Fluorescence resonance energy transfer between sites in G‐actin

Intramonomer fluorescence resonance energy transfer spectroscopy was employed to investigate the spatial relationship between labels attached to the residues Cys‐10, Tyr‐69, Cys‐374, the high‐affinity metal binding site and the nucleotide binding site in G‐actin. The separation between the fluorescence donor 5‐(dimethyl‐amino)naphthalene‐1‐sulphonyl (Dns) chloride (dansyl chloride) used to label Tyr‐69 and the acceptor 4‐dimethylaminophenylazophenyl‐4′‐maleimide (DABM) used to label Cys‐374 was found to be 3.6 nm. The distance separating Dns on Tyr‐69 from DABM on Cys‐10 was found to be 2.7 nm. The distance separating the acceptor DABM bound to Cys‐374 from the fluorescence donor formycin A 5′‐triphosphate (FTP) occupying the nucleotide binding site was determined to be 3.0 nm. A slightly larger separation was determined between the FTP site and DABM attached to Cys‐10. In this case a value of 3.2 nm was obtained. The distance separating Dns on Tyr‐69 from Co 2+ in the high‐affinity metal binding site was determined to be 1.1 nm. Finally, the separation of FTP, now acting as donor, from the Dns molecule attached to Tyr‐69 and acting as the acceptor was determined to be 2.1 nm. The likely relationship between these label sites on actin is represented by a model which is used to assist in the determination of the actin structure, with particular reference to the environment of the metal and nucleotide binding sites.