Intrinsic fluorescence of actin

Abstract

A study has been made of the intrinsic fluorescence associated with G-and F-actin, with EDTA-and heat-denatured actin, and with actin in 8 M urea. A small decrease in the Trp and an increase in the Tyr contribution is associated with the polymerization of G-to F-actin. An appreciable red shift of the fluorescence spectrum occurs when G-or F-actin is denatured, indicating increased exposure of Trp to the aqueous environment, This change in fluorescence produced by the addition of EDTA can be used as a quick measure of the fraction of denatured species associated with a given actin preparation. Attempts to regenerate native actin from denatured actin have been unsuccessful. The fluorescence A ctin, one of the major protein components of the myofibril involved in the process of muscle contraction, is capable of existing in different states. G-actin, the monomeric native form of actin, has a tightly bound nucleotide and a tightly bound Ca2+ or Mgz+ normally found associated with it. If the nucleotide or metal ion is removed, the protein irreversibly denatures (Asakura, 1961 ;Maruyama and Gergely, 1961). This denatured form of actin (d-actin) appears, on the basis of optical rotatory dispersion and circular dichroism studies (Nagy and Jencks, 1962;, to be only partially unfolded compared with actin in 8 M urea (u-actin). F-actin, the fibrous form which exists in the muscle, can be formed from G-actin but not form d-actin by a reversible salt-induced polymerization. Although this reversible polymerization does not appear to have a major role in muscle contraction, localized dislocations of the actin filament may have some importance (Oosawa et al., 1961 ;Asakura et al., 1963;Szent-Gyorgyi and Prior, 1966).The purpose of the present study is to characterize the intrinsic fluorescence properties of the different states of actin in order to further obtain information regarding conformational differences. We found that small differences exist between G-and F-actin with respect to the contributions of both Tyr and Trp to the intrinsic fluorescence when proper corrections for light scattering were made. Studies of the temperature dependence of the fluorescence indicate qualitative and quantitative differences between G-and F-actin in their stability toward heat denaturation. The large fluorescence spectral shift that occurs when G-or F-actin is denatured indicates an increase in average exposure of Trp side chains to the aqueous solvent. The quenching of the fluorescence of G-and F-actin due to the binding of Cu2+ appears to involve energy transfer to a new absorption band associated with the

Conformational changes in subdomain I of actin induced by proteolytic cleavage within the DNase I‐binding loop: energy transfer from tryptophan to AEDANS

“…These data, taken together, provide evidence for high conformational coupling within the 'small' domain, which is responsible for interaction with many actin-binding proteins. The position of the intrinsic tryptophan residues in the focus of these transitions could explain the high sensitivity of tryptophan fluorescence to actin polymerization and modifications of the polypeptide chain [19,20].…”

The limiting rate of the ATP‐mediated dissociation of actin from rabbit skeletal muscle myosin subfragment 1

“…Earlier studies have measured the reaction directly using slow muscle F actin was prepared from an acetone powder as in [8] and concentrations were determined from E%$ = 11.08 cm-' and M, = 42000 [9]. Myosin subfragment 1 was prepared from rabbit skeletal muscle as in [lo] and concentrations are quoted on the basis of M, = 115000 and Eir8 = 7.9 cm-'.…”

Fluorescence energy transfer between the myosin subfragment‐1 isoenzymes and F‐actin in the absence and presence of nucleotides

“…ml-' . cm-' [35]. Labelled protein concentrations were determined by the microtannin turbidity method [36] using S1 and actin samples of known concentrations as standards.…”