Journal: Nature Communications

Article Title: Cryo-electron microscopy structures of pyrene-labeled ADP-P i - and ADP-actin filaments

doi: 10.1038/s41467-020-19762-1

Figure Lengend Snippet: a Phosphate slows the labeling of ADP-actin filaments with N-(1-pyrene)iodoacetamide. Solutions of Mg-ADP-actin filaments were polymerized from 5 µM monomers overnight at 4 °C in 100 mM KCl; 1 mM MgCl 2 ; 10 mM imidazole, pH 7.0; 0.3 mM ADP; 3 mM NaN 3 and preincubated at room temperature with the same volume of either water, 20 mM potassium phosphate or 20 mM potassium sulfate before adding 50 µM N-(1-pyrene)iodoacetamide. The basal fluorescence is from the free N-(1-pyrene)iodoacetamide, and the fluorescence increase is due to the conjugation of N-(1-pyrene)iodoacetamide to the sidechain of C374 in actin filaments. b Effect of phosphate in the buffer on the fluorescence of Mg-ADP-pyrenyl-actin filaments. The fluorescence change in each data point was calculated by subtracting the fluorescence of 120 µL of Mg-ADP-pyrenyl-actin filaments (polymerized from 5 µM monomers) preincubated with 30 µL of water from the fluorescence of 120 µL of Mg-ADP-pyrenyl-actin filaments (polymerized from 5 µM monomers) preincubated with the same volume of phosphate or sulfate. Each sample was incubated for ~1 hour before the measurements. Error bars indicate the standard deviations of five readings on the same sample. The Y-axes are in arbitrary units (A.U.). Data are presented as mean values +/− SD. Source data are provided as a Source Data file.

Article Snippet: The fluorescence graphs (Fig. ) were plotted with OriginLab.

Techniques: Labeling, Fluorescence, Conjugation Assay, Incubation