Localization of the phalloidin and nucleotide‐binding sites on actin

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Abstract
Phalloidin was found to block nucleotide exchange in F‐actin, without interfering with nucleotide hydrolysis. This inhibition of nucleotide exchange occurs under conditions in which monomers are able to exchange. The distance separating a fluorescent chromophore attached to phalloidin from the nucleotide on actin was determined using fluorescence resonance energy‐transfer spectroscopy. They are separated by less than 1.0 nm. Added confirmation of the close proximity of phalloidin to nucleotide was obtained by extracting a small peptide‐ATP complex from an actin digest. The peptide comprises residues 114–118, which are from the same region as the residues that others have shown to crosslink to phalloidin [Vandekerckhove et al. (1985) EMBO J. 4, 2815–2818]. The results suggest that phalloidin has two major effects. It traps actin monomers in a conformation which appears to be distinct from G‐actin and it stabilizes the structure of F‐actin, an event accompanied by the trapping of ADP.