Since the fluorescent reagent N-(1-pyrene)iodoacetamide was first used to label
skeletal muscle actin in 1981, the pyrene-labeled actin has become the most
widely employed tool to measure the kinetics of actin polymerization and the
interaction between actin and actin-binding proteins. Here we report
high-resolution cryo-electron microscopy structures of actin filaments with
N-1-pyrene conjugated to cysteine 374 and either ADP (3.2 Å) or ADP-phosphate
(3.0 Å) in the active site. Polymerization buries pyrene in a hydrophobic
cavity between subunits along the long-pitch helix with only minor differences
in conformation compared with native actin filaments. These structures explain
how polymerization increases the fluorescence 20-fold, how myosin and cofilin
binding to filaments reduces the fluorescence, and how profilin binding to actin
monomers increases the fluorescence.
