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

Abstract

lntramonomer fluorescence resonance energy transfer spectroscopy was employed to investigate the spatial relationship between labels attached to the residues Cys-1 0, 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-(dimethylamin0)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.The nucleotide binding site in actin undertakes a central role in the maintenance of the structural integrity of the monomer [l]. In this respect, the nucleotide site in actin differs from the sites in other nucleotide-binding proteins, such as myosin, which appear to display homologous primary structural patterns [2].Although the nucleotide site in G-actin is normally occupied by ATP, other nucleotides which possess useful fluorescent properties can be substituted with very little reduction in the high binding constant displayed by ATP, equal to 7.5 x 10' M-' [3]. Among these is 1,P-ethenoadenosine 5'-triphosphate (EATP) which binds with only a fivefold reduction in affinity [3] and formycin A 5'-triphosphate (FTP) which possesses an affinity about threefold lower than ATP The triphosphate moiety of the bound ATP has been localized to residues 114-118 in actin [5] with the residues 11 6 -11 8 and 121 -124 found to be strongly involved in the binding of ATP and tripolyphosphate [6].The rate of exchange of the G-actin-bound nucleotide is reduced 30-fold in the presence of DNase I [7] while the nucleotide appears to be non-exchangeable following the labelling of Tyr-69 with Dns residues within the actin segment 50-69 [9, 101. The DNase I binding site appears to be largely confined to the outermost rim of the N-terminal side of the cleft between the two major domains in actin [ll]. The same X-ray diffraction study has indicated that the triphosphate moiety of ATP is located deep within the base of the cleft [ l l ] and is sterically blocked by the binding of phalloidin in the cleft [S]. The relationship be...

Structural Implications of the Chemical Modification of Cys10 on Actin

“…These considerations prompted the attempts to label Cys 10 on actin with reporter groups for spectroscopic studies. Specific modification of this residue appeared to be achieved on Cys 374 pre-blocked G-actin in the presence of 2.0 M urea (Barden et al, 1987). However, the distances determined by FRET between Cys 10 and other sites of actin were inconsistent with the atomic structure of G-actin suggesting that the exposure to urea might have altered its structure irreversibly (O'Donoghue et al, 1992).…”

“…Because of its reactivity and accessibility to reagents Cys 374 on the actin C-terminus has been the main site for attachment of fluorescent and spin probes on ␣-actin. Other sites of actin labeling, Lys-61 and Tyr-69, although useful for fluorescence energy transfer (FRET) measurements (Barden et al, 1987;Miki et al, 1992), are involved in actin polymerization (Miki, 1987;Holmes et al, 1990;Chantler and Gratzer, 1975) and their derivatization affects actin properties. The labeling of Gln-41 on actin with a dansyl probe appears to have no effect on actomyosin interactions (Kim et al, 1998), but the transglutaminase mediated reaction does not offer the same labeling flexibility and choices as cysteine modifications do.…”