Cryo-electron microscopy structures of pyrene-labeled ADP-Pi- and ADP-actin filaments

Steven Z Chou,Thomas D Pollard

doi:10.1038/s41467-020-19762-1

Abstract

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.

Highlights

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 actinbinding proteins
We provide high-resolution cryo-electron microscopy structures of pyrene-labeled ADP-Pi- and ADP-actin filaments, which explain these mechanisms and show how the presence of pyrene modifies the filament structure in small ways and why other dyes coupled to C374 compromise polymerization
Image processing of electron micrographs of 411,301 particles of Mg-ADP-Pi-pyrenyl-actin filaments produced an electron potential map with an overall resolution of 3.0 Å and 240,254 particles of Mg-ADP-pyrenyl-actin filaments gave a map with an overall resolution of 3.2 Å (Supplementary Fig. 2; Supplementary Table 1)

Summary

Introduction

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 actinbinding proteins. 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. Many of these cellular processes are achieved by the polymerization and depolymerization of actin filaments and the interaction between actin and actin-binding proteins To study these molecular events, many fluorescent reagents have been developed for labeling actin either covalently or non-covalently. We report that phosphate-binding increases the fluorescence of Mg-ADP-pyrenyl-actin filaments

Methods

Results

Conclusion