Fluorescence resonance energy transfer between sites in G-actin. The spatial relationship between Cys-10, Tyr-69, Cys-374, the high-affinity metal and the nucleotide.
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-(dimethylamino)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 Co2+ 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.
Barden
J A
JA
Department of Anatomy, University of Sydney, New South Wales, Australia.
dos Remedios
C G
CG
D000199:Actins / Q000032:analysis*; D000345:Affinity Labels; D001665:Binding Sites; D003545:Cysteine / Q000032:analysis; D004735:Energy Transfer; D008433:Mathematics; D008670:Metals / Q000032:analysis*; D009711:Nucleotides / Q000032:analysis*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000032:analysis
D000199:Actins; D000345:Affinity Labels; D008670:Metals; D009711:Nucleotides; D014443:Tyrosine; D003545:Cysteine