Characterization of Skeletal Muscle Actin Labeled with the Triplet Probe ERYTHROSIN‐5‐IODOACETAMIDE (original) (raw)
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Intrinsic fluorescence of actin
Biochemistry, 1972
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 . This denatured form of actin (d-actin) appears, on the basis of optical rotatory dispersion and circular dichroism studies , 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 .
European Journal of Biochemistry, 1987
Modification of Lys-61 in actin with fluorescein-5-isothiocyanate (FITC) blocks actin polymerization Biochim. Biophys. Acta 791, 57-62]. FITC-labelled actin recovered its ability to polymerize on addition of phalloidin. The polymers had the same characteristic helical thread-like structure as normal F-actin and the addition of myosin subfragment-1 to the polymers formed the characteristic arrowhead structure in electron microscopy. The polymers activated the ATPase activity of myosin subfragment-I as efficiently as normal F-actin. These results indicate that Lys-61 is not directly involved in an actin-actin binding region nor in myosin binding site.
European Journal of Biochemistry, 1983
The interaction of regulated and unregulated actin of myosin-free ghost single fibre with myosin subfragment-1 free of 5,5'-dithiobis(2-nitrobenzoic acid) light chains was investigated by polarized microphotometry. The anisotropy of intrinsic tryptophan fluorescence of regulated actin is Ca2 +-dependent and has a maximal value at low (pCa2 7) and a minimal value at high ( p C a I 6) concentrations of Ca2+. The interaction of myosin subfragment-1 with actin induces cooperative changes in actin structure, which manifest themselves in a decrease in the anisotropy of tryptophan fluorescence. The cooperativity of conformational changes in actin, induced by myosin subfragment-l , is high for regulated actin in the absence of Ca2+ and low for unregulated and regulated actin in the presence of Ca2+. The data obtained suggest that the decrease of the flexibility of actin filaments, induced by tropomyosin or by Caunsaturated troponin-tropomyosin complex, results in increased cooperativity of conformational changes of actin induced by myosin subfragment-1.
Interaction of actin with dansyl-tropomyosin
FEBS Letters, 1986
chloride (dansyl chloride) reacts with rabbit skeletal muscle tropomyosin (TM) to yield a highly fluorescent product, DNS-TM. The extent of modification of TM can be regulated over a wide range, 0.3-15.5 dansyl groups per TM, depending upon the temperature and duration of the reaction. However, under all conditions employed, about 15 different fluorescent tryptic peptides of TM were produced. DNS-TM undergoes end-to-end polymerization at low ionic strengths, but to a somewhat lesser extent than unlabelled TM does. DNS-TM also binds muscle F-actin. This interaction may be followed fluorimetrically by observing a blue-shift in emission maximum, an increase in fluorescence intensity or an increase in fluorescence polarization of the DNS-TM complex with F-actin. Actin Tropomyosin Fluorescence polarization Polymerization Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/86/$3.50 0 1986 Federation of European Biochemical Societies
Tropomyosin-Troponin Regulation of Actin Does Not Involve Subdomain 2 Motions
Journal of Biological Chemistry, 2001
Dynamic properties of F-actin structure prompted suggestions (Squire, J. M., and Morris, E. P. (1998) FASEB J. 12, 761-771) that actin subdomain 2 movements play a role in thin-filament regulation. Using fluorescently labeled yeast actin mutants Q41C, Q41C/C374S, and D51C/C374S and azidonitrophenyl putrescine (ANP) Gln 41 -labeled ␣-actin, we monitored regulation-linked changes in subdomain 2. These actins had fully regulated acto-S1 ATPase activities, and emission spectra of regulated Q41C AEDANS /C374S and D51C AEDANS /C374S filaments did not reveal any calciumdependent changes. Fluorescence energy transfer in these F-actins mostly occurred from Trp 340 and Trp 356 to 5-(2((acetyl)amino)ethyl)amino-naphthalene-1-sulfonate (AEDANS)-labeled Cys 41 or Cys 51 of adjacent same strand protomers. Our results show that fluorescence energy transfer between these residues is similar in the mostly blocked (؊Ca 2؉ ) and closed (؉Ca 2؉ ) states. Ca 2؉ also had no effect on the excimer band in the pyrenelabeled Q41C-regulated actin, indicating virtually no change in the overlap of pyrenes on Cys 41 and Cys 374 . ANP quenching of rhodamine phalloidin fluorescence showed that neither Ca 2؉ nor S1 binding to regulated ␣-actin affects the phalloidin-probe distance. Taken together, our results indicate that transitions between the blocked, closed, and open regulatory states involve no significant subdomain 2 movements, and, since the cross-linked ␣-actin remains fully regulated, that subdomain 2 motions are not essential for actin regulation.
