Fig. 1: Comparison of the binding properties of different families of fluorescent ATP analogs to monomeric actin (G-actin) by steady-state fluorescence anisotropy.
From: A functional family of fluorescent nucleotide analogues to investigate actin dynamics and energetics
ATP adenosine triphosphate, εATP N6-etheno-ATP, EDA-ATP 2′(3′)-O-(N-(2-(amino)ethyl)carbamoyl)-ATP. a Skeletal formulas of the families of fluorescent nucleotides tested in this study. Purple, orange, and red ovals indicate positions of the fluorophore. b Binding ability of different ATTO-488 fluorescent ATP analogs (0.2 µM) to G-actin (2 µM) in NFG + MEI buffer, revealed by measuring changes in steady-state anisotropy values 30 min after the initiation of the experiment. Light (resp. dark) gray are values obtained in the absence (resp. presence) of G-actin. Bar graphs indicate mean values and standard deviations. n = 20 for each condition. c Cut-through view of an actin monomer (3DAW, chain A) with bound ATP. Purple, orange, and red ovals represent the positions of the modifications in the ATP molecules shown in panel a. In the case of EDA-ATP (red) and γ-[(6-Amino)hexyl]-ATP (orange), the fluorescent modifications are conjugated to ATP in a way that they point towards the interior of actin, leading to a probable steric clash, whereas in the case of N6-(6-Amino)hexyl-ATP, the fluorescent modification (purple) is expected to stick out from the nucleotide-binding pocket of actin. d One of the theoretically possible configurations of the N6-(6-Amino)hexyl-ATP molecule (cyan), when bound to G-actin (green). Due to the flexible linker between the ATP moiety and the dye, the molecule is expected to obtain multiple different orientations (represented by a funnel). Circled numbers mark the actin subdomains. e Crystal structure of the complex of rabbit muscle N6-(6-Amino)hexyl-ATP-actin (green) and an ADF-H domain from mouse twinfilin (yellow). f Comparison of the positions of nucleotide moieties of ATP-ATTO-488 (gray sticks) in actin (green cartoon) from the crystal structure (6YP9) with the crystal structure of same proteins (3DAW) in the presence of unlabeled ATP (cyan sticks) and actin (cyan cartoon). The sphere represents the metal ion bound to the nucleotide (magenta – this structure, green – 3DAW). Please note that the protein structures and the positions of nucleotides are nearly identical in both cases. Source data are provided as a Source Data file.
