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

Autor:	Chou SZ; Department of Molecular Cellular and Developmental Biology, Yale University, PO Box 208103, New Haven, CT, 06520-8103, USA., Pollard TD; Department of Molecular Cellular and Developmental Biology, Yale University, PO Box 208103, New Haven, CT, 06520-8103, USA. thomas.pollard@yale.edu.; Department of Molecular Biophysics and Biochemistry, Yale University, PO Box 208103, New Haven, CT, 06520-8103, USA. thomas.pollard@yale.edu.; Department of Cell Biology, Yale University, PO Box 208103, New Haven, CT, 06520-8103, USA. thomas.pollard@yale.edu.
Jazyk:	angličtina
Zdroj:	Nature communications [Nat Commun] 2020 Nov 19; Vol. 11 (1), pp. 5897. Date of Electronic Publication: 2020 Nov 19.
DOI:	10.1038/s41467-020-19762-1
Abstrakt:	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.
Databáze:	MEDLINE
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