phalloidine and 5-iodoacetamidofluorescein

To investigate how TM stabilization induced by the Gly126Arg mutation in skeletal α-TM or in smooth muscle β-TM affects the flexibility of TMs and their position on troponin-free thin filaments, we labelled the recombinant wild type and mutant TMs with 5-IAF and F-actin with FITC-phalloidin, incorporated them into ghost muscle fibres and studied polarized fluorescence at different stages of the ATPase cycle. It has been shown that in the myosin- and troponin-free filaments the Gly126Arg mutation causes a shift of TM strands towards the outer domain of actin, reduces the number of switched on actin monomers and decreases the rigidity of the C-terminus of α-TM and increases the rigidity of the N-terminus of β-TMs. The binding of myosin subfragment-1 to the filaments shifted the wild type TMs towards the inner domain of actin, decreased the flexibility of both terminal parts of TMs, and increased the number of switched on actin monomers. Multistep alterations in the position of α- and β-TMs and actin monomers in the filaments and in the flexibility of TMs and F-actin during the ATPase cycle were observed. The Gly126Arg mutation uncouples a correlation between the position of TM and the number of the switched on actin monomers in the filaments.

Topics: Actins; Adenosine Triphosphatases; Amino Acid Substitution; Fluorescein-5-isothiocyanate; Fluoresceins; Humans; Muscle, Skeletal; Muscle, Smooth; Mutation; Phalloidine; Protein Structure, Secondary; Tropomyosin

In helically symmetric protein assemblies, fluorescence resonance energy transfer (FRET) spectroscopy can be used to determine the radial coordinates of fluorescent probes attached to specific amino acid side chains. This is done by separately labeling monomers with donor and acceptor probes, mixing them in different proportions, allowing the mixtures to self-assemble, and then measuring the fluorescence intensity. Provided the helical symmetry is known, and provided the donor- and acceptor-labeled monomers associate randomly, the radial coordinate of the probes can be calculated. Using different probe pairs, two different research groups have employed this method to show that the Cys-374 site of the actin filament (F-actin) is located at a radius of either 35-40 A [Taylor, D.L., Reidler, J., Spudich, J.A., & Stryer, L. (1981) J. Cell Biol. 89, 362-367] or 20-25 A [Kasprzak, A.A., Takashi, R., & Morales, M.F. (1988) Biochemistry 27, 4512-4522]. We have reinvestigated these disparate radius determinations using the same probe pairs employed by these authors with a wide range of acceptor molar fractions. Our results suggest that labeling actin with probes makes the association of monomers significantly nonrandom. This may be avoided by polymerizing actin in the presence of phalloidin. The nonrandomness also can be modeled using stochastic simulation. Taking the average diameters of the probes into account, we conclude that in phalloidin-stabilized F-actin, Cys-374 lies at a radius of (17-18) +/- 5 A. This value is consistent with radial coordinates determined by electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Actins; Amino Acid Sequence; Animals; Cysteine; Electrophoresis, Polyacrylamide Gel; Energy Transfer; Fluoresceins; Fluorescent Dyes; Models, Chemical; Molecular Sequence Data; Muscle, Skeletal; Naphthalenesulfonates; Phalloidine; Polymers; Rabbits; Reference Standards; Spectrometry, Fluorescence; Sulfhydryl Reagents