Biochemistry, 1999
We have investigated how Ca 2+ or Mg 2+ bound at the high-affinity cation binding site in F-actin modulates the dynamic response of these filaments to ATP hydrolysis by attached myosin head fragments (S1). Rotational motions of the filaments were monitored using steady-state phosphorescence emission anisotropy of the triplet probe erythrosin-5-iodoacetamide covalently attached to cysteine 374 of actin. The anisotropy of filaments containing only Ca 2+ increased from 0.080 to 0.137 upon binding S1 in a rigor complex and decreased to 0.065 in the presence of ATP, indicating that S1 induced additional rotational motions in the filament during ATP hydrolysis. The comparable anisotropy values for Mg 2+containing filaments were 0.067, 0.137, and 0.065, indicating that S1 hydrolysis did not induce measurable rotational motions in these filaments. Phalloidin, a fungal toxin which stabilizes F-actin and increases its rigidity, increased the anisotropy of F-actin containing either Ca 2+ or Mg 2+ but not the anisotropy of the 1:1 S1-actin complexes of these filaments. Mg 2+-containing filaments with phalloidin bound also displayed increased rotational motions during S1 ATP hydrolysis. A strong positive correlation between the phosphorescence anisotropy of F-actin under specific conditions and the extent of the rotational motions induced by S1 during ATP hydrolysis suggested that the long axis torsional rigidity of F-actin plays a crucial role in modulating the dynamic response of the filaments to ATP hydrolysis by S1. Cooperative responses of F-actin to dynamic perturbations induced by S1 during ATP hydrolysis may thus be physically mediated by the torsional rigidity of the filament.
C-terminal actin-binding sites of smooth muscle caldesmon switch actin between conformational states
The International Journal of Biochemistry & Cell Biology, 2001
Caldesmon is a component of the thin filaments of smooth muscles where it is believed to play an essential role in regulating the thin filaments' interaction with myosin and hence contractility. We studied the effects of caldesmon and two recombinant fragments CaDH1 (residues 506-793) and CaDH2 (residues 683-767) on the structure of actin-tropomyosin by making measurements of the fluorescence polarisation of probes specifically attached to actin. CaDH1, like the parent molecule caldesmon, is an inhibitor of actin-tropomyosin interaction with myosin whilst CaDH2 is an activator. The F-actin in permeabilised and myosin free rabbit skeletal muscle 'ghost' fibres was labelled by tetramethyl rhodamine-isothiocyanate (TRITC)-phalloidin or fluorescein-5%-isothiocyanate (FITC) at lysine 61. Fluorescence polarisation measurements were made and the parameters b A , b E , [ 1/2 and N were calculated. b A and b E are angles between the fiber axis and the absorption and emission dipoles, respectively; [ 1/2 is the angle between the F-actin filament axis and the fiber axis; N is the relative number of randomly oriented fluorophores. Actin-tropomyosin interaction with myosin subfragment-1 induced changes in the parameters of the polarised fluorescence that are typical of strong binding of myosin to actin and of the 'on' conformational state of actin. Caldesmon and CaDH1 (as well as troponin in the absence of Ca 2 + ) diminished the effect of S-1, whereas CaDH2 (as well as troponin in the presence of Ca 2 + ) enhanced the effect of S1. Thus the structural evidence correlates with biochemical evidence that C-terminal actin-binding sites of caldesmon can modulate the structural transition of actin monomers between 'off' (caldesmon and CaDH1) and 'on' (S-1 and CaDH2) states in a manner analogous to troponin.
Biophysical Journal, 1996
Actin labeled at Gln-41 with dansyl ethylenediamine (DED) via transglutaminase reaction was used for monitoring the interaction of myosin subfragment 1 (Si) with the His-40-Gly-42 site in the 38-52 loop on F-actin. Proteolytic digestions of F-actin with subtilisin and trypsin, and acto-Sl ATPase measurements on heat-treated F-actin revealed that the labeling of Gln-41 had a stabilizing effect on subdomain 2 and the actin filaments. DED on Gln-41 had no effect on the values of Km and Vmax of the acto-Sl ATPase and the sliding velocities of actin filaments in the in vitro motility assays. This suggests either that Si does not bind to the 40-42 site on actin or that such binding is not functionally important. The binding of monoclonal antidansyl IgG to DED-F-actin did not affect acto-Si binding in the absence of nucleotides, indicating that the 40-42 site does not contribute much to rigor acto-Si binding. Myosin-induced changes in subdomain 2 on actin were manifested through an increase in the fluorescence of DED-F-actin, a decrease in the accessibility of the probe to collisional quenchers, and a partial displacement of antidansyl IgG from actin by Si. It is proposed that these changes in the 38-52 loop on actin originate from S1 binding to other myosin recognition sites on actin.