Fig. 2: Structural basis of mutation-induced fluorescence lifetimes variation within the FAST variants.

a, b Correlations between the lifetimes of major long-lived exponential components revealed after the measurement of FAST-fluorogens’ fluorescence decays (τ₁ from Table 1) and the rates of radiative (a) and non-radiative (b) transitions calculated for individual protein-fluorogen pairs. The data sets include original FAST (circles) and its variants (R52K, triangles; F62L, diamonds; P68K, squares; and P68T, crosses) with four ligands: HBR-2,5-DM (blue), HBR-DOM2 (orange), HMBR (magenta), and 25DOM-HBI-2T (red). Dashed lines represent the correlation curve obtained for each ligand. c–f PyMOL generated models showing the possible effect of mutations, constructed based on the spatial structure of the nanoFAST/HBR-DOM2 complex (PDB code A8O0)³⁵. The sidechain of the original FAST amino acid is shown in gray, and the result of the mutation is colored. The ligand molecule is shown in cyan.