Figure 1.

Fluorescence properties of the dyes Alexa488 and Alexa647 tethered to proteins are sample-dependent due to variations of the local dye environment. Average fluorescence lifetimes, ⟨τ⟩_x, and residual anisotropies, r_∞, of the fluorophores Alexa647 and Alexa488 attached via maleimide or hydroxylamine click chemistry to different amino acids of various proteins (human guanylate binding protein 1, T4 lysozyme, postsynaptic density protein 95, lipase foldase of Pseudomonas aeruginosa and the cyclin-dependent kinase inhibitor 1B). (A) For each sample, the species weighted averaged lifetime ⟨τ⟩_x and r_∞ are shown as dots overlaid by a Gaussian kernel density estimation.⁵⁶ The fluorescence parameters are compiled in Table S1 for Alexa647 and in Table S2 for Alexa488 together with individual fluorescence lifetimes from a detailed decay analysis. Using radiative lifetimes of τ_F = 3.1 ns and τ_F = 4.5 ns for Alexa647⁵⁷ and Alexa488, respectively, the relative brightnesses, ⟨τ⟩_x/τ_F, were calculated. The average values of all Alexa647 and Alexa488 samples are ⟨τ⟩_x/τ_F = 0.43 ± 0.07 and ⟨τ⟩_x/τ_F = 0.76 ± 0.11, respectively. The average residual anisotropies of Alexa647 and Alexa488 for all samples are ⟨r_∞⟩ = 0.25 ± 0.07 and ⟨r_∞⟩ = 0.18 ± 0.05, respectively. (B) The fluorescence intensity decays of the Alexa488 samples were formally resolved into two components τ₁ and τ₂ with the respective fractions x₁ and x₂ = 1 – x₁. For each sample the lifetimes and fractions are shown as open circles overlaid with a Gaussian-kernel density estimation (green). The average lifetimes of the populations are τ₁ = 3.9 ± 0.2 ns and τ₂ = 1.0 ± 0.5 ns with species fractions of x₁ = 0.8 ± 0.1 and x₂ = 0.2 ± 0.1, respectively. The presented data are summarized in Table S2.