ANS (15 μm) constructed from MEM analysis. Symbols: square, <1 ns DAS (λ_max∼535 nm); circle, 2–5 ns DAS (λ_max∼478 nm); triangle, >8 ns DAS (λ_max∼465 nm). Solid line represents steady-state fluorescence spectrum (λ_max∼472 nm). Dashed lines are log-normal fitting of DAS components.

Figure 7

The average lifetimes of the fluorescence for the mixture of apo-G59W (5 μm) and ANS (15 μm) at different wavelengths from triple exponential decay analysis. Free ANS (0.25 ns) is excluded from the calculation of τ_aver.

Figure 8

The average lifetimes (the long- and short-lifetime components) of fluorescence for the mixture of apo-G59W (5 μm) and ANS (15 μm) at different wavelengths from MEM decay analysis. Symbols: triangle, long-lifetime component; circle, short-lifetime component.

Figure 9

The constructed TRES (a) and TRANES (b) for the mixture of apoG59W (5 μm) and ANS (15 μm) at various time delays. Symbols: dash dot line and circle, 1 ns; dash line and triangle, 5 ns; solid line and square, 10 ns; dot line and star, 20 ns. Symbols are not shown in (b). TRANES show two isoemissive points, 497 nm and 438.6 nm (left and right dashed vertical lines, respectively). The spectra are shown in the wave number scale to be consistent with the literature.

Figure 10

The constructed TRES (a) and TRANES (b) for the mixture of apo-WTL (5 μm) and ANS (15 μm). Symbols: dash dot line and circle, 1 ns; dash line and triangle, 5 ns; solid line and square, 10 ns; dot line and star, 20 ns. Symbols are not shown in (b). TRANES show two isoemissive points, 500.5 and 436.7 nm (left and right dashed vertical lines, respectively). The spectra are shown in wave number scale to be consistent with the literature.

Figure 11

The correlation of fluorescence lifetimes for mutant TL (5 μm) ANS (15 μm) complexes with fractional intensities (a) and λ_max (b). Symbols: triangle, circle and square represents long, short and free ANS lifetime components.