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

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.

SUBMITTER: Chou SZ

PROVIDER: S-EPMC7677365 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Cryo-electron microscopy structures of pyrene-labeled ADP-P<sub>i</sub>- and ADP-actin filaments.

Chou Steven Z SZ Pollard Thomas D TD

Nature communications 20201119 1

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 b ...[more]

PMID: 33214556

